Diindolylmethane (DIM) Information Resource Center

This section is updated regularly. Paper abstracts are presented in chronological order from most recent.

Anticancer Drugs. 2020 Dec 10. doi: 10.1097

3,3'-diindolylmethane exerts antiproliferation and apoptosis induction by TRAF2-p38 axis in gastric cancer.

Ye Y(1), Ye F(1)(2), Li X(1), Yang Q(3), Zhou J(4), Xu W(5), Aschner M(6), Lu R(1)(7), Miao S(8).

Author information: (1)Department of Preventive Medicine and Public Health Laboratory Science, School of Medicine, Jiangsu University, Zhenjiang. (2)Department of Clinical Laboratory Center, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing. (3)Department of Pathology, Zhenjiang First People's Hospital, Zhenjiang. (4)Department of Molecular Cell Biology and Toxicology, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing. (5)Key Laboratory of Medical Science and Laboratory Medicine of Jiangsu Province, School of Medicine, Jiangsu University, Zhenjiang, China. (6)Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, USA. (7)Center for Experimental Research, Affiliated Kunshan Hospital to Jiangsu University School of Medicine, Kunshan, Suzhou, Jiangsu. (8)Department of Health Care, Zhenjiang Fourth Peoples Hospital, Zhenjiang, China.

3,3'-diindolylmethane (DIM), an active phytochemical derivative extracted from cruciferous vegetables, possesses anticancer effects. However, the underlying anticancer mechanism of DIM in gastric cancer remains unknown. Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), one of the signal transduction proteins, plays critical role in proliferation and apoptosis of human gastric cancer cells, but there are still lack of practical pharmacological modulators for potential clinical application. Here, we further explored the role of TRAF2 in inhibiting cell proliferation and inducing apoptosis by DIM in human gastric cancer BGC-823 and SGC-7901 cells. After treating BGC-823 and SGC-7901 cells with DIM for 24 h, cell proliferation, apoptosis and TRAF2-related protein were measured. Our findings showed that DIM inhibited the expressions of TRAF2, activated p-p38 and its downstream protein p-p53, which were paralleled with DIM-triggered cells proliferation, inhibition and apoptosis induction. These effects of DIM were reversed by TRAF2 overexpression or p38 mitogen-activated protein kinase (MAPK)-specific inhibitor (SB203580). Taken together, our data suggest that regulating TRAF2/p38 MAPK signaling pathway is essential for inhibiting gastric cancer proliferation and inducing apoptosis by DIM. These findings broaden the understanding of the pharmacological mechanism of DIM's action as a new modulator of TRAF2, and provide a new therapeutic target for human gastric cancer.

Mol Immunol. 2020 Dec 10:S0161-5890(20)30543-5. doi: 10.1016/j.molimm.2020.11.013. Online ahead of print.

3,3'-Diindolylmethane alleviates acute atopic dermatitis by regulating T cell differentiation in a mouse model.

Wu X(1), Liu J(1), Chen C(1), Huang Z(1), Zang Y(1), Chen J(1), Dong L(1), Zhang J(2), Ding Z(3).

Author information: (1)State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Xianlin Avenue 163, Nanjing, 210023, Jiangsu, China. (2)State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Xianlin Avenue 163, Nanjing, 210023, Jiangsu, China; Collaborative Innovation Center of Chemistry for Life Sciences, Nanjing University, Hankou Road 22, Nanjing, 210093, Jiangsu, China. Electronic address: [email protected]. (3)State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Xianlin Avenue 163, Nanjing, 210023, Jiangsu, China; Changzhou High-Tech Research Institute of Nanjing University, Changzhou, 213164, China. Electronic address: [email protected].

Atopic dermatitis is a severe, chronic relapsing inflammatory disease of the skin with family clustering. It is characterized into acute phase, which is dominated by T helper 2-type immune responses, and chronic phase, which is dominated by T helper 1-type immune responses. Studies have shown that 3,3'-diindolylmethane not only has antitumor effects but also can relieve symptoms of inflammatory diseases by inhibiting the nuclear factor-κB signaling pathway and regulating T cell differentiation. To study the effect of 3,3'-diindolylmethane on atopic dermatitis and the underlying mechanism, a mouse model of acute atopic dermatitis was established using 2,4-dinitrofluorobenzene. After intraperitoneal injection of 3,3'-diindolylmethane, skin erythema and edema in mice were significantly alleviated. Furthermore, 3,3'-diindolylmethane reduced immune activation, probably by inhibiting the secretion of thymic stromal lymphopoietin by keratinocytes. 3,3'-Diindolylmethane also promoted the differentiation of regulatory T cells and inhibited the activation of T helper 2 and T helper 17 cells to reduce atopic dermatitis-related immune responses. However, it showed no significant effect on the differentiation of T helper 1 cells. These results indicate that 3,3'-diindolylmethane has a significant inhibitory effect on T helper 2 cells in the acute phase of atopic dermatitis. Our findings may provide not only more insights into the pathological mechanism of AD, but also a new candidate medicine for it.

J Med Chem. 2020 Dec 2. doi: 10.1021/acs.jmedchem.0c01378. Online ahead of print.

Modulation of the G-Protein-Coupled Receptor 84 (GPR84) by Agonists and Antagonists.

Chen LH(1), Zhang Q(2), Xie X(2), Nan FJ(1)(3).

Author information: (1)State Key Laboratory of Drug Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. (2)CAS Key Laboratory of Receptor Research, National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. (3)Yantai Institute of Materia Medica, Shandong 264000, China.

Since the discovery of medium-chain fatty acids as GPR84 ligands, significant advancements have been made in the development of GPR84 agonists and antagonists. Most agonists have lipid-like structures except for 3,3'-diindolylmethane (DIM), which acts as an allosteric agonist. GPR84 activation in macrophages leads to increased cytokine secretion, chemotaxis, and phagocytosis, revealing the proinflammatory role of GPR84 associated with various inflammatory responses. Three GPR84 antagonists (S)-2-((1,4-dioxan-2-yl)methoxy)-9-(cyclopropylethynyl)-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one (GLPG1205), sodium 2-(3-pentylphenyl)acetate (PBI-4050), and sodium 2-(3,5-dipentylphenyl)acetate (PBI-4547) have displayed therapeutic effects in animal models of several inflammatory and fibrotic diseases and are being evaluated in clinical studies. Although GLPG1205 has failed in a clinical trial for ulcerative colitis, it is undergoing another phase II clinical study for idiopathic pulmonary fibrosis. Further studies are needed to resolve the GPR84 structure, identify more endogenous ligands, elucidate their physiological and pathological roles, and fulfill the therapeutic potential of GPR84 antagonists and agonists.

J Med Food. 2020 Dec;23(12):1248-1258. doi: 10.1089/jmf.2020.4704. Epub 2020 Nov 25.

PTEN/Akt Signaling-Mediated Activation of the Mitochondrial Pathway Contributes to the 3,3'-Diindolylmethane-Mediated Antitumor Effect in Malignant Melanoma Cells.

Wang X(1), Zhao Y(1), Yu M(1), Xu Y(2).

Author information: (1)Department of Plastic Surgery and Renmin Hospital of Wuhan University, Wuhan, China. (2)Department of Otolaryngology, Renmin Hospital of Wuhan University, Wuhan, China.

3,3'-diindolylmethane (DIM) has an anticancer activity, but the role DIM plays on malignant melanoma cells and its specific mechanism is unclear. We studied the biological effects of DIM on malignant melanoma cells and the related mechanism and the results showed that DIM significantly suppressed cell proliferation and induced apoptosis in malignant melanoma cells. In addition, the expression levels of phosphatase and tensin homolog deleted on chromosome ten (PTEN), Bax, Bid, cleaved caspase-3, and cleaved caspase-9 were increased after DIM treatment. In A2058 PTENmut cells, DIM-mediated inhibition of proliferation and DIM-induced apoptosis were attenuated. Additionally, the overexpression and knockdown of PTEN could regulate such effects of DIM in malignant melanoma cells. Furthermore, DIM exerted growth-inhibiting and apoptosis-inducing effects in vivo. This study demonstrated that DIM has antitumor effect in human malignant melanoma cells through the mitochondrial apoptotic pathway activated by PTEN/Akt signaling.

DOI: 10.1089/jmf.2020.4704 PMID: 33237846

PLoS One. 2020 Nov 11;15(11):e0242198. doi: 10.1371/journal.pone.0242198. eCollection 2020.

Anti-inflammatory activity of diindolylmethane alleviates Riemerella anatipestifer infection in ducks.

Fernandez-Colorado CP(1)(2), Cammayo PLT(2), Flores RA(2), Nguyen BT(2), Kim WH(3), Kim S(2), Lillehoj HS(3), Min W(2).

Author information: (1)Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, College, Laguna, Philippines. (2)College of Veterinary Medicine & Institute of Animal Medicine, Gyeongsang National University, Jinju, Republic of Korea. (3)Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD, United States of America.

3,3'-Diindolylmethane (DIM) is found in cruciferous vegetables and is used to treat various inflammatory diseases because of its potential anti-inflammatory effects. To investigate effects of DIM in Riemerella anatipestifer-infected ducks which induce upregulation of inflammatory cytokines, ducks were treated orally with DIM at dose of 200 mg/kg/day and infected the following day with R. anatipestifer. Infected and DIM-treated ducks exhibited 14% increased survival rate and significantly decreased bacterial burden compared to infected untreated ducks. Next, the effect on the expression level of inflammatory cytokines (interleukin [IL]-17A, IL-17F, IL-6, IL-1β) of both in vitro and in vivo DIM-treated groups was monitored by quantitative reverse-transcription PCR (qRT-PCR). Generally, the expression levels of the cytokines were significantly reduced in DIM-treated splenic lymphocytes stimulated with killed R. anatipestifer compared to stimulated untreated splenic lymphocytes. Similarly, the expression levels of the cytokines were significantly reduced in the spleens and livers of DIM-treated R. anatipestifer-infected ducks compared to infected untreated ducks. This study demonstrated the ameliorative effects of DIM in ducks infected with R. anatipestifer. Thus, DIM can potentially be used to prevent and/or treat R. anatipestifer infection via inhibition of inflammatory cytokine expression.

Cell Immunol. 2020 Dec;358:104238. doi: 10.1016/j.cellimm.2020.104238. Epub 2020 Oct 17.

DIM mitigates the development of experimental autoimmune encephalomyelitis by maintaining the stability and suppressive function of regulatory T cells.

Yang S(1), Tan L(1), Chen Y(2), Liu A(1), Hong M(1), Peng Z(3).

Author information: (1)Department of Neurology, First Affiliated Hospital of Guangdong Pharmaceutical College, Guangzhou 510000, China. (2)Department of Neurology, The People's Hospital of Linying County, Luohe 462600, China. (3)Department of Neurology, First Affiliated Hospital of Guangdong Pharmaceutical College, Guangzhou 510000, China. Electronic address: [email protected].

Recent studies have revealed that indoles, dietary ligands of the aryl hydrocarbon receptor (AhR), have immunomodulatory characteristics of balancing the differentiation of regulatory T cells (Tregs) and Th17 cells in multiple autoimmune diseases. In this study, we aimed to investigate the potency of the indole, 3,3'-diindolylmethane (DIM), on the stability and suppressive function of Tregs in experimental autoimmune encephalomyelitis (EAE). Furthermore, we used the AhR antagonist CH223191 to verify that DIM exerts its effects on Tregs through the activation of AhR. We found that DIM treatment significantly alleviated the severity of EAE by maintaining the stability and suppressive function of Tregs instead of facilitating the differentiation of Tregs. Thus, these DIM-treated Tregs might indirectly inhibit the generation of Th17 cells and the production of proinflammatory cytokines. And we confirmed the critical role of AhR in the EAE model. Our study further investigated the mechanisms by which dietary indoles promote Treg activity in the EAE model. DIM may act as a novel therapeutic to restrain autoimmune inflammation in multiple sclerosis.

Biochimie. 2020 Dec;179:217-227. doi: 10.1016/j.biochi.2020.10.007. Epub 2020 Oct 22.

Breast cancer organoid model allowed to reveal potentially beneficial combinations of 3,3'-diindolylmethane and chemotherapy drugs.

Nikulin SV(1), Alekseev BY(2), Sergeeva NS(2), Karalkin PA(2), Nezhurina EK(2), Kirsanova VA(2), Sviridova IK(2), Akhmedova SA(2), Volchenko NN(2), Bolotina LV(2), Osipyants AI(3), Hushpulian DM(4), Topchiy MA(5), Asachenko AF(6), Koval AP(7), Shcherbo DS(7), Kiselev VI(8), Mikhaylenko DS(9), Schumacher U(10), Poloznikov AA(11).

Author information: (1)Faculty of Biology and Biotechnologies, Higher School of Economics, Moscow, 101000, Russia; P. A. Hertsen Moscow Oncology Research Center, Branch of the National Medical Research Radiological Center, Ministry of Health of the Russian Federation, Moscow, 125284, Russia. (2)P. A. Hertsen Moscow Oncology Research Center, Branch of the National Medical Research Radiological Center, Ministry of Health of the Russian Federation, Moscow, 125284, Russia. (3)School of Biomedicine, Far Eastern Federal University, Vladivostok, 690091, Russia. (4)School of Biomedicine, Far Eastern Federal University, Vladivostok, 690091, Russia; Institute of Nanotechnology of Microelectronics, 32A Leninsky Prospekt, Moscow, 119991, Russia. (5)A. V. Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Moscow, 119991, Russia. (6)A. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilov str. 28, Moscow, 119991, Russia. (7)Molecular Oncology Laboratory, Institute of Translational Medicine, Pirogov Russian National Research Medical University, Moscow, 117997, Russia. (8)National Medical Research Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov, Moscow, 117997, Russia. (9)Institute of Molecular Medicine, Biomedical Science and Technology Park, I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, 119991, Russia; Research Centre for Medical Genetics, Moscow, 115522, Russia. (10)Institute of Anatomy and Experimental Morphology, University Medical Center, Hamburg-Eppendorf, Hamburg, 20246, Germany. (11)Faculty of Biology and Biotechnologies, Higher School of Economics, Moscow, 101000, Russia; P. A. Hertsen Moscow Oncology Research Center, Branch of the National Medical Research Radiological Center, Ministry of Health of the Russian Federation, Moscow, 125284, Russia. Electronic address: [email protected].

Epigenetic alterations represent promising therapeutic targets in cancer treatment. Recently it was revealed that small molecules have the potential to act as microRNA silencers. Capacity to bind the discrete stem-looped structure of pre-miR-21 and prevent its maturation opens opportunities to utilize such compounds for the prevention of initiation, progression, and chemoresistance of cancer. Molecular simulations performed earlier identified 3,3'-diindolylmethane (DIM) as a potent microRNA-21 antagonist. However, data on DIM and microRNA-21 interplay is controversial, which may be caused by the limitations of the cell lines.

Life Sci. 2020 Nov 15;261:118348. doi: 10.1016/j.lfs.2020.118348. Epub 2020 Aug 26.

Inhibition of endogenous hydrogen sulfide biosynthesis enhances the anti-cancer effect of 3,3'-diindolylmethane in human gastric cancer cells.

Ye F(1), Li X(2), Sun K(3), Xu W(4), Shi H(5), Bian J(6), Lu R(7), Ye Y(8).

Author information: (1)Department of Preventive Medicine and Public Health Laboratory Science, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China; Department of Clinical Laboratory Center, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing, Zhejiang, China. (2)Department of Preventive Medicine and Public Health Laboratory Science, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China. (3)Department of Gastrointestinal Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu, China. (4)Key Laboratory of Medical Science and Laboratory Medicine of Jiangsu Province, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China. (5)School of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu, China. (6)Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. (7)Department of Preventive Medicine and Public Health Laboratory Science, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China; Center for Experimental Research, Affiliated Kunshan Hospital, Jiangsu University, Kunshan, Suzhou, Jiangsu, China. (8)Department of Preventive Medicine and Public Health Laboratory Science, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China. Electronic address: [email protected].

AIMS: 3,3'-Diindolylmethane (DIM) has limited anti-cancer effects in gastric cancer. Hydrogen sulfide (H2S) plays an important role in the tumor development and therapy, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), two key endogenous H2S biosynthesis enzymes, can affect endogenous H2S levels and alter cancer treatment. Our main objective was to investigate whether the aminooxyacetic acid (AOAA) and DL-Propargylglycine (PAG), two specific inhibitors of CBS and CSE, could assist DIM to exert a stronger anti-cancer effects in gastric cancer BGC-823 and SGC-7901 cells. MATERIALS AND METHODS: Cell proliferation was assayed by MTT and cell colony-forming assay. Apoptosis and migration were detected by Hoechst staining and scratch test respectively. Western blot was used to evaluate the expression of proteins related to proliferation, apoptosis and migration. KEY FINDINGS: Combination of AOAA or PAG with DIM synergistically inhibited proliferation and migration, increased apoptosis in gastric cancer cells. The p38-p53 axis was also further activated by the combination of AOAA or PAG with DIM. Exogenous H2S from sodium hydrosulfide, attenuated the efficacy of DIM in cancer cells by reducing the activation level of p38-p53 axis. Taken together, AOAA or PAG inhibited the expression of endogenous H2S biosynthesis enzymes and effectively enhanced susceptibility of gastric cancer to DIM through activating p38-p53 axis. SIGNIFICANCE: The current study highlight more precise requirements for the clinical application of sulfur-containing anti-cancer drugs, and open a new way to enhance the sensitivity of DIM in chemotherapy of gastric cancer.

J Microencapsul. 2020 Nov;37(7):528-541. doi: 10.1080/02652048.2020.1815882. Epub 2020 Sep 1.

Ethylcellulose microparticles enhance 3,3'-diindolylmethane anti-hypernociceptive action in an animal model of acute inflammatory pain.

Mattiazzi J(1), Sari MHM(1), Araujo PCO(2), Englert AV(1), Nadal JM(3), Farago PV(3), Nogueira CW(2), Cruz L(1).

Author information: (1)Programa de Pós-graduação em Ciências Farmacêuticas, Laboratório de Tecnologia Farmacêutica, Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Federal University of Santa Maria, Santa Maria, Brazil. (2)Programa de Pós-graduação em Bioquímica Toxicológica, Laboratório de Síntese, Reatividade e Avaliação Farmacológica e Toxicológica de Organocalcogênios, Departamento de Biologia Molecular, Centro de Ciências Naturais e Exatas, Federal University of Santa Maria, Santa Maria, Brazil. (3)Programa de Pós-graduação em Ciências Farmacêuticas, State University of Ponta Grossa, Brazil.

AIM: The present work aimed at the DIM-loaded microparticles development and anti-hypernociceptive action evaluation. METHOD: The formulations were prepared by O/W solvent emulsion-evaporation method and characterised by particle diameter, content and DIM encapsulation efficiency, drug release profile, thermal behaviour and physicochemical state. The anti-hypernociceptive action was evaluated in the animal model of acute inflammatory pain. RESULT: The MPs had a mean diameter in the micrometric range (368 ± 31 μm), narrow size distribution, DIM content of 150 mg/g, encapsulation efficiency around 84% and prolonged compound release. Evaluations of the association form of DIM to MPs demonstrated the feasibility of the systems to incorporate DIM and increases its thermal stability. An improvement in the anti-hypernociceptive action of DIM was observed by its microencapsuation, because it was increased and prolonged. CONCLUSION: Therefore, the MPs developed represent a promising formulation for oral administration of the DIM in the treatment of inflammatory pain.

Apoptosis. 2020 Oct;25(9-10):747-762. doi: 10.1007/s10495-020-01631-3. Epub 2020 Aug 20.

Neuroprotective effect of 3,3'-Diindolylmethane against perinatal asphyxia involves inhibition of the AhR and NMDA signaling and hypermethylation of specific genes.

Rzemieniec J(1), Bratek E(2), Wnuk A(1), Przepiórska K(1), Salińska E(2), Kajta M(3).

Author information: (1)Laboratory of Molecular Neuroendocrinology, Department of Experimental Neuroendocrinology, Maj Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343, Krakow, Poland. (2)Department of Neurochemistry, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, 02-106, Warsaw, Poland. (3)Laboratory of Molecular Neuroendocrinology, Department of Experimental Neuroendocrinology, Maj Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343, Krakow, Poland. [email protected].

Each year, 1 million children die due to perinatal asphyxia; however, there are no effective drugs to protect the neonatal brain against hypoxic/ischemic damage. In this study, we demonstrated for the first time the neuroprotective capacity of 3,3'-diindolylmethane (DIM) in an in vivo model of rat perinatal asphyxia, which has translational value and corresponds to hypoxic/ischemic episodes in human newborns. Posttreatment with DIM restored the weight of the ipsilateral hemisphere and normalized cell number in the brain structures of rats exposed to perinatal asphyxia. DIM also downregulated the mRNA expression of HIF1A-regulated Bnip3 and Hif1a which is a hypoxic marker, and the expression of miR-181b which is an indicator of perinatal asphyxia. In addition, DIM inhibited apoptosis and oxidative stress accompanying perinatal asphyxia through: downregulation of FAS, CASP-3, CAPN1, GPx3 and SOD-1, attenuation of caspase-9 activity, and upregulation of anti-apoptotic Bcl2 mRNA. The protective effects of DIM were accompanied by the inhibition of the AhR and NMDA signaling pathways, as indicated by the reduced expression levels of AhR, ARNT, CYP1A1, GluN1 and GluN2B, which was correlated with enhanced global DNA methylation and the methylation of the Ahr and Grin2b genes. Because our study provided evidence that in rat brain undergoing perinatal asphyxia, DIM predominantly targets AhR and NMDA, we postulate that compounds that possess the ability to inhibit their signaling are promising therapeutic tools to prevent stroke.

Appl Biochem Biotechnol. 2020 Dec;192(4):1208-1223. doi: 10.1007/s12010-020-03378-8. Epub 2020 Jul 25.

Modulatory Effect of Indoles on the Expression of miRNAs Regulating G1/S Cell Cycle Phase in Breast Cancer Cells.

El-Daly SM(1)(2), Gamal-Eldeen AM(3)(4)(5), Gouhar SA(6), Abo-Elfadl MT(3)(4), El-Saeed G(6).

Author information: (1)Medical Biochemistry Department, Medical Research Division, National Research Centre, 33 El Buhouth St. Dokki, Cairo, 12622, Egypt. [email protected]. (2)Cancer Biology and Genetics Laboratory, Centre of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt. [email protected]. (3)Cancer Biology and Genetics Laboratory, Centre of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt. (4)Biochemistry Department, National Research Centre, Cairo, Egypt. (5)Clinical Laboratory Department, College of Applied Medical Sciences, Taif University, Taif, Kingdom of Saudi Arabia. (6)Medical Biochemistry Department, Medical Research Division, National Research Centre, 33 El Buhouth St. Dokki, Cairo, 12622, Egypt.

Indole-3-carbinol (I3C) is a naturally occurring glucosinolate found in Brassica vegetables that is usually converted in gastric acidic environment to the efficient metabolite 3,3'-diindolylmethane (DIM). Both indoles (I3C and DIM) are known chemopreventive agents for various cancers including breast cancer. This study aimed to investigate the influence of both indoles on the tumor suppressor miRNAs (let-7a-e, miR-15a, miR-16, miR-17-5p, miR-19a, and miR-20a) and oncomiRs (miR-181a, miR-181b, miR-210, miR-221, and miR-106a), which are controlling the cell cycle key regulators: cyclin-dependent kinases (CDKs), CDK inhibitor p27Kip1, and cyclin D1. Our results indicated that both indoles generally elevated the expression of the tumor suppressor miRNAs let-7a-e, miR-19a, miR-17-5p, and miR-20a and decreased the expression of the oncomiR list. Both indoles were able to significantly suppress the expression of CDK4 and CDK6 as well as the apoptotic markers Bcl-2 and survivin. Both indoles decreased cyclin-D1 protein, where I3C decreased cytoplasmic and nuclear cyclin-D1 significantly. Cytoplasmic and nuclear P27Kip1 showed overexpression following treatment with I3C higher than that detected following DIM treatment. This study provides a mechanistic elucidation of the previously reported cell cycle arrest by I3C and DIM in breast cancer cells suggesting that this effect could be through modulation of miRNAs expression that, in turn, regulates the genetic network controlling the G1/S phase in cell cycle progression.

Int J Mol Sci. 2020 Jun 30;21(13):4642. doi: 10.3390/ijms21134642.

Chemopreventive Agent 3,3'-Diindolylmethane Inhibits MDM2 in Colorectal Cancer Cells.

Gao X(1), Liu J(1), Cho KB(1), Kedika S(1), Guo B(1).

Author information: (1)Department of Pharmacology and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77204, USA.

3,3'-Diindolylmethane (DIM) is a naturally derived chemopreventive compound. It comes from glucobrassicin, an indole glucosinolate enriched in cruciferous vegetables, and is formed in the acidic environment of the stomach after ingestion. Mouse double minute 2 homolog (MDM2) is an important, multi-functional oncogenic protein and it has been well recognized for its negative regulation of the tumor suppressor protein p53. We discovered a novel mechanism of action of DIM, that it directly inhibits MDM2 in multiple colorectal cancer (CRC) cell lines. Treatment with DIM decreased MDM2 at messenger RNA (mRNA) and protein levels, inhibited cancer cell proliferation, and induced cell cycle arrest and apoptosis. DIM-induced decrease of MDM2 is p53-independent and is partly mediated by proteasome degradation of MDM2, as blocking of the proteasome activity reversed MDM2 protein inhibition. Overexpression of MDM2 blocked DIM's effects in growth suppression and apoptosis induction. When combined with imidazoline MDM2 inhibitors (Nutlin-3a and Idasanutlin/RG-7388), synergism was observed in cancer cell growth inhibition. In summary, our data support a new mechanism of action for DIM in direct inhibition of MDM2. The identification of MDM2 as a novel DIM target may help develop a new strategy in CRC prevention.

Integr Med (Encinitas). 2019 Oct;18(5):36-39.

Hormonal Regulation In Pcos Using Acupuncture And Herbal Supplements: A Case Report And Review Of The Literature.

Alois M(1), Estores IM(2).

Author information: (1)Assistant Professor in the Division of General Internal Medicine and UF Health Integrative Medicine Program Department of Medicine University of Florida College of Medicine located in Gainesville, Florida, USA. (2)Medical Director UF Health Integrative Medicine Program and Associate Professor Division of General Internal Medicine and Division of Physical Medicine and Rehabilitation at University of Florida College of Medicine, located in Gainesville, Florida, USA.

Polycystic ovarian syndrome (PCOS) is a common cause of menstrual irregularity and hyperandrogenism in women of reproductive age. Conventional treatment is centered around the use of oral contraceptive pills (OCPs) to regulate menstrual cycles, protect against endometrial hyperplasia, and manage clinically evident androgen excess. Many women prefer to avoid OCPs due to concerns about risks and adverse effects, or simply because they desire a non-pharmacologic approach. We present the case of a young woman with a strong preference for a natural approach to her care who presented with PCOS manifesting in menstrual irregularity, acne, and hirsutism. In this case, acupuncture as well as the botanicals Vitex agnus-castus and diindolylmethane were used over a 10-month period. The patient regained menstrual cyclicity, and clinical and biochemical hyperandrogenism normalized. This report underscores the need for an integrative approach to the management of multifactorial disorders such as PCOS and highlights the basic science and clinical data supporting the use of acupuncture, Vitex agnus-castus, and diindolylmethane in patients with this condition.

J Exp Clin Cancer Res. 2020 Jun 16;39(1):113. doi: 10.1186/s13046-020-01618-7.

3,3'-Diindolylmethane modulates aryl hydrocarbon receptor of esophageal squamous cell carcinoma to reverse epithelial-mesenchymal transition through repressing RhoA/ROCK1-mediated COX2/PGE(2) pathway.

Zhu P(1), Yu H(2), Zhou K(1), Bai Y(1), Qi R(3), Zhang S(4).

Author information: (1)Department of Thoracic Surgery, The First Hospital of China Medical University, No.155 North Nanjing Street, Shenyang, 110001, China. (2)Department of Gynecology and Obstetrics, Clinical Medical School, Shandong University, 44# Wenhua Xi Road, Jinan, 250012, China. (3)Department of Dermatology, The First Hospital of China Medical University, No.155 North Nanjing Street, Shenyang, 110001, China. [email protected]. (4)Department of Thoracic Surgery, The First Hospital of China Medical University, No.155 North Nanjing Street, Shenyang, 110001, China. [email protected].

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive tumors in the world. Aryl hydrocarbon receptor (AHR) has been reported to promote tumor metastasis and epithelial-mesenchymal transition (EMT) is a vital process of conferring cancer cells capabilities of migration and invasion. However, the mechanism by which modulation of AHR can inhibit tumor metastasis remains unknown. Thus, we aim to investigate the underlying mechanism regarding reversing EMT process of ESCC through modulation of AHR. METHODS: We used AHR selective modulator 3,3'-diindolylmethane (DIM) to treat ESCC cell lines TE1 and KYSE150 so as to examine alterations of migration and invasion by wound healing and Transwell assay. Western blotting (WB) and qPCR were performed to detect relative genes and proteins changes regarding EMT process. Cell transfection was utilized for confirming pathways involved in DIM-induced reversal of EMT and in vivo assay was conducted for verification of the underlying mechanism. Co-IP assay was conducted for detecting protein-protein interactions. RESULTS: AHR was overexpressed in ESCC and modulation of AHR by DIM could inhibit migration and invasion as well as downregulate mesenchymal cell markers β-Catenin, Vimentin and Slug and upregulate epithelial cell marker Claudin-1. Meanwhile, synergically overexpression of AHR, RhoA and ROCK1 correlated with poor clinical outcomes. DIM could inhibit COX2/PGE2 pathway by targeting AHR, and COX2 selective inhibitor Celecoxib could suppress EMT and metastasis. Results of PGE2 treatment were opposite to that of Celecoxib. Meanwhile, blockade of RhoA/ROCK1 pathway also exerted prohibitive effects on EMT and metastasis. WB results showed COX2/PGE2 pathway could be regulated by RhoA/ROCK1 pathway and DIM could inhibit RhoA/ROCK1 pathway through modulation of AHR. In vivo assay verified the results in vitro. Co-IP results showed DIM could modulate AHR to reverse EMT directly through inhibition of interaction between AHR and EGFR (epidermal growth factor receptor) so as to block RhoA/ROCK1-mediated COX2/PGE2 pathway which was connected by NF-κB. CONCLUSIONS: In brief, modulation of AHR by DIM can reverse EMT process and inhibit metastasis of ESCC through repressing RhoA/ROCK1-mediated COX2/PGE2 pathway.

Eur J Pharm Sci. 2020 Aug 1;151:105379. doi: 10.1016/j.ejps.2020.105379. Epub 2020 May 27.

Gellan gum-based hydrogel containing nanocapsules for vaginal indole-3-carbinol delivery in trichomoniasis treatment.

Osmari BF(1), Giuliani LM(1), Reolon JB(1), Rigo GV(2), Tasca T(2), Cruz L(3).

Author information: (1)Laboratório de Tecnologia Farmacêutica, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil. (2)Grupo de Pesquisa em Tricomonas, GPTrico(,) Laboratório de Pesquisa em Parasitologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. (3)Laboratório de Tecnologia Farmacêutica, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.. Electronic address: [email protected].

Trichomonas vaginalis infection is the STI most common worldwide. Indole-3-carbinol (I3C) is a phytochemical presenting promising biological activities. In this study, design, formulation, and evaluation of a vaginal hydrogel containing I3C-loaded nanocapsules for the treatment of trichomoniasis have been investigated. Nanocapsules of Eudragit® RS100 and rosehip oil containing I3C (NC-I3C) were prepared by interfacial deposition of preformed polymer method. In vitro evaluations showed that free I3C (IC50 = 3.36 µg/mL) was able to reduce the trophozoites viability at higher concentrations (3.13 and 6.25 µg/mL), while nanoencapsulation (IC50 = 2.09 µg/mL) reduced the viability at all concentrations evaluated. Comparing free and nanoencapsulated I3C, we observe that nanoencapsulation improved anti-T. vaginalis activity. In order to obtain a formulation for vaginal administration, hydrogels (HG-NC-I3C) were prepared by thickening the NC-I3C with gellan gum. HG-NC-I3C presented particle size below 195 nm, low polydispersity index (<0.2), I3C content = 0.50 ± 0.01 mg/g, pH = 7.05, non-Newtonian pseudoplastic flow behavior and exhibited mucoadhesion to cow's vaginal mucosa. Evaluation of irritation potential by chorioallantoic membrane method indicated that the formulations are considered non-irritating. Besides that, permeation through the cow's vaginal mucosa showed that nanoencapsulation promoted I3C controlled release. This way, the developed HG-NC-I3C can be considered a promising approach for trichomoniasis treatment through vaginal administration.

Carcinogenesis. 2020 Oct 15;41(10):1395-1401. doi: 10.1093/carcin/bgaa050.

3,3-Diindolylmethane (DIM): a nutritional intervention and its impact on breast density in healthy BRCA carriers. A prospective clinical trial.

Yerushalmi R(1)(2), Bargil S(1), Ber Y(3), Ozlavo R(3), Sivan T(3), Rapson Y(2)(4), Pomerantz A(1), Tsoref D(1), Sharon E(5), Caspi O(1)(2), Grubsrein A(2)(4), Margel D(2)(3).

Author information: (1)Davidoff Cancer Center, Rabin Medical Center-Beilinson Campus, Petach Tikva, Israel. (2)Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. (3)Division of Urology, Petach Tikva, Israel. (4)Imaging Department, Petach Tikva, Israel. (5)Division of Surgery, Hospital for Women, Rabin Medical Center-Beilinson Campus, Petach Tikva, Israel.

Women who carry the BRCA mutation are at high lifetime risk of breast cancer, but there is no consensus regarding an effective and safe chemoprevention strategy. A large body of evidence suggests that 3,3-diindolylmethane (DIM), a dimer of indole-3-carbinol found in cruciferous vegetables, can potentially prevent carcinogenesis and tumor development. The primary aim of this prospective single-arm study was to investigate the effect of DIM supplementation on breast density, a recognized predictive factor of breast cancer risk. Participants were 23 healthy female BRCA carriers (median age 47 years; 78% postmenopausal) who were treated with oral DIM 100 mg × 1/day for 1 year. The amount of fibroglandular tissue (FGT) and background parenchymal enhancement (BPE) on magnetic resonance imaging (MRI) performed before and after the intervention was scored by two independent expert radiologists using the Breast Imaging and Reporting Data System. The results showed a decrease in the average score for FGT amount from 2.8 ± 0.8 at the onset to 2.65 ± 0.84 after 1 year (P = 0.031), with no significant change in BPE (P = 0.429). A group of DIM-untreated age- and menopausal-status-matched women from the BRCA clinic did not show a significant change in FGT amount (P = 0.33) or BPE (P = 0.814) in a parallel year. Mean estradiol level decreased from 159 to 102 pmol/l (P = 0.01), and mean testosterone level decreased from 0.42 to 0.31 pmol/l (P = 0.007). Side effects were grade 1. In conclusion, 1 year's supplementation with DIM 100 mg × 1/day in BRCA carriers was associated with a significant decline in FGT amount on MRI. Larger randomized studies are warranted to corroborate these findings.

Sci Rep. 2020 May 14;10(1):7969. doi: 10.1038/s41598-020-64729-3.

Synthesis of diindolylmethane (DIM) bearing thiadiazole derivatives as a potent urease inhibitor.

Taha M(1), Rahim F(2), Khan AA(2), Anouar EH(3), Ahmed N(4), Shah SAA(5)(6), Ibrahim M(7), Zakari ZA(8)(9).

Author information: (1)Department of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia. [email protected]. (2)Department of Chemistry, Hazara University, Mansehra, 21300, Khyber Pakhtunkhwa, Pakistan. (3)Department of Chemistry, College of Science and Humanities in Al-Kharj, Prince Sattam bin Abdulaziz University, Al-Kharj, 11942, Saudi Arabia. (4)Department of Pharmacy, Quaid-i-Azam University, Islamabad, 45320, Pakistan. (5)Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia, 42300, D. E., Selangor, Malaysia. (6)Faculty of Pharmacy, Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia, 42300, Darul Ehsan, Selangor, Malaysia. (7)Department of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia. (8)Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia. [email protected]. (9)Halal Institute Research Institute, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia. [email protected].

The current study describes synthesis of diindolylmethane (DIM) derivatives based-thiadiazole as a new class of urease inhibitors. Diindolylmethane is natural product alkaloid reported to use in medicinal chemistry extensively. Diindolylmethane-based-thiadiazole analogs (1-18) were synthesized and characterized by various spectroscopic techniques 1HNMR, 13C-NMR, EI-MS and evaluated for urease (jack bean urease) inhibitory potential. All compounds showed excellent to moderate inhibitory potential having IC50 value within the range of 0.50 ± 0.01 to 33.20 ± 1.20 µM compared with the standard thiourea (21.60 ± 0.70 µM). Compound 8 (IC50 = 0.50 ± 0.01 µM) was the most potent inhibitor amongst all derivatives. Structure-activity relationships have been established for all compounds. The key binding interactions of most active compounds with enzyme were confirmed through molecular docking studies.

Health Phys. 2020 Dec;119(6):746-757. doi: 10.1097/HP.0000000000001257.

Effect of 3,3'-Diindolylmethane on Pulmonary Injury Following Thoracic Irradiation in CBA Mice.

Laiakis EC(1)(2), McCart EA(3), Deziel A(1), Rittase WB(3), Bouten RM(3), Jha J(4), Wilkins WL(5), Day RM(3), Fornace AJ Jr(1)(2).

Author information: (1)Department of Oncology, Georgetown University, Washington, DC 20057. (2)Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, DC 20057. (3)Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814. (4)Current address: Rise Therapeutics, Rockville, MD 20850. (5)Department of Laboratory Animal Research, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.

The molecule 3,3'-diindolylmethane (DIM) is small, a major bioactive metabolite of indole-3 carbinol (13C), and a phytochemical compound from cruciferous vegetables released upon exposure to the gut acid environment. DIM is a proposed anti-cancer agent and was previously demonstrated to prevent radiation damage in the bone marrow and the gastrointestinal tract. Here we investigated the effect of DIM on radiation-induced injury to the lung in a murine model through untargeted metabolomics and gene expression studies of select genes. CBA mice were exposed to thoracic irradiation (17.5 Gy). Mice were treated with vehicle or DIM (250 mg kg, subcutaneous injection) on days -1 pre-irradiation through +14 post-irradiation. DIM induced a significant improvement in survival by day 150 post-irradiation. Fibrosis-related gene expression and metabolomics were examined using lung tissue from days 15, 45, 60, 90, and 120 post-irradiation. Our qRT-PCR experiments showed that DIM treatment reduced radiation-induced late expression of collagen Iα and the cell cycle checkpoint proteins p21/waf1 (CDKN1A) and p16ink (CDKN2A). Metabolomic studies of lung tissue demonstrated a significant dampening of radiation-induced changes following DIM treatment. Metabolites associated with pro-inflammatory responses and increased oxidative stress, such as fatty acids, were suppressed by DIM treatment compared to irradiated samples. Together these data suggest that DIM reduces radiation-induced sequelae in the lung.

Int J Nanomedicine. 2020 Mar 31;15:2259-2268. doi: 10.2147/IJN.S238256. eCollection 2020.

Nanoformulated Bioactive Compounds Derived from Different Natural Products Combat Pancreatic Cancer Cell Proliferation.

Mousa DS(1), El-Far AH(2), Saddiq AA(3), Sudha T(4), Mousa SA(4).

Author information: (1)Yale University, New Haven, CT, USA. (2)Department of Biochemistry, Faculty of Veterinary Medicine, Damanhour University, Damanhour 22511, Egypt. (3)Faculty of Science, Department of Biology, University of Jeddah, Jeddah 21589, Saudi Arabia. (4)Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY 12144, USA.

PURPOSE: This study was designed to determine the potential effect of nanoencapsulated bioactive compounds from different natural sources on human pancreatic cancer. BACKGROUND: Pancreatic cancer carries the highest fatality rate among all human cancers because of its high metastatic potential and late presentation at the time of diagnosis. Hence there is a need for improved methods to prevent and treat it. Natural products, such as 3, 3'-diindolylmethane (DIM) and ellagic acid (EA) demonstrated anticancer efficacy against various cancer types. However, DIM is insoluble. Hence, using nanotechnology to encapsulate these compounds in combination with EA might improve their physical and chemical properties and their delivery to the cancer cells. METHODS: Human pancreatic cancer cells, namely SUIT2-luciferase transfected, were used to examine the effects of DIM or EA and their nanoformulation in poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) [PLGA-PEG] nanoparticles (NPs) on SUIT2-luciferase cell viability/proliferation over 24 hrs. Additionally, effects on tumor weight and angiogenesis were determined using the chick chorioallantoic membrane (CAM) tumor implant model. RESULTS: Both DIM and EA PLGA-PEG NPs resulted in rapid suppression of pancreatic cancer cell viability/proliferation within 24 hrs (P < 0.01), while the non-encapsulated DIM and EA did not show any significant effect on SUIT2 cancer cell viability or cell proliferation (MTT assay). In the CAM pancreatic cancer cell (SUIT2) implant model, results showed a greater suppression of tumor weight (P < 0.01), tumor cell viability, and tumor angiogenesis (P < 0.01) for DIM NPs and EA NPs and their combinations versus DIM or EA alone. CONCLUSION: Nanoformulation of DIM and EA resulted in a more effective suppression of pancreatic cancer cell viability, pancreatic tumor weight, implanted cancer cell viability, and tumor angiogenesis as compared with these bioactive compounds alone.

J BUON. 2020 Jan-Feb;25(1):202-211.

Selective efficacy and safety of first line and non front line antineoplasic agent combinations determined with individualized assays on colorectal cancer primary cultures.

N Garza-Trevino E(1), G Martínez Rodríguez H, S Rodríguez González M, Delgado González P, F González Guerrero J, S García González I, Flores Gutiérrez JP, Soto Domínguez A, Said Fernández S.

Author information: (1)Autonomous University of Nuevo Leon (UANL), Medicine School, (FM), Department of Biochemistry and Molecular Medicine, Monterrey, N.L.Mexico.

PURPOSE: To evaluate the efficacy of first-line and not-conventional antineoplastic drug combinations in colorectal adenocarcinoma primary cultures (CRAC PCs). METHODS: The efficacy and safety of 21 drug combinations (DCs) were evaluated using a simplified adenosine triphosphate-based chemotherapy response assay (sATP-CRA). The efficacy of each DC was reported as the percentage of cell death (PCD) produced on each of 12 CRAC-PCs, and the safety of each DC was evaluated as a safety window (SW). The SW was calculated as the quotient of PCD-CRAC PC/PCD-hMSC-TA (human mesenchymal stem cells derived from adipose tissue). Nine DCs contained 5-fluorouracil and oxaliplatin, and 1-3 non-front line drugs (NFLDs [carboplatin, doxorubicin, cisplatin, aspirin, or 3,3' diindolylmethane]). The other 11 DCs only contained 2-4 NFLDs. RESULTS: The efficacy and safety each DC were highly variable and depended on each CRAC PC and DC. The usefulness of DCs was considered as a combination of PCD >20 and an SW >0.6: 13 /21 DCs (62%) met the requirements of efficacy and safety on 7/12 CRAC PCs (58.3%). CONCLUSIONS: The resistance to 5-fluorouracil/oxaliplatin of CRAC PCs and the usefulness of seven new DCs strongly suggest the convenience of performing ex vivo individualized assays to evaluate DCs, and implement new and more useful treatments, instead of submitting patients to standardized chemotherapies in a blinded manner. Approaches such as this and properly evaluated in clinical assays could increase the life expectancy of patients with cancer and improve their quality of life.

PMID: 32277633

Int J Mol Sci. 2020 Mar 17;21(6):2048. doi: 10.3390/ijms21062048.

Indole-3-Carbinol Derivative DIM Mitigates Carbon Tetrachloride-Induced Acute Liver Injury in Mice by Inhibiting Inflammatory Response, Apoptosis and Regulating Oxidative Stress.

Munakarmi S(1), Chand L(1), Shin HB(2), Jang KY(3), Jeong YJ(1)(2).

Author information: (1)Laboratory of Liver Regeneration, Biomedical Research Institute, Chonbuk National University Medical School, Jeonju 54907, Korea. (2)Department of Surgery, Chonbuk National University Hospital, Jeonju 54907, Korea. (3)Department of Pathology, Chonbuk National University Hospital, Jeonju 54907, Korea.

3,3'-Diindolylmethane (DIM), a metabolic product of indole-3-carbinol extracted from cruciferous vegetables exhibits anti-inflammatory and anti-cancer properties. Earlier, the product has been demonstrated to possess anti-fibrotic properties; however, its protective effects on liver injury have not been clearly elucidated. In this study, we postulated the effects and molecular mechanisms of action of DIM on carbon tetrachloride (CCl4)-induced liver injury in mice. Acute liver injury was induced by a single intraperitoneal administration of CCl4 (1 ml/kg) into mice. DIM was injected via subcutaneous route for three days at various doses (2.5, 5 and 10 mg/kg) before CCl4 injection. Mice were sacrificed and serum was collected for quantification of serum transaminases. The liver was collected and weighed. Treatment with DIM significantly reduced serum transaminases levels (AST and ALT), tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS). CCl4- induced apoptosis was inhibited by DIM treatment by the reduction in the levels of cleaved caspase-3 and Bcl2 associated X protein (Bax). DIM treated mice significantly restored Cytochrome P450 2E1, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in CCl4 treated mice. In addition, DIM downregulated overexpression of hepatic nuclear factor kappa B (NF-κB) and inhibited CCl4 mediated apoptosis. Our results suggest that the protective effects of DIM against CCl4- induced liver injury are due to the inhibition of ROS, reduction of pro-inflammatory mediators and apoptosis.

J Neuroimmunol. 2020 Feb 20;342:577195. doi: 10.1016/j.jneuroim.2020.577195. Online ahead of print.

Laquinimod and 3,3'-diindolylemethane alleviate neuropathological events and neurological deficits in a mouse model of intracerebral hemorrhage.

Matsumoto K(1), Kinoshita K(1), Yoshimizu A(1), Kurauchi Y(1), Hisatsune A(2), Seki T(1), Katsuki H(3).

Author information: (1)Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences and School of Pharmacy, Kumamoto University, Kumamoto 862-0973, Japan. (2)Priority Organization for Innovation and Excellence, Kumamoto University, Kumamoto 860-8555, Japan; Program for Leading Graduate Schools "HIGO (Health life science: Interdisciplinary and Glocal Oriented) Program", Kumamoto University, Kumamoto 862-0973, Japan. (3)Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences and School of Pharmacy, Kumamoto University, Kumamoto 862-0973, Japan. Electronic address: [email protected].

We examined the effects of compounds shown to activate aryl hydrocarbon receptor (AhR) signaling on a mouse model of intracerebral hemorrhage (ICH). Daily oral administration of laquinimod (25 mg/kg) or 3,3'-diindolylmethane (250 mg/kg) from 3 h after ICH induction improved motor functions, prevented the decrease of neurons within the hematoma, and attenuated activation of microglia/macrophages and astrocytes in the perihematomal region as well as infiltration of neutrophils into the hematoma. Elevated expression of AhR was detected in microglia and neutrophils, and both drugs inhibited upregulation of interleukin-6 and CXCL1. These results propose AhR as a therapeutic target for ICH.

ACS Omega. 2019 Dec 23;5(1):394-405. doi: 10.1021/acsomega.9b02904. eCollection 2020 Jan 14.

Nanofibrils of a Cu(II)-Thiophenyltriazine-Based Porous Polymer: A Diverse Heterogeneous Nanocatalyst.

Kundu SK(1), Kayet A(1), Baidya R(1), Satyanarayana L(2), Maiti DK(1).

Author information: (1)Department of Chemistry, University of Calcutta, 92 A.P.C. Road, Kolkata 700009, India. (2)Analytical Department, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad 500007, India.

Herein, we report knitting of a thiophenyltriazine-based porous organic polymer (TTPOP) with high surface area and high abundance of nitrogen and sulfur sites, synthesized through a simple one-step Friedel-Crafts reaction of 2,4,6-tri(thiophen-2-yl)-1,3,5-triazine and formaldehyde dimethyl acetal in the presence of anhydrous FeCl3, and thereafter grafting of Cu(OAc)2·H2O in the porous polymer framework to achieve the potential catalyst (CuII-TTPOP). TTPOP and CuII-TTPOP were characterized thoroughly utilizing solid-state 13C-CP MAS NMR, Fourier transform infrared, wide-angle powder X-ray diffraction, thermogravimetric analysis, and X-ray photoelectron spectroscopy and surface imaging by transmission electron microscopy and field emission scanning electron microscopy. The porosity of the nanomaterials was observed in the surface imaging and verified through conducting N2 gas adsorption techniques. Keeping in mind the tremendous importance of C-C and C-N coupling and cyclization processes, the newly synthesized CuII-TTPOP was employed successfully for a wide range of organic catalytic transformations under mild conditions to afford directly valuable diindolylmethanes and spiro-analogues, phthalimidines, propargyl amines, and their sugar-based chiral compounds with high yields using readily available substrates. The highly stable new heterogeneous catalyst showed outstanding sustainability, robustness, simple separation, and recyclability.

J Cell Commun Signal. 2020 Jun;14(2):175-192. doi: 10.1007/s12079-019-00535-5. Epub 2020 Jan 10.

Modulation of aryl hydrocarbon receptor inhibits esophageal squamous cell carcinoma progression by repressing COX2/PGE2/STAT3 axis.

Zhu P(1), Zhou K(1), Lu S(2), Bai Y(1), Qi R(3), Zhang S(4).

Author information: (1)Department of Thoracic Surgery, The First Hospital of China Medical University, No.155 North Nanjing Street, Shenyang, 110001, China. (2)Department of Otolaryngology, Dermatology, Pathology, University of Colorado Anschutz Medical Campus, 12700 E 19th Avenue, Aurora, CO, 80045, USA. (3)Department of Dermatology, The First Hospital of China Medical University, No.155 North Nanjing Street, Shenyang, 110001, China. [email protected]. (4)Department of Thoracic Surgery, The First Hospital of China Medical University, No.155 North Nanjing Street, Shenyang, 110001, China. [email protected].

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors with poor prognosis. Aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor and emerging evidence shows it is associated with tumor initiation and promotion. However, the relationship between AHR and ESCC is not clear and it is meaningful to explore whether AHR could be a therapeutic target. In the present study, immunohistochemistry was performed to determine AHR expression levels in ESCC tissues. Knockdown of AHR expression in ESCC cell lines genetically and modulation of AHR by 3, 3'-diindolylmethane (DIM) pharmacologically both in vitro and in vivo were utilized to examine the corresponding alterations in cell growth, migration and invasion. Our study indicated that AHR expression levels were elevated in ESCC and associated with poor prognosis. Both knockdown and modulation of AHR inhibited tumor progression through down-regulating expression levels of PCNA, Bcl-2, Cyclin D1, MMP1, MMP2, MMP9 and up-regulating expression levels of Bax, Cleaved-Caspase 3. Our findings also indicated that repressing COX2/PGE2/STAT3 axis exerted inhibitory effects on ESCC both in vitro and in vivo assays. Taken together, AHR plays the key role in ESCC progression and targeting AHR as a therapeutic strategy with DIM is deserved for further exploration.

Phytochem Anal. 2020 Jul;31(4):522-530. doi: 10.1002/pca.2916. Epub 2020 Jan 8.

Rapid measurement of indole levels in Brassica vegetables using one millilitre binary organic extraction solvent and capillary electrophoresis-UV analysis.

Suparman(1)(2), Inpota P(1), Phonchai A(3), Wilairat P(4), Chantiwas R(1).

Author information: (1)Department of Chemistry and Centre of Excellence for Innovation in Chemistry and Flow Innovation-Research for Science and Technology Laboratories (FIRST Labs), Faculty of Science, Mahidol University, Rama VI Rd, Bangkok, 10400, Thailand. (2)Department of Chemistry, Faculty of Pharmacy, University of Muhammadiyah Purwokerto, Jl. Raya Dukuh Waluh, Purwokerto, 53182, Indonesia. (3)Department of Applied Science, Faculty of Science, Prince of Songkla University, 15 Karnjanavanich Rd, Hat Yai, Songkhla, 90110, Thailand. (4)National Doping Control Centre, Mahidol University, Rama VI Rd, Bangkok, 10400, Thailand.

INTRODUCTION: Brassica vegetables contain high levels of indole compounds which have been found to provide health benefits, especially as cancer-preventive agents. An efficient and rapid method using solvent extraction with capillary electrophoresis (CE) and ultraviolet (UV) detection was developed for the determination of four major indoles from four types of Brassica vegetables. MATERIALS AND METHODS: Freeze-dried samples of four Brassica vegetables, i.e. broccoli, cauliflower, Chinese cabbage and cabbage, were selected. Hence, 1 mL of the binary solvent dimethylformamide (DMF)-methanol, 4:1 (v/v), was used for sample extraction. The extracts were diluted with the running buffer and directly analysed using CE with UV detection of four indole compounds. RESULTS: The binary solvent DMF-methanol, 4:1 (v/v) was selected from studies of the extraction efficiency of standard indoles spiked in ivy gourd (as the negative control sample) and using diphenylamine as the internal standard. Recovery was 80(±10)-120(±3)% for the four indoles: indole-3-carbinol (I3C), indole-3-acetonitrile (I3A), indole-3-acetic acid (IAA), and 3,3'-diindolylmethane (DIM). For direct analysis suitable dilution of the extract with the running buffer was required. The linear range of the quantitation is 0.75-25.0 μg/mL, limit of detection (LOD) of 0.14-0.52 μg/mL and r2 > 0.998. The amount of indole in the Brassica vegetables are in the order I3C > > IAA, I3A > DIM. CONCLUSION: A rapid method for extraction and quantitation of four indoles in four Brassica vegetables using CE with UV detection was developed. It has the potential as an efficient technique for generating data for use in agricultural and nutritional studies.

Org Lett. 2020 Feb 7;22(3):827-831. doi: 10.1021/acs.orglett.9b04272. Epub 2020 Jan 8.

CF(3)SO(2)Na-Mediated, UV-Light-Induced Friedel-Crafts Alkylation of Indoles with Ketones/Aldehydes and Bioactivities of Products.

Yang T(1), Lu H(2), Shu Y(2), Ou Y(1), Hong L(2), Au CT(3), Qiu R(1).

Author information: (1)State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering , Hunan University , Changsha 410082 , P. R. China. (2)College of Biology , Huazhong University of Science and Technology , Wuhan 430074 , Hubei , P. R. China. (3)College of Chemistry and Chemical Engineering , Hunan Institute of Engineering , Xiangtan 411104 , Hunan , P. R. China.

A concise, one-step route to produce 3,3'-diindolylmethanes (DIMs) from simple indoles and ketones or aldehydes is reported. The key step is the ready formation of indole derivatives that involves the in situ conversion of CF3SO2Na reagent to ·CF3 under oxygen or air (1.0 atm) and UV irradiation. It is disclosed that most of the obtained DIMs show anticancer activities in human bladder cancer cell lines EJ and T24.

DOI: 10.1021/acs.orglett.9b04272 PMID: 31913641

Antioxidants. 2019 Dec 18;9(1):3. doi: 10.3390/antiox9010003.

3,3'-Diindolylmethane Promotes BDNF and Antioxidant Enzyme Formation via TrkB/Akt Pathway Activation for Neuroprotection against Oxidative Stress-Induced Apoptosis in Hippocampal Neuronal Cells.

Lee BD(1), Yoo JM(2)(3), Baek SY(1), Li FY(1), Sok DE(4), Kim MR(1).

Author information: (1)Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Korea. (2)Korean Medicine-Application Center, Korea Institute of Oriental Medicine, Daegu 41062, Korea. (3)Korean Medicine R&D Team 1, National Institute for Korean Medicine Development, Gyeongsan 38540, Korea. (4)College of Pharmacy, Chungnam National University, Daejeon 34134, Korea.

3,3'-Diindolylmethane (DIM), a metabolite of indole-3-carbinol present in Brassicaceae vegetables, possesses various health-promoting effects. Nonetheless, the effect of DIM on neurodegenerative diseases has not been elucidated clearly. In this study, we hypothesized DIM may protect neuronal cells against oxidative stress-induced apoptosis by promoting the formation of brain-derived neurotrophic factor (BDNF) and antioxidant enzymes through stabilizing the activation of the tropomyosin-related kinase receptor B (TrkB) cascade and we investigated the effect of DIM on oxidative stress-mediated neurodegenerative models. DIM protected neuronal cells against oxidative stress-induced apoptosis by regulating the expression of apoptosis-related proteins in glutamate-treated HT-22 cells. Additionally, DIM improved the expression of BDNF and antioxidant enzymes, such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinine oxidoreductase-1, by promoting the activation of the TrkB/protein kinase B (Akt) pathway in the cells. Consistent with in vitro studies, DIM attenuated memory impairment by protecting hippocampal neuronal cells against oxidative damage in scopolamine-treated mice. Conclusionally, DIM exerted neuroprotective and antioxidant actions through the activation of both BDNF production and antioxidant enzyme formation in accordance with the TrkB/Akt pathway in neuronal cells. Such an effect of DIM may provide information for the application of DIM in the prevention of and therapy for neurodegenerative diseases.

Molecules. 2019 Dec 6;24(24):4474. doi: 10.3390/molecules24244474.

Amelioration of Hyperglycemia-Induced Nephropathy by 3,3'-Diindolylmethane in Diabetic Mice.

Choi KM(1), Yoo HS(1).

Author information: (1)College of Pharmacy, Chungbuk National University, Osongsaengmyeong 1-ro, Heungduk-gu, Cheongju 28160, Korea.

Type 1 diabetes mellitus (insulin-dependent diabetes) is characterized by hyperglycemia caused by an insulin deficiency. Diabetic nephropathy is a major complication of hyperglycemia. 3,3'-diindolylmethane (DIM)-a natural compound produced from indole-3-carbinol, found in cruciferous vegetables-enhances glucose uptake by increasing the activation of the insulin signaling pathway in 3T3-L1 adipocytes. In this study, we investigated whether DIM could improve insulin-dependent diabetes and nephropathy in streptozotocin (STZ)-induced diabetic mice. In mice, STZ induced hyperglycemia, hunger, thirst, and abnormally increased kidney weight and serum creatinine, which is a renal functional parameter. DIM decreased STZ-increased high blood glucose levels and food and water intake in diabetic mice. DIM also improved diabetic nephropathy by inhibiting the expression of PKC-α, the marker of albuminuria, and TGF-β1, an indicator of renal hypertrophy, in diabetic mice. Our findings suggest that DIM may ameliorate hyperglycemia and diabetic nephropathy through the inhibition of PKC-α and TGF-β1 signaling.

Bull Exp Biol Med. 2019 Nov;168(1):45-47. doi: 10.1007/s10517-019-04642-6. Epub 2019 Nov 25.

An Experimental Study of the Effect of Diindolylmethane on Alveolocyte and Hepatocyte Adhesion Strength in Mice.

Kiselev VI(1), Sukhikh GT(1), Pchelintseva OI(2), Udut VV(3), Kuznetsov IN(1), Drukh VM(4).

Author information: (1)V. I. Kulakov National Medical Research Center of Obstetrics, Gynecology, and Perinatology, Ministry of Health of the Russian Federation, Moscow, Russia. (2)MiraxBioFarma, Moscow, Russia. [email protected]. (3)E. D. Goldberg Research Institute of Pharmacology and Regenerative Medicine, Tomsk National Research Medical Center, Russian Academy of Sciences, Tomsk, Russia. (4)MiraxBioFarma, Moscow, Russia.

We studied the effect of diindolylmethane in a dose of 600 mg/kg on the change in adhesion strength of alveolocytes and hepatocytes in CBA mice. Diindolylmethane was administered intragastrically to experimental animals for 10 days, controls intragastrically received an equivalent volume of saline. At the end of the therapeutic period, mice treated with diindolylmethane showed a significant increase in the adhesion strength of alveolocytes by 16% (p=0.003) and hepatocytes by 61% (p=0.0001) in comparison with the control group, which indicates the antipromotor activity of diindolylmethane.

DOI: 10.1007/s10517-019-04642-6 PMID: 31761981

Front Pharmacol. 2019 Oct 9;10:1167. doi: 10.3389/fphar.2019.01167. eCollection 2019.

Anti-Cancer Effects of 3, 3'-Diindolylmethane on Human Hepatocellular Carcinoma Cells Is Enhanced by Calcium Ionophore: The Role of Cytosolic Ca(2+) and p38 MAPK.

Jiang Y(1)(2), Fang Y(1)(3), Ye Y(1), Xu X(4), Wang B(5), Gu J(6), Aschner M(7), Chen J(4), Lu R(1)(8).

Author information: (1)Department of Preventive Medicine and Public Health Laboratory Science, School of Medicine, Jiangsu University, Zhenjiang, China. (2)Department of Pathology, Kunshan Hospital of Traditional Chinese Medicine, Suzhou, China. (3)Department of Gastroenterology, The First People's Hospital of Taicang City, Taicang Affiliated Hospital of Soochow University, Suzhou, China. (4)Department of General Surgery, Affiliated Kunshan Hospital, Jiangsu University School of Medicine, Suzhou, China. (5)Department of Digestive Disease, Affiliated Kunshan Hospital, Jiangsu University School of Medicine, Suzhou, China. (6)Institute of Life Science, Jiangsu University, Zhenjiang, China. (7)Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, United States. (8)Center for Experimental Research, Affiliated Kunshan Hospital, Jiangsu University School of Medicine, Suzhou, China.

Purpose: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol (I3C) in the Brassica species of cruciferous vegetables, has anticancer effects, but its exact underlying mechanism of action is unknown. We explored the roles of cytosolic free calcium ([Ca2+]i) and p38 MAPK in the anti-cancer effects of DIM in human hepatocellular carcinoma cells. Methods: Cell proliferation was measured with a Cell Counting Kit-8 (CCK-8) and the clonogenic formation assay. Cell apoptosis was examined by flow cytometric analysis and Hoechst dye staining. Cleaved-caspase3, cleaved-PARP, Bax, total, and phosphorylated p38 MAPK were assayed by western blotting. [Ca2+]i was measured with Fluo-3/AM by fluorescence microscopy. A23187, a calcium ionophore, was used to increase [Ca2+]i levels. Results: DIM inhibited cell proliferation in both SMMC-7721 and HepG2 cells in a concentration- and time-dependent manner. DIM also enhanced phosphorylation of p38 MAPK (p-p38), which was attenuated by SB203580. The proliferation inhibition and apoptosis induction by DIM were also blunted. In addition, DIM increased [Ca2+]i in HCC cells, and this effect was inhibited by the calcium chelator, BAPTA-AM, resulting in reduced p-p38 MAPK activation and apoptosis in DIM-treated cells, though the proliferation inhibition by DIM was unchanged. However, the DIM-induced cell proliferation inhibition and apoptosis were significantly enhanced by A23187, a selective calcium ionophore, which was attributed to exaggerated p-p38 MAPK. Conclusions: The calcium ionophore enhanced DIM-induced anti-cancer effects in hepatocellular carcinoma cells, secondary to [Ca2+]i-dependent activation of p38 MAPK. Treatment with a combination of DIM and calcium ionophore may offer a new approach to enhance the chemotherapeutic efficacy in liver cancer.

DOI: 10.3389/fphar.2019.01167 PMCID: PMC6795059 PMID: 31649538

Cell Metab. 2019 Dec 3;30(6):1141-1151.e5. doi: 10.1016/j.cmet.2019.08.021. Epub 2019 Sep 19.

Trimethylamine N-Oxide Binds and Activates PERK to Promote Metabolic Dysfunction.

Chen S(1), Henderson A(2), Petriello MC(3), Romano KA(4), Gearing M(2), Miao J(2), Schell M(5), Sandoval-Espinola WJ(6), Tao J(7), Sha B(7), Graham M(8), Crooke R(8), Kleinridders A(5), Balskus EP(6), Rey FE(4), Morris AJ(3), Biddinger SB(9).

Author information: (1)Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China; Division of Endocrinology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. (2)Division of Endocrinology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. (3)Division of Cardiovascular Medicine, College of Medicine, University of Kentucky, Lexington, KY, USA; Lexington Veterans Affairs Medical Center, Lexington, KY, USA. (4)Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA. (5)German Institute of Human Nutrition, Central Regulation of Metabolism, 14558 Nuthetal, Germany; German Center for Diabetes Research, 85764 München-Neuherberg, Germany. (6)Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. (7)Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA. (8)Ionis Pharmaceuticals, Carlsbad, CA, USA. (9)Division of Endocrinology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. Electronic address: [email protected].

The gut-microbe-derived metabolite trimethylamine N-oxide (TMAO) is increased by insulin resistance and associated with several sequelae of metabolic syndrome in humans, including cardiovascular, renal, and neurodegenerative disease. The mechanism by which TMAO promotes disease is unclear. We now reveal the endoplasmic reticulum stress kinase PERK (EIF2AK3) as a receptor for TMAO: TMAO binds to PERK at physiologically relevant concentrations; selectively activates the PERK branch of the unfolded protein response; and induces the transcription factor FoxO1, a key driver of metabolic disease, in a PERK-dependent manner. Furthermore, interventions to reduce TMAO, either by manipulation of the gut microbiota or by inhibition of the TMAO synthesizing enzyme, flavin-containing monooxygenase 3, can reduce PERK activation and FoxO1 levels in the liver. Taken together, these data suggest TMAO and PERK may be central to the pathogenesis of the metabolic syndrome.

DOI: 10.1016/j.cmet.2019.08.021 PMID: 31543404

Int J Mol Sci. 2019 Aug 12;20(16):3917. doi: 10.3390/ijms20163917.

Preparation and Characterization of Whey Protein Isolate-DIM Nanoparticles.

Khan A(1)(2), Wang C(1)(2), Sun X(3), Killpartrick A(4), Guo M(5)(6).

Author information: (1)Key Laboratory of Dairy Science, Northeast Agriculture University, Harbin, Heilongjiang 150030, China. (2)Department of Food Science, College of Food Science and Engineering, Jilin University, Changchun 30062, China. (3)Department of Food Science and Engineering, Northeast Agriculture University, Harbin, Heilongjiang 150030, China. (4)Food Science Corporation, Inc. Williston, VT 05495, USA. (5)Key Laboratory of Dairy Science, Northeast Agriculture University, Harbin, Heilongjiang 150030, China. [email protected]. (6)College of Agriculture and Life Sciences, The University of Vermont, Burlington, VT 05405, USA. [email protected].

3,3'-Diindolylmethane (DIM) is a bioactive compound found in Cruciferous vegetables that possesses health benefits such as antioxidant, anticancer, and anti-inflammatory effects. However, hydrophobicity and photolabile limit its pharmaceutical applications. This study aims to prepare and characterize DIM-encapsulated whey protein isolate (WPI) nanoparticles mixed at different ratios of WPI and DIM using the combined heating-ultrasound method. Results showed that all the samples showed adequate physicochemical characteristics: The mean particle size of the nanoparticles could be controlled down to 96-157 nm depending on the DIM to WPI ratio used in the preparation with a low polydispersity index (<0.5), higher negative values of zeta potential (>-40 mV) as well as with greater encapsulation efficiency (>82%). Flow behavior indices showed the shear-thinning Non-Newtonian or pseudoplastic (n < 1) behavior of the nanoparticles. The thermal properties were characterized by differential scanning calorimetry (DSC), which showed that DIM was successfully entrapped in WPI nanoparticles. The secondary structure of WPI was changed after DIM incorporation; electrostatic interaction and hydrogen bonding were major facilitating forces for nanoparticles formation, confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Transmission electron microscopy (TEM) micrographs showed that all the samples had a smooth surface and spherical structure. The wall material (WPI) and encapsulation method provide effective protection to DIM against UV light and a broad range of physiologically relevant pH's (2.5, 3.5, 4.5, 5.5, and 7). In conclusion, whey protein isolate (WPI)-based nanoparticles are a promising approach to encapsulate DIM and overcome its physicochemical limitations with improved stability.

DOI: 10.3390/ijms20163917 PMCID: PMC6721066 PMID: 31408980

Front Immunol. 2019 Jul 23;10:1620. doi: 10.3389/fimmu.2019.01620. eCollection 2019.

A Novel Phytochemical, DIM, Inhibits Proliferation, Migration, Invasion and TNF-α Induced Inflammatory Cytokine Production of Synovial Fibroblasts From Rheumatoid Arthritis Patients by Targeting MAPK and AKT/mTOR Signal Pathway.

Du H(1), Zhang X(1), Zeng Y(1), Huang X(1), Chen H(1), Wang S(1), Wu J(2), Li Q(2), Zhu W(3), Li H(1), Liu T(1), Yu Q(2), Wu Y(1), Jie L(2).

Author information: (1)School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, China. (2)Department of Rheumatology and Clinical Immunology, Zhujiang Hospital, Southern Medical University, Guangzhou, China. (3)Department of Toxicology, Guangzhou Center for Disease Control and Prevention, Guangzhou, China.

In rheumatoid arthritis(RA) pathogenesis, activated RA fibroblast-like synoviocytes (RA-FLSs) exhibit similar proliferative features as tumor cells and subsequent erosion to cartilage will eventually lead to joint destruction. Therefore, it is imperative to search for compounds, which can effectively inhibit the abnormal activation of RA-FLSs, and retard RA progression.3'3-Diindolylmethane (DIM), the major product of the acid-catalyzed oligomerization of indole-3-carbinol from cruciferous vegetables, has been reported to be functionally relevant to inhibition of migration, invasion and carcinogenesis in some solid tumors. In this study, we explored the anti-proliferation, anti-metastasis and anti-inflammation effects of DIM on RA-FLSs as well as the underlying molecular mechanisms. To do this, primary RA-FLSs were isolated from RA patients and an animal model. Cell proliferation, migration and invasion were measured using CCK-8, scratch, and Transwell assays, respectively. The effects of DIM on Matrix metalloproteinases (MMPs) and some inflammatory factors mRNA and key molecules such as some inflammatory factors and those involved in aberrantly-activated signaling pathway in response to tumor necrosis factor α(TNF-α), a typical characteristic mediator in RA-FLS, were quantitatively measured by real-time PCR and western blotting. Moreover, the effect of DIM on adjuvant induced arthritis(AIA) models was evaluated with C57BL/6 mice in vivo. The results showed that DIM inhibited proliferation, migration and invasion of RA-FLS in vitro. Meanwhile, DIM dramatically suppressed TNF-α-induced increases in the mRNA levels of MMP-2, MMP-3, MMP-8, and MMP-9; as well as the proinflammatory factors IL-6, IL-8, and IL-1β. Mechanistic studies revealed that DIM is able to suppress phosphorylated activation not only of p38, JNK in MAPK pathway but of AKT, mTOR and downstream molecules in the AKT/mTOR pathway. Moreover, DIM treatment decreased expression levels of proinflammatory cytokines in the serum and alleviated arthritis severity in the knee joints of AIA mice. Taken together, our findings demonstrate that DIM could inhibit proliferation, migration and invasion of RA-FLSs and reduce proinflammatory factors induced by TNF-α in vitro by blocking MAPK and AKT/mTOR pathway and prevent inflammation and knee joint destruction in vivo, which suggests that DIM might have therapeutic potential for RA.

DOI: 10.3389/fimmu.2019.01620 PMCID: PMC6663984 PMID: 31396207

Org Biomol Chem. 2019 Sep 7;17(33):7747-7759. doi: 10.1039/c9ob01249d. Epub 2019 Aug 6.

Controlling the regioselectivity of the ring opening of spiro-epoxyoxindoles for efficient synthesis of C(3)-N(1')-bisindoles and C(3)-N(1')-diindolylmethane.

Hajra S(1), Maity S(2), Roy S(1), Das D(3).

Author information: (1)Centre of Biomedical Research, Sanjay Gandhi Post-Graduate Institute of Medical Sciences Campus, Raebareli Road, Lucknow 226014, India. [email protected]. (2)Centre of Biomedical Research, Sanjay Gandhi Post-Graduate Institute of Medical Sciences Campus, Raebareli Road, Lucknow 226014, India. [email protected] and Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur 721302, India. (3)Dept. of Chemical Sciences, Indian Institute of Science Education and Research Mohali, Manauli PO 140306, Punjab, India.

An efficient strategy for the construction of both C(3)-N(1') bisindoles and C(3)-N(1') diindolylmethane has been explored via proper tuning of the nucleophilicity of indoline/indole to spiro-epoxyoxindole. Lewis acid-catalyzed highly regio- as well as chemoselective coupling at the C-3 centre of spiro-epoxyoxindoles with indolines furnishes C(3)-N(1') bisindoles whereas base mediated and Lewis acid-catalyzed regioselective coupling at the less hindered site of spiro-epoyoxindoles with indoles via the SN2 mechanism provides C(3)-N(1') diindolylmethane.

DOI: 10.1039/c9ob01249d PMID: 31386749

J Agric Food Chem. 2019 Aug 21;67(33):9277-9285. doi: 10.1021/acs.jafc.9b03039. Epub 2019 Aug 6.

3,3'-Diindolylmethane Improves Intestinal Permeability Dysfunction in Cultured Human Intestinal Cells and the Model Animal Caenorhabditis elegans.

Kim JY(1), Le TAN(1), Lee SY(1), Song DG(1), Hong SC(1), Cha KH(1), Lee JW(2)(3), Pan CH(1)(3), Kang K(1)(3).

Author information: (1)Natural Product Informatics Research Center , Korea Institute of Science and Technology , Gangneung , Gangwon-do 25451 , Republic of Korea. (2)Natural Products Research Center , Korea Institute of Science and Technology , Gangneung , Gangwon-do 25451 , Republic of Korea. (3)Division of Bio-Medical Science & Technology, KIST School , Korea University of Science and Technology (UST) , Seoul 02792 , Republic of Korea.

3,3'-Diindolylmethane (DIM), a digestive metabolite originating from cruciferous vegetables, has dietary potential for the treatment of various human intestinal diseases. Although intestinal permeability dysfunction is closely related to the initiation and progression of human intestinal inflammatory diseases (IBDs), the effect of DIM on intestinal permeability is unclear. We evaluated the effect of DIM on the intestinal permeability of human intestinal cell monolayers and the animal model Caenorhabditis elegans, which were treated with IL-1β and Pseudomonas aeruginosa, respectively, to mimic IBD conditions. DIM substantially restored the intestinal permeability of differentiated Caco-2 cells by enhancing the expression of tight junction proteins (including occludin and ZO-1). Compared to the IL-1β single treatment (551.0 ± 49.0 Ω·cm2), DIM (10 μM) significantly increased the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers (919.0 ± 66.4 Ω·cm2, p < 0.001). DIM also ameliorated the impaired intestinal permeability and extended the lifespan of C. elegans fed P. aeruginosa. The mean lifespan of DIM-treated worms (10.8 ± 1.3 days) was higher than that of control-treated worms (9.7 ± 1.1 days, p < 0.01). Thus, DIM is a potential nutraceutical candidate for the treatment of leaky gut syndrome by improving intestinal permeability.

DOI: 10.1021/acs.jafc.9b03039 PMID: 31353906 [Indexed for MEDLINE]

Mol Imaging Biol. 2020 Jun;22(3):593-601. doi: 10.1007/s11307-019-01399-2.

Synthesis and Evaluation of Diindole-Based MRI Contrast Agent for In Vivo Visualization of Necrosis.

Zhang L(1)(2)(3), Liu L(1)(2)(3), Zhang D(1)(3), Jin Q(1)(3), Gao M(1)(3), Wu T(1)(2)(3), Feng Y(1)(3)(4), Ni Y(1)(3)(4), Yin Z(5), Zhang J(6)(7).

Author information: (1)Laboratories of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, No.100, Shizi Street, Hongshan Road, Qixia District, Nanjing, 210028, Jiangsu Province, People's Republic of China. (2)Department of TCMs Pharmaceuticals, School of TCM & State Key Laboratory of Natural Medicines, China Pharmaceutical University, No.24, Tongjiaxiang, Gulou District, Nanjing, 210009, Jiangsu Province, People's Republic of China. (3)Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210028, Jiangsu Province, People's Republic of China. (4)Theragnostic Laboratory, KU Leuven, Campus Gasthuisberg, 3000, Leuven, Belgium. (5)Department of TCMs Pharmaceuticals, School of TCM & State Key Laboratory of Natural Medicines, China Pharmaceutical University, No.24, Tongjiaxiang, Gulou District, Nanjing, 210009, Jiangsu Province, People's Republic of China. [email protected]. (6)Laboratories of Translational Medicine, Jiangsu Province Academy of Traditional Chinese Medicine, No.100, Shizi Street, Hongshan Road, Qixia District, Nanjing, 210028, Jiangsu Province, People's Republic of China. [email protected]. (7)Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, 210028, Jiangsu Province, People's Republic of China. [email protected].

PURPOSE: Noninvasive imaging of cell necrosis can provide an early evaluation of tumor response to treatments. Here, we aimed to design and synthesize a novel diindole-based magnetic resonance imaging (MRI) contrast agent (Gd-bis-DOTA-diindolylmethane, Gd-DIM) for assessment of tumor response to therapy at an early stage. PROCEDURES: The oil-water partition coefficient (Log P) and relaxivity of Gd-DIM were determined in vitro. Then, its necrosis avidity was examined in necrotic cells in vitro and in rat models with microwave ablation-induced muscle necrosis (MAMN) and ischemia reperfusion-induced liver necrosis (IRLN) by MRI. Visualization of tumor necrosis induced by combretastatin A-4 disodium phosphate (CA4P) was evaluated in rats bearing W256 orthotopic liver tumor by MRI. Finally, DNA binding assay was performed to explore the possible necrosis-avidity mechanism of Gd-DIM. RESULTS: The Log P value and T1 relaxivity of Gd-DIM is - 2.15 ± 0.01 and 6.61 mM-1 s-1, respectively. Gd-DIM showed predominant necrosis avidity in vitro and in vivo. Clear visualization of the tumor necrosis induced by CA4P was achieved at 60 min after administration of Gd-DIM. DNA binding study indicated that the necrosis-avidity mechanism of Gd-DIM may be due to its binding to exposed DNA in necrotic cells. CONCLUSION: Gd-DIM may serve as a promising necrosis-avid MRI contrast agent for early assessment of tumor response to therapy.

DOI: 10.1007/s11307-019-01399-2 PMID: 31332630
Bioorg Med Chem Lett. 2019 Aug 15;29(16):2345-2348. doi: 10.1016/j.bmcl.2019.06.014. Epub 2019 Jun 12.

Discovery of 2,3'-diindolylmethanes as a novel class of PCSK9 modulators.

Winston-McPherson GN(1), Xie H(1), Yang K(1), Li X(1), Shu D(2), Tang W(3).

Author information: (1)School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, United States. (2)School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, United States; Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706, United States. (3)School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, United States; Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, WI 53706, United States. Electronic address: [email protected].

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of low density lipoprotein receptor (LDLR). Anti-PCSK9 agents have been approved for the treatment of hypercholesterolemia. We recently discovered a series of small-molecule PCSK9 modulators that contains a relatively small pharmacophore of 2,3'-diindolylmethane with molecular weights around only 250. These molecules can significantly lower the amount of PCSK9 protein in a cell-based phenotypic assay. Our SAR studies yielded compound 16 with a IC50-value of 200 nM. No obvious cytotoxicity was observed at concentrations below 50 µM.

Int J Mol Sci. 2019 Jun 15;20(12):2939. doi: 10.3390/ijms20122939.

Phytochemicals as Modulators of Long Non-Coding RNAs and Inhibitors of Cancer-Related Carbonic Anhydrases.

Saghafi T(1), Taheri RA(2), Parkkila S(3)(4), Emameh RZ(5).

Author information: (1)Department of Energy and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), 14965/161, Tehran, Iran. [email protected]. (2)Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, P.O.Box 14965/161 Tehran, Iran. [email protected]. (3)Faculty of Medicine and Health Technology, Tampere University, FI-33520 Tampere, Finland. [email protected]. (4)Fimlab Laboratories Ltd. and Tampere University Hospital, FI-33520 Tampere, Finland. [email protected]. (5)Department of Energy and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), 14965/161, Tehran, Iran. [email protected].

Long non-coding RNAs (lncRNAs) are classified as a group of transcripts which regulate various biological processes, such as RNA processing, epigenetic control, and signaling pathways. According to recent studies, lncRNAs are dysregulated in cancer and play an important role in cancer incidence and spreading. There is also an association between lncRNAs and the overexpression of some tumor-associated proteins, including carbonic anhydrases II, IX, and XII (CA II, CA IX, and CA XII). Therefore, not only CA inhibition, but also lncRNA modulation, could represent an attractive strategy for cancer prevention and therapy. Experimental studies have suggested that herbal compounds regulate the expression of many lncRNAs involved in cancer, such as HOTAIR (HOX transcript antisense RNA), H19, MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), PCGEM1 (Prostate cancer gene expression marker 1), PVT1, etc. These plant-derived drugs or phytochemicals include resveratrol, curcumin, genistein, quercetin, epigallocatechin-3-galate, camptothcin, and 3,3'-diindolylmethane. More comprehensive information about lncRNA modulation via phytochemicals would be helpful for the administration of new herbal derivatives in cancer therapy. In this review, we describe the state-of-the-art and potential of phytochemicals as modulators of lncRNAs in different types of cancers.

Molecules. 2019 Jun 7;24(11):2151. doi: 10.3390/molecules24112151.

Interactions between β-Lactoglobulin and 3,3'-Diindolylmethane in Model System.

Wang C(1)(2), Zhou X(3), Wang H(4), Sun X(5), Guo M(6)(7).

Author information: (1)Key Laboratory of Dairy Science, Northeast Agricultural University, Harbin 150030, Heilongjiang, China. [email protected]. (2)Department of Food Science, College of Food Science and Engineering, Jilin University, Changchun 130062, Jilin, China. [email protected]. (3)Key Laboratory of Dairy Science, Northeast Agricultural University, Harbin 150030, Heilongjiang, China. [email protected]. (4)Key Laboratory of Dairy Science, Northeast Agricultural University, Harbin 150030, Heilongjiang, China. [email protected]. (5)Key Laboratory of Dairy Science, Northeast Agricultural University, Harbin 150030, Heilongjiang, China. [email protected]. (6)Key Laboratory of Dairy Science, Northeast Agricultural University, Harbin 150030, Heilongjiang, China. [email protected]. (7)Department of Nutrition and Food Sciences, College of Agriculture and Life Sciences, University of Vermont, Burlington, VT 05405, USA. [email protected].

The compound 3,3'-diindolylmethane (DIM) has a broad spectrum of anticancer activities. However, low stability and bioavailability limit its application. Elucidating interactions between DIM and β-lactoglobulin (β-LG) may be useful for fabricating whey protein-based protecting systems. Interaction with DIM increased the diameter and absolute zeta potential value of β-LG. UV-absorption spectra suggested that there was a complex of DIM and β-LG. β-LG showed enhanced fluorescence intensity by complexing with DIM with a binding constant of 6.7 × 105 M-1. Upon interaction with DIM, β-LG was decreased in secondary structure content of helix and turn while increased in β-sheet and unordered. FT-IR spectra and molecular docking results indicated the roles of hydrophobic interaction and hydrogen bond for the formation of DIM and β-LG nanocomplexes. Data suggested that β-LG may be a good vehicle for making a protein-based DIM protection and delivery system due to the tight binding of DIM to β-LG.

Colloids Surf B Biointerfaces. 2019 Sep 1;181:295-304. doi: 10.1016/j.colsurfb.2019.05.063. Epub 2019 May 25.

3,3'-Diindolylmethane nanoencapsulation improves its antinociceptive action: Physicochemical and behavioral studies.

Mattiazzi J(1), Marcondes Sari MH(1), Brum TB(1), Araújo PCO(2), Nadal JM(3), Farago PV(3), Nogueira CW(2), Cruz L(4).

Author information: (1)Programa de Pós-graduação em Ciências Farmacêuticas, Laboratório de Tecnologia Farmacêutica, Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, 97105-900, RS, Brazil. (2)Programa de Pós-graduação em Bioquímica Toxicológica, Laboratório de Síntese, Reatividade e Avaliação Farmacológica e Toxicológica de Organocalcogênios, Departamento de Biologia Molecular, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, 97105-900, RS, Brazil. (3)Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Estadual de Ponta Grossa, 84030-900, PR, Brazil. (4)Programa de Pós-graduação em Ciências Farmacêuticas, Laboratório de Tecnologia Farmacêutica, Departamento de Farmácia Industrial, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Santa Maria, 97105-900, RS, Brazil. Electronic address: [email protected].

This study aimed to characterize the physicochemical properties of 3,3'-diindolylmethane (DIM)-loaded nanocapsules (NCs) as well as the antinociceptive effect using distinct animal models (hot plate test, formalin-induced nociception and complete Freud's adjuvant induced paw inflammation). The DIM-loaded NCs (composed by primula oil and ethylcellulose) were characterized using differential scanning calorimetry, thermogravimetric analysis, Fourier-transformed infrared spectroscopy, X-ray diffractometry and scanning electron microscopy. The physicochemical characterization demonstrated that DIM could be molecularly dispersed into the NCs, whose size was nanometric with a spherical shape. An improvement in DIM thermal stability was achieved by its encapsulation and there were no interactions among the formula components. For the nociceptive evaluation, male adult Swiss mice were pretreated with the NCs or free DIM by the intragastric route at the dose of 10 mg/Kg (time-response curve), 5 or 2.5 mg/Kg (dose-response curve). The behavioral tests were performed over an experimental period of 0.5-8 h. Both free and nanoencapsulated DIM reduced the mechanical hypernociception induced by CFA, mitigated nociceptive behavior of formalin-induced neurogenic and inflammatory pain and increased paw withdrawal latency assessed by the hot-plate test. Importantly, the DIM nanoencapsulation promoted a rapid initiation and prolonged the bioactive antinociceptive action (up to 8 h) as well as reduced the effective dose in comparison to its free form. In summary, this study reported that the NCs had adequate nanometric size, increased DIM stability and its antinociceptive action in different animal models, suggesting that the formulation may be a possible therapeutic alternative to the management of pain and inflammatory-related pathologies.

Mol Med Rep. 2019 Jun;19(6):5115-5122. doi: 10.3892/mmr.2019.10178. Epub 2019 Apr 22.

3,3'‑Diindolylmethane mitigates lipopolysaccharide‑induced acute kidney injury in mice by inhibiting NOX‑mediated oxidative stress and the apoptosis of renal tubular epithelial cells.

He J(1), Huang T(2), Zhao L(2).

Author information: (1)Department of Nephrology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China. (2)Department of Emergency, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China.

3,3'‑Diindolylmethane (DIM) is a naturally derived indole compound found in the Brassica family of vegetables. DIM has several beneficial effects, including anti‑cancer, anti‑inflammatory and anti‑angiogenic functions. However, the effects of DIM on acute kidney injury (AKI) stimulated by lipopolysaccharide (LPS) are poorly studied. In this present study, male BALB/c mouse models of AKI were established using intraperitoneal injections of 10 mg/kg LPS. DIM (40 mg/kg) was administered intraperitoneally 24 and 2 h before LPS exposure. The results indicated that DIM significantly mitigated histopathological changes in the kidneys and improved the levels of blood urea nitrogen and serum creatinine. DIM also suppressed the LPS‑induced production of reactive oxygen species and cell apoptosis. Furthermore, DIM treatment significantly decreased the expression of NADPH oxidase 2 (NOX2) and NOX4 in LPS‑treated mice. Therefore, DIM may exert its renoprotective actions by inhibiting NOX‑mediated oxidative stress and the apoptosis of renal tubular epithelial cells.

Nutr Cancer. 2019;71(6):992-1006. doi: 10.1080/01635581.2019.1577979. Epub 2019 Apr 29.

3,3'-Diindolylmethane Inhibits TNF-α- and TGF-β-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells.

Lee J(1).

Author information: (1)a Department of Food and Nutrition , Chosun University , Gwangju , Korea.

Epithelial-mesenchymal transition (EMT) is the initial event required by cancer cells. Thus, inhibition of the EMT process could have potential benefits for preventing the spread of cancers. The phytochemicals have been reported to have inhibitory activity against the EMT process in breast cancers, but the mechanism behind this effect has not been fully elucidated. 3,3'-Diindolylmethane (DIM) is a major indole derived from bioactive compounds in cruciferous vegetables. In this study, we examined the effects of DIM cotreatment together with TNF-α/TGF-β on the EMT process as well as the mechanisms underlying its effects on human breast cancer cells. DIM significantly enhanced the mRNA and protein expression of E-cadherin and occludin in MCF-7 cells. The protein expression levels of E-cadherin and occludin in MCF-7 cells were significantly decreased after TNF-α/TGF-β treatment alone, but these effects were reversed by the DIM co-treatment. Furthermore, DIM with TNF-α/TGF-β co-treatment attenuated the phosphorylation of Smad2/3 and ERK1/2 proteins. DIM significantly inhibited the TNF-α/TGF-β-induced migration of breast cancer cells. Taken together, the results indicated that DIM effectively suppressed EMT processes through the inhibition of TNF-α/TGF-β-associated signaling pathways in breast cancer cells. Thus, DIM may be a novel preventive and/or therapeutic approach for the treatment of breast cancers.

Biopharm Drug Dispos. 2019 May;40(5-6):188-194. doi: 10.1002/bdd.2182. Epub 2019 May 20.

Strain-specific altered nicotine metabolism in 3,3'-diindolylmethane (DIM) exposed mice.

Bloom AJ(1), Upadhyaya P(2), Kharasch ED(3).

Author information: (1)Department of Genetics, Washington University, St Louis, MO. (2)Masonic Cancer Center, University of Minnesota, Minneapolis, MN. (3)Department of Anesthesiology, Duke University School of Medicine, Durham, NC.

Two indole compounds, indole-3-carbinol (I3C) and its acid condensation product, 3,3'-diindolymethane (DIM), have been shown to suppress the expression of flavin-containing monooxygenases (FMO) and to induce some hepatic cytochrome P450s (CYPs) in rats. In liver microsomes prepared from rats fed I3C or DIM, FMO-mediated nicotine N-oxygenation was decreased, whereas CYP-mediated nicotine metabolism to nicotine iminium and subsequently to cotinine was unchanged. Therefore, it was hypothesized that in mice DIM would also suppress nicotine N-oxygenation without affecting CYP-mediated nicotine metabolism. Liver microsomes were produced from male and female C57BL/6 J and CD1 mice fed 2500 parts per million (ppm) DIM for 14 days. In liver microsomes from DIM-fed mice, FMO-mediated nicotine N-oxygenation did not differ from the controls, but CYP-mediated nicotine metabolism was significantly increased, with results varying by sex and strain. To confirm the effects of DIM in vivo, control and DIM-fed CD1 male mice were injected subcutaneously with nicotine, and the plasma concentrations of nicotine, cotinine and nicotine-N-oxide were measured over 30 minutes. The DIM-fed mice showed greater cotinine concentrations compared with the controls 10 minutes following injection. It is concluded that the effects of DIM on nicotine metabolism in vitro and in vivo differ between mice and rats and between mouse strains, and that DIM is an effective inducer of CYP-mediated nicotine metabolism in commonly studied mouse strains.

 

Front Immunol. 2019 Mar 26;10:560. doi: 10.3389/fimmu.2019.00560. eCollection 2019.

Indole Treatment Alleviates Intestinal Tissue Damage Induced by Chicken Coccidiosis Through Activation of the Aryl Hydrocarbon Receptor.

Kim WH(1), Lillehoj HS(1), Min W(2).

Author information: (1)Animal Biosciences and Biotechnology Laboratory, U. S. Department of Agriculture, Beltsville Agricultural Research Center, ARS, Beltsville, MD, United States. (2)College of Veterinary Medicine and Institute of Animal Medicine, Gyeongsang National University, Jinju, South Korea.

Indoles, as the ligands of aryl hydrocarbon receptor (AhR), have been shown to possess immune-modulating property in terms of the balancing between regulatory T cells (Treg) and T helper 17 cells (Th17) activities. In the present study, we examined the effects of dietary indoles, 3,3'-diindolylmethane (DIM) and indole-3-carbinol (I3C), on CD4+T cell population and functions in chickens. Furthermore, the effects of dietary DIM treatment on chicken coccidiosis caused by an apicomplexan parasite were investigated. Dietary treatment of healthy chickens with DIM and I3C induced increased CD4+CD25+ (Treg) cells and the mRNA expression of IL-10, while decreasing number of CD4+IL-17A+ (Th17) cells and Th17-related cytokines transcripts expression in the intestine. In addition, we explored the role of AhR in indole-treated splenic lymphocytes by using AhR antagonist and our results suggested that DIM is a ligand for chicken AhR. In chicken coccidiosis, treatment of DIM increased the ratio of Treg/Th17 cells and significantly reduced intestinal lesion although no significant changes in body weight and fecal oocyst production were noted compared to non-treated control group. These results indicate that DIM is likely to affect the ratios of Treg/Th17 reducing the level of local inflammatory response induced by Eimeria or facilitate repairing process of inflamed gut following Eimeria infection. The results described herein are thus consistent with the concept that AhR ligand modulates the T cell immunity through the alteration of Treg/Th17 cells with Treg dominance. To our knowledge, present study is the first scientific report showing the effects of dietary indole on T cell immunity in poultry species.

Bioorg Chem. 2019 Jun;87:484-494. doi: 10.1016/j.bioorg.2019.03.061. Epub 2019 Mar 22.

Synthesis and biological evaluation of new bisindole-imidazopyridine hybrids as apoptosis inducers.

Sunkari S(1), Bonam SR(2), Rao AVS(3), Riyaz S(4), Lakshma Nayak V(3), Kumar HMS(2), Kamal A(5), Nagendra Babu B(6).

Author information: (1)Centre for Semio Chemicals, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India; Academy of Scientific and Innovative Research, New Delhi 110 025, India. (2)Academy of Scientific and Innovative Research, New Delhi 110 025, India; Vaccine Immunology Laboratory, Natural Product Chemistry Division, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India. (3)Centre for Semio Chemicals, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India. (4)Department of Chemistry, Jawaharlal Nehru Technological University, Hyderabad 500085, India. (5)Centre for Semio Chemicals, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India; Academy of Scientific and Innovative Research, New Delhi 110 025, India; School of Pharmaceutical Education and Research (SPER), Jamia Hamdard, New Delhi 110062, India. Electronic address: [email protected]. (6)Centre for Semio Chemicals, CSIR-Indian Institute of Chemical Technology (IICT), Hyderabad 500007, India; Academy of Scientific and Innovative Research, New Delhi 110 025, India. Electronic address: [email protected].

A series of diindolylmethanes (5a-t) were designed, synthesized, and examined for their cytotoxicity against four human cancer cell lines like prostate (DU-145), lung (A549), breast (MCF-7) and cervical cancer (HeLa). These results revealed that among all the hybrids, two (5k and 5r) were identified and exhibited significant cytotoxic effect against A549 cancer cells with IC50 values of 1.65 ± 0.3 and 1.80 ± 0.8 µM respectively. To investigate the reasons for the cytotoxic activity, the conventional biological assays were carried out with 5k and 5r on the A549 cancer cells. Both hybrids led to the arrest of A549 cell lines at the G2/M phase of the cell cycle and strongly induced apoptosis. Further the apoptotic effects of 5k and 5r were confirmed by ROS, annexin-V FITC, and mitochondrial membrane potential. Moreover, structure-activity relationships were elucidated with various substitutions on these hybrids.

4Cells. 2019 Mar 7;8(3):220. doi: 10.3390/cells8030220.

Activation of COUP-TFI by a Novel Diindolylmethane Derivative.

Yoon K(1)(2), Chen CC(3), Orr AA(4), Barreto PN(5), Tamamis P(6), Safe S(7)(8).

Author information: (1)Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX 77030, USA. [email protected]. (2)Division of Translational Science, National Cancer Center, Goyang-si, Gyeonggi-do 10408, Korea. [email protected]. (3)Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA. [email protected]. (4)Department of Chemical Engineering, Texas A&M University, College Station, TX 77843, USA. [email protected]. (5)Department of Chemical Engineering, Texas A&M University, College Station, TX 77843, USA. [email protected]. (6)Department of Chemical Engineering, Texas A&M University, College Station, TX 77843, USA. [email protected]. (7)Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX 77030, USA. [email protected]. (8)Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA. [email protected].

Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan receptor and member of the nuclear receptor superfamily. Among a series of methylene substituted diindolylmethanes (C-DIMs) containing substituted phenyl and heteroaromatic groups, we identified 1,1-bis(3'-indolyl)-1-(4-pyridyl)-methane (DIM-C-Pyr-4) as an activator of COUP-TFI. Structure activity studies with structurally diverse heteroaromatic C-DIMs showed that the pyridyl substituted compound was active and the 4-pyridyl substituent was more potent than the 2- or 3-pyridyl analogs in transactivation assays in breast cancer cells. The DIM-C-Pyr-4 activated chimeric GAL4-COUP-TFI constructs containing full length, C- or N-terminal deletions, and transactivation was inhibited by phosphatidylinositol-3-kinase and protein kinase A inhibitors. However, DIM-C-Pyr-4 also induced transactivation and interactions of COUP-TFI and steroid receptor coactivators-1 and -2 in mammalian two-hybrid assays, and ligand-induced interactions of the C-terminal region of COUP-TFI were not affected by kinase inhibitors. We also showed that DIM-C-Pyr-4 activated COUP-TFI-dependent early growth response 1 (Egr-1) expression and this response primarily involved COUP-TFI interactions with Sp3 and to a lesser extent Sp1 bound to the proximal region of the Egr-1 promoter. Modeling studies showed interactions of DIM-C-Pyr-4 within the ligand binding domain of COUP-TFI. This report is the first to identify a COUP-TFI agonist and demonstrate activation of COUP-TFI-dependent Egr-1 expression.

Noncoding RNA Res. 2018 Nov 4;3(4):213-225. doi: 10.1016/j.ncrna.2018.11.001. eCollection 2018 Dec.

Interplay of non-coding RNAs and approved antimetabolites such as gemcitabine and pemetrexed in mesothelioma.

Biersack B(1).

Author information: (1)Organic Chemistry Laboratory, University of Bayreuth, Universitätsstraße 30, 95447, Bayreuth, Germany.

Erratum in Noncoding RNA Res. 2020 Nov 07;5(4):219.

Gemcitabine and pemetrexed are clinically approved antimetabolites for the therapy of mesothelioma diseases. These drugs are often applied in combination with platinum complexes and other drugs. The activity of antimetabolites depended on the expression levels of certain non-coding RNAs, in particular, of small microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). The development of tumor resistance towards antimetabolites was regulated by non-coding RNAs. An overview of the interplay between gemcitabine/pemetrexed antimetabolites and non-coding RNAs in mesothelioma is provided. Further to this, various non-coding RNA-modulating agents are discussed which displayed positive effects on gemcitabine or pemetrexed treatment of mesothelioma diseases. A detailed knowledge of the connections of non-coding RNAs with antimetabolites will be constructive for the design of improved therapies in future.

DOI: 10.1016/j.ncrna.2018.11.001 PMCID: PMC6257890 PMID: 30809600

J Inorg Biochem. 2019 May;194:1-6. doi: 10.1016/j.jinorgbio.2019.02.005. Epub 2019 Feb 13.

Anticancer properties of a new non-oxido vanadium(IV) complex with a catechol-modified 3,3'-diindolylmethane ligand.

Dankhoff K(1), Ahmad A(2), Weber B(1), Biersack B(3), Schobert R(4).

Author information: (1)Inorganic Chemistry IV, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany. (2)USA Mitchell Cancer Institute, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA. (3)Organic Chemistry Laboratory, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany. Electronic address: [email protected]. (4)Organic Chemistry Laboratory, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany.

In order to identify new active drug candidates against cancer diseases we investigated the tumor cell growth inhibition, formation of reactive oxygen species, mitochondrial membrane damage, cell cycle arrest and DNA binding activity of a new bis(triethylammonium) tris[1,1-bis(indol-3-yl)-1-(3,4-catecholate)methane]vanadate(IV) complex. It exhibited significant antiproliferative activity against various cancer cell lines, showed a stronger DNA binding than cisplatin and led to mitochondrial damage, a formation of reactive oxygen species, and a cell cycle arrest in the G2/M phase of cancer cells.

Molecules. 2019 Feb 15;24(4):702. doi: 10.3390/molecules24040702.

Physicochemical and Microstructural Properties of Polymerized Whey Protein Encapsulated 3,3'-Diindolylmethane Nanoparticles.

Khan A(1), Wang C(2), Sun X(3), Killpartrick A(4), Guo M(5)(6).

Author information: (1)Department of Food Science, College of Food Science and Engineering, Jilin University, Changchun 130062, China. [email protected]. (2)Department of Food Science, College of Food Science and Engineering, Jilin University, Changchun 130062, China. [email protected]. (3)Department of Food Science, College of Food Science and Engineering, Jilin University, Changchun 130062, China. [email protected]. (4)Food Science Corporation, Inc., Williston, VT 05495, USA. [email protected]. (5)College of Agriculture and Life Sciences, The University of Vermont, Burlington, VT 05405, USA. [email protected]. (6)Department of Food Science, Northeast Agriculture University, Harbin 150030, China. [email protected].

The fat-soluble antioxidant 3,3'-diindolylmethane (DIM), is a natural phytochemical found in Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts. The stability of this compound is a major challenge for its applications. Polymerized whey protein (PWP)-based DIM nanoparticles were prepared at different mass ratios of protein and DIM by mixing PWP and DIM followed by ultrasound treatment for 4 min. All the nanoparticles were studied for particle size, zeta potential, rheological and microstructural properties, and storage stability. The mean particle size of the PWP-based nanoparticles was significantly increased (p < 0.05) by the addition of DIM at different mass ratios, ranging from 241.33 ± 14.82 to 270.57 ± 15.28 nm. Zeta potential values of all nanoparticles were highly negative (greater than ±30 mV), suggesting a stable solution due its electrostatic repulsive forces. All samples exhibited shear thinning behavior (n < 1), fitted with Sisko model (R² > 0.997). Fourier Transform Infrared (FTIR)spectra revealed that the secondary structure was changed and the absorption intensity for hydrogen bonding got stronger by further incorporating DIM into PWP. Transmission electronic microscopy (TEM) images showed spherical and smooth surface shape of the PWP-based nanoparticles. DIM encapsulated by PWP showed enhanced stability at 4, 37 and 55 °C for 15 days evidenced by changes in mean particle size and color (a*-value and b*-value) compared with control (DIM only). In conclusion, the polymerized whey protein based 3,3'-diindolylmethane nanoparticles are stable and the encapsulation may protect the core material from oxidation.

 Apoptosis. 2019 Jun;24(5-6):435-452. doi: 10.1007/s10495-019-01522-2.

The neuroprotective action of 3,3'-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy that depends on HDAC and AhR/CYP1A1 but not ERα/CYP19A1 signaling.

Rzemieniec J(1), Wnuk A(1), Lasoń W(2), Bilecki W(3), Kajta M(4).

Author information: (1)Department of Experimental Neuroendocrinology, Laboratory of Molecular Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smetna Street 12, 31-343, Krakow, Poland. (2)Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smetna Street 12, 31-343, Krakow, Poland. (3)Department of Pharmacology, Laboratory of Pharmacology and Brain Biostructure, Institute of Pharmacology, Polish Academy of Sciences, Smetna Street 12, 31-343, Krakow, Poland. (4)Department of Experimental Neuroendocrinology, Laboratory of Molecular Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smetna Street 12, 31-343, Krakow, Poland. [email protected].

There are no studies examining the effects of 3,3'-diindolylmethane (DIM) in neuronal cells subjected to ischemia. Little is also known about the roles of apoptosis and autophagy as well as AhR and ERα signaling and HDACs in DIM action. We demonstrated for the first time the strong neuroprotective capacity of DIM in mouse primary hippocampal cell cultures exposed to ischemia at early and later stages of neuronal development. The protective effects of DIM were mediated via inhibition of ischemia-induced apoptosis and autophagy that was accompanied by a decrease in AhR/CYP1A1 signaling and an increase in HDAC activity. DIM decreased the levels of pro-apoptotic factors, i.e., Fas, Caspase-3, and p38 mitogen-activated protein kinase (MAPK). DIM also reduced the protein levels of autophagy-related Beclin-1 (BECN1) and microtubule-associated proteins 1A/1B light chain (LC3), partially reversed the ischemia-induced decrease in Nucleoporin 62 (NUP62) and inhibited autophagosome formation. In addition, DIM completely reversed the ischemia-induced decrease in histone deacetylase (HDAC) activity in hippocampal neurons. Although DIM inhibited AhR/CYP1A1 signaling, it did not influence the protein expression levels of ERα and ERα-regulated CYP19A1 which are known to be controlled by AhR. This study demonstrated for the first time, that the neuroprotective action of 3,3'-diindolylmethane against ischemia involves an inhibition of apoptosis and autophagy and depends on AhR/CYP1A1 signaling and HDAC activity, thus creating the possibility of developing new therapeutic strategies that target neuronal degeneration at specific molecular levels.

 Bioorg Med Chem Lett. 2019 Apr 15;29(8):1007-1011. doi: 10.1016/j.bmcl.2019.02.010. Epub 2019 Feb 11.

Pharmacokinetic evaluation of medicinally important synthetic N,N' diindolylmethane glucoside: Improved synthesis and metabolic stability.

Magotra A(1), Gour A(1), Sharma DK(2), Dash AK(3), Singh G(1), Mukherjee D(4), Nandi U(5).

Author information: (1)PK-PD, Toxicology and Formulation Division, CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir 180 001, India; Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir 180 001, India. (2)Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir 180 001, India; Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester 01605, USA. (3)Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir 180 001, India; School of Pharmaceutical Sciences, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh 173 229, India. (4)Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir 180 001, India; Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir 180 001, India. Electronic address: [email protected]. (5)PK-PD, Toxicology and Formulation Division, CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir 180 001, India; Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Integrative Medicine, Jammu, Jammu and Kashmir 180 001, India. Electronic address: [email protected].

An improved route for the synthesis of N,N'-diindolyl methane (DIM) glycosides has been developed by using Fe/Al pillared clay catalyst. In-silico pharmacokinetics followed by in-vitro studies like aqueous solubility, lipophilicity, P-glycoprotein (P-gp) dependent ATPase activity, permeability, plasma protein binding, RBC partitioning, metabolic stability in different liver microsomes and its in-vitro-in-vivo extrapolation were conducted for the most potent derivative namely NGD16. The compound was found to have low solubility, optimum lipophilicity, no P-gp inhibitory activity, intermediate permeability, high plasma protein binding, low RBC partitioning, acceptable metabolic stability in rat liver microsomes (RLM) as well as human liver microsomes (HLM) with intermediate hepatic extraction ratio.

Cancer Lett. 2019 Feb 1. pii: S0304-3835(19)30051-5. doi:

10.1016/j.canlet.2019.01.031. [Epub ahead of print]

 

3,3'-Diindolylmethane inhibits patient-derived xenograft colon tumor growth by

targeting COX1/2 and ERK1/2.

 

Tian X(1), Liu K(2), Zu X(1), Ma F(1), Li Z(3), Lee M(4), Chen H(5), Li Y(5),

Zhao Y(3), Liu F(1), Oi N(5), Bode AM(5), Dong Z(6), Kim DJ(7).

 

3,3'-Diindolymethane (DIM) is a dimeric condensation product of indole-3-carbinol

(I3C) that is found in broccoli and cabbage. Although DIM has been reported to

exhibit anticancer properties against multiple tumor types, the direct target

proteins of DIM have not been fully investigated. In the present study, we report

that DIM is a novel COX1/2 and ERK1/2 inhibitor that suppresses growth of colon

cancer in vitro and in vivo. To identify possible molecular targets of DIM, 11

potential candidate proteins were validated by an in vitro kinase or enzyme

assay. We found that DIM directly inhibits COX1/2 and ERK1/2 protein activities

in vitro. Additionally, the PGE2 production (COX-mediated metabolite) and

phosphorylated RSK expression (ERK1/2 direct downstream kinase) were strongly

suppressed by DIM in colon cancer cells. The inhibition of cell growth by DIM is

dependent on the expression of COX1/2 or ERK1/2 proteins. Notably, oral

administration of DIM suppressed patient-derived xenograft colon tumor growth in

an in vivo mouse model. Overall these results suggest that DIM is a potent and

dual COX1/2 and ERK1/2 inhibitor that might be used for chemotherapy against

colon cancer.

 

Chem Biodivers. 2019 Feb 4. doi: 10.1002/cbdv.201900028. [Epub ahead of print]

 

Pentafluorophenyl substitution of natural diindolylmethane strongly enhances

growth inhibition and apoptosis induction in various cancer cell lines.

 

Ahmad A(1), Dandawate P(2), Schruefer S(3), Padhye S(2), Sarkar FH(4), Schobert

R(3), Biersack B(5).

 

Diindolylmethane (DIM) is a known antitumoral compound formed by

digestion of indole-3-carbinol, an ingredient of various Brassica vegetables. Out

of a series of nine fluoroaryl derivatives of 1, three pentafluorophenyl

derivatives 2c, 2h and 2i were identified that exhibited a two to five times

greater anti-proliferative effect and an increased apoptosis induction when

compared with 1 in the following carcinoma cell lines: BxPC-3 pancreas, LNCaP

prostate, C4-2B prostate, PC3 prostate and the triple-negative MDA-MB-231 breast

carcinoma. Compound 2h was particularly efficacious against androgen-refractory

C4-2B prostate cancer cells (IC50 = 6.4 µM) and 2i against androgen-responsive

LNCaP cells (IC50 = 6.2 µM). In addition, 2c and 2h exhibited distinct activity

in three cancer cell lines resistant to 1.

 

Breast Cancer. 2019 Jan 25. doi: 10.1007/s12282-019-00950-x. [Epub ahead of

print]

 

3, 3'-Diindolylmethane-encapsulated chitosan nanoparticles accelerate molecular

events during chemical carcinogen-induced mammary cancer in Sprague Dawley rats.

 

Isabella S(1), Mirunalini S(2).

 

BACKGROUND: 3, 3'-Diindolylmethane (DIM) is a dietary indole compound; its

medical application was limited because of poor bioavailability, unsatisfying

dispersity, and rapid metabolism. To conquer this problem, nanoformulation of DIM

was synthesized and investigated its mechanism-based chemotherapeutic potential.

METHODS: 7,12-Dimethylbenz(a)anthracene (DMBA) 25 mg/kg b.wt initiated mammary

carcinogenesis in rats, the investigational tumor model that closely resembles

human mammary cancer. Rats had accessed after 8 weeks of tumor formation, DIM

10 mg/kg b.wt. and DIM@CS-NP 0.5 mg/kg b.wt. were administrated orally for 8

weeks.

RESULTS: The treatment with DIM@CS-NP 0.5 mg/kg b.wt. on DMBA-induced

tumor-bearing rats was down-regulated Cyclin D1, Bcl-2 expression, and

up-regulated proapoptotic proteins such as Bax, p53, Cytochrome-C, Caspase-9, and

Caspase-3 as compared to DIM 10 mg/kg b.wt. In addition, the mRNA expressions of

Cyclin D1, Bcl-2 decreased and increased Bax, p53 expression, in

immunohistochemical analysis decreased expressions of Cyclin D1 and PCNA in the

treatment of DIM@CS-NP 0.5 mg/kg b.wt. compared to DIM 10 mg/kg b.wt.

Histological analysis of tumor tissues shows abnormal in collagen deposition in

with Masson's trichrome (MT) and Picrosirius red (PR) staining, the treatment of

DIM@CS-NP 0.5 mg/kg b.wt. reduced the collagen deposition as compared to DIM

10 mg/kg b.wt.

CONCLUSION: Our results clearly provide evidence that DIM@CS-NP exerts

chemotherapeutic effect than DIM in DMBA model of mammary cancer by hold back

anomalous tumor cell proliferation and inducing apoptosis to intervene through

alterations of up-regulated and down-regulated molecules. Taken together, the

data provide new evidence for mechanism action of DIM@CS-NP on mammary cancer.

 

AAPS PharmSciTech. 2019 Jan 7;20(2):49. doi: 10.1208/s12249-018-1240-8.

 

Incorporation of 3,3'-Diindolylmethane into Nanocapsules Improves Its

Photostability, Radical Scavenging Capacity, and Cytotoxicity Against Glioma

Cells.

 

Mattiazzi J(1), Sari MHM(1), Lautenchleger R(1), Dal Prá M(2), Braganhol E(2),

Cruz L(3)(4).

 

3,3'-Diindolylmethane (DIM) is a phytochemical that presents health benefits

(antitumor, antioxidant, and anti-inflammatory effects). However, it is water

insoluble and thermo- and photolabile, restraining its pharmaceutical

applications. As a strategy to overcome such limitations, this study aimed the

development and characterization of DIM-loaded nanocapsules (NCs) prepared with

different compositions as well as the in vitro assessment of scavenging activity

and cytotoxicity. The formulations were obtained using the interfacial deposition

of preformed polymer method and were composed by Eudragit® RS100 or

ethylcellulose as polymeric wall and primula or apricot oil as the core. All the

formulations had adequate physicochemical characteristics: nanometric size

(around 190 nm), low polydispersity index (< 0.2), pH value at acid range, high

values of zeta potential, drug content, and encapsulation efficiency (~ 100%).

Besides, nanoencapsulation protected DIM against UVC-induced degradation and

increased the scavenging activity assessed by the

2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) and

1-1-diphenyl-2-picrylhydrazyl methods. The developed DIM-loaded nanocapsules were

further evaluated regarding the in vitro release profile and cytotoxicity against

a human glioblastoma cell line (U87 cells). The results demonstrated that the

nanoencapsulation promoted a sustained release of the bioactive compound (in the

range of 58-78% after 84 h) in comparison to its free form (86% after 12 h), as

well as provided a superior cytotoxic effect against the U87 cells in the highest

concentrations. Therefore, our results suggest that nanoencapsulation could be a

promising approach to overcome the DIM physicochemical limitations and

potentialize its biological properties.

 

Arch Toxicol. 2018 Dec 19. doi: 10.1007/s00204-018-2380-z. [Epub ahead of print]

 

Ultrasensitivity dynamics of diverse aryl hydrocarbon receptor modulators in a

hepatoma cell line.

 

Hoffman TE(1), Acerbo ER(1), Carranza KF(1), Gilberto VS(1), Wallis LE(1),

Hanneman WH(2).

 

The aryl hydrocarbon receptor (AhR) is a nuclear receptor that facilitates a wide

transcriptional response and causes a variety of adaptive and maladaptive

physiological functions. Such functions are entirely dependent on the type of

ligand activating it, and therefore, the nuances in the activation of this

receptor at the single-cell level have become a research interest for different

pharmacological and toxicological applications. Here, we investigate the

activation of the AhR by diverse classes of compounds in a Hepa1c1c7-based murine

hepatoma cell line. The exogenous compounds analyzed produced different levels of

ultrasensitivity in AhR activation as measured by XRE-coupled EGFP production and

analyzed by both flow cytometric and computational simulation techniques.

Interestingly, simulation experiments reported herein were able to reproduce and

quantitate the natural single-cell stochasticity inherent to mammalian cell lines

as well as the ligand-specific differences in ultrasensitivity. Classical AhR

modulators 2,3,7,8-tetrachlorodibenzodioxin (10- 1-105 pM), PCB-126

(10- 1-107 pM), and benzo[a]pyrene (10- 1-107 pM) produced the greatest levels of

single-cell ultrasensitivity and most maximal responses, while consumption-based

ligands indole-3-carbinol (103-109 pM), 3,3'-diindolylmethane (103-108 pM), and

cannabidiol (103-108 pM) caused low-level AhR activation in more purely graded

single-cell fashions. All compounds were tested and analyzed over a 24 h period

for consistency. The comparative quantitative results for each compound are

presented within. This study aids in defining the disparity between different

types of AhR modulators that produce distinctly different physiological outcomes.

In addition, the simulation tool developed for this study can be used in future

studies to predict the quantitative effects of diverse types of AhR ligands in

the context of pharmacological therapies or toxicological concerns.

 

BMC Struct Biol. 2018 Dec 6;18(1):15. doi: 10.1186/s12900-018-0095-2.

 

Comparative analysis of interactions between aryl hydrocarbon receptor ligand

binding domain with its ligands: a computational study.

 

Chitrala KN(1), Yang X(1), Nagarkatti P(1), Nagarkatti M(2).

 

BACKGROUND: Aryl hydrocarbon receptor (AhR) ligands may act as potential

carcinogens or anti-tumor agents. Understanding how some of the residues in AhR

ligand binding domain (AhRLBD) modulate their interactions with ligands would be

useful in assessing their divergent roles including toxic and beneficial effects.

To this end, we have analysed the nature of AhRLBD interactions with

2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), 6-formylindolo[3,2-b]carbazole

(FICZ), indole-3-carbinol (I3C) and its degradation product,

3,3'-diindolylmethane (DIM), Resveratrol (RES) and its analogue, Piceatannol

(PTL) using molecular modeling approach followed by molecular dynamic

simulations.

RESULTS: Results showed that each of the AhR ligands, TCDD, FICZ, I3C, DIM, RES

and PTL affect the local and global conformations of AhRLBD.

CONCLUSION: The data presented in this study provide a structural understanding

of AhR with its ligands and set the basis for its functions in several pathways

and their related diseases.

 

Mini Rev Med Chem. 2018 Nov 16. doi: 10.2174/1389557518666181116120145. [Epub

ahead of print]

 

Molecular targets, anti-cancer properties and potency of synthetic

indole-3-carbinol derivatives.

 

Noroozi MK(1), Mahmoodi M(1), Jafarzadeh A(1), Darekordi A(2), Hajizadeh MR(1),

Hassanshahi G(1).

 

The indole-3-carbinol (I3C) displays anti-proliferative and anti-cancer

activities against human cancer cells. Cellular proliferation is an important

event associated with cancer development and continued progression. This manifest

is described by altered expression and/or functions of cell cycle related

proteins. The constitutive activation of many signal transduction pathways also

stimulates cell growth. The immediate stages in tumor development are accompanied

by a fibrogenic response and the progression of a hypoxic environment, is in

favor of the survival and proliferatory functions of cancer stem cells. The main

part for the survival strategy in cancer cells may manifest through altering cell

metabolism. The growth and metastasis may be supported by increased generation of

appropriate hormones (in hormone dependent cancers), by promoting the development

of angiogenesis, with epithelial to mesenchymal transition. This may be

facilitated by progression of autophagy phenomenon, as well as by taking cues

from neighboring stromal cells. Several signaling pathways in association with

various factors specific for cellular viability, including hypoxia inducible

factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like

growth factor 1 (IGF-1) receptor, Human foreskin fibroblasts (HFF-1), Wnt, cell

cycle associated proteins, as well as androgen and estrogen receptor signaling

are reported to be inhibited by I3C. These evidences, in combination with

bioinformatics data represent very important information for describing signaling

pathways in parallel with molecular targets that may provide early diagnostic

markers and/or critical targets for designing and development of novel

therapeutic regimes alone or combined with drugs, to prevent tumor formation and

further progression. Very particularly, I3C and its dimeric product, 3,

3'-diindolylmethane (DIM), have been widely investigated for their importance

against a number of human cancers both in vitro and in vivo. We aimed the present

manuscript, to review an in-depth study of the anticancer properties and the

miscellaneous mechanisms underlying the anticarcinogenicity, thereby broadening

I3C therapeutic marvel.

 

BMC Pharmacol Toxicol. 2018 Nov 9;19(1):71. doi: 10.1186/s40360-018-0262-x.

 

3,3'-Diindolylmethane protects cardiomyocytes from LPS-induced inflammatory

response and apoptosis.

 

Luo Q(1)(2)(3), Yang A(1)(2)(3), Cao Q(1)(2)(3), Guan H(4)(5)(6).

 

BACKGROUND: 3,3'-Diindolylmethane (DIM) has been extensively studied as a

potential therapeutic drug with free radical scavenging, antioxidant and

anti-angiogenic effects. However, whether DIM has similar effects on

cardiomyocytes remains unknown. Here we evaluated DIM's influence on inflammation

and apoptosis of H9C2 cardiomyocytes induced by LPS and to explore the possible

mechanism of the effects.

METHODS: H9C2 cells were incubated with DIM (10, 20 and 30 μM) with or without

LPS for 24 h. The cytotoxicity of DIM was detected by CCK-8. The levels of tumour

necrosis factor (TNF)-α and interleukin (IL)-6 were then measured using RT-qPCR

and ELISA. Cell apoptosis rate and reactive oxygen species (ROS) content after

DIM treatment were measured by flow cytometry. Expressions of NFκB, P-NFκB, IκBa,

P-IκBa, Bax and Bcl-2 after DIM treatment were detected by western blot. The rate

of NFκB nuclear translocation after DIM treatment was determined by

immunocytochemical analysis.

RESULTS: LPS stimulation promoted TNF-α and IL-6 mRNA expression. After treatment

with various concentrations of DIM (10, 20 and 30 μM), TNF-α and IL-6 mRNA

expression was clearly impaired, especially in the LPS + DIM30(μM) group. ELISA

was used to measure TNF-α and IL-6 concentrations in cellular supernatant, and

the result was verified to be consistent with RT-qPCR. Additionally, DIM

treatment significantly blocked LPS-induced oxidative stress and inhibited

LPS-induced apoptosis in H9C2 cardiomyocytes according to the results detected by

flow cytometry. Moreover, compared with LPS alone, DIM significantly inhibited

the LPS-induced phosphorylation of NFκB (p-NFκB) and Bax expression and increased

Bcl-2 expression.

CONCLUSIONS: DIM may have a protective effect for H9C2 cardiomyocytes against

LPS-induced inflammatory response and apoptosis. DIM may be a new insight into

the treatment of septic cardiomyopathy.

 

J Oleo Sci. 2018;67(11):1473-1481. doi: 10.5650/jos.ess18077.

 

A Scandium Arylsulfonate-based Coordination Polymer as a Heterogeneous Catalyst

for the Friedel-Crafts Reaction of Indoles with Aldehydes.

 

Tungjiratthitikan P(1), Furuno H(2).

 

The scandium sulfonate-based coordination polymer Sc2(BPDS)3, which is easily

prepared by mixing scandium triisopropoxides and biphenyl-4,4'-disulfonic acid

(BPDSA), is an air-stable and storable solid that effectively catalyzes the

Friedel-Crafts reaction of indoles with aromatic aldehydes under heterogeneous

conditions to afford various aryl(diindolyl)methanes. The catalyst can be reused

without significant loss of activity after separation from the reaction mixture

by simple centrifugation followed by drying.

 

Free Radic Biol Med. 2019 Jan;130:244-255. doi:

10.1016/j.freeradbiomed.2018.10.410. Epub 2018 Oct 21.

 

Amelioration of whole abdominal irradiation-induced intestinal injury in mice

with 3,3'-Diindolylmethane (DIM).

 

Lu L(1), Jiang M(2), Zhu C(2), He J(2), Fan S(3).

 

Ionizing radiation-induced intestinal injury is a catastrophic disease with

limited effective therapies. 3,3'-Diindolylmethane (DIM), a potent antioxidant

agent, has previously been shown to ameliorate hematopoietic injury in a murine

model of total body radiation injury, but its effects on ionizing

radiation-induced intestinal damage are not clear. Here, we demonstrate that

administration of DIM not only protects mice against whole abdominal irradiation

(WAI)-induced lethality and weight loss but also ameliorates crypt-villus

structural and functional injury of the small intestine. In addition, treatment

with DIM significant enhances WAI-induced reductions in Lgr5+ ISCs and their

progeny cells, including lysozyme+ Paneth cells, Villin+ enterocytes and Ki67+

instantaneous amplifying cells, thus promoting small intestine repair following

WAI exposure. Notably, the expression of Nrf2 increased, while the number of

apoptotic cells and the expression of γH2AX decreased in the small intestines of

DIM-treated mice compared to mice treated with vehicle following WAI. In vitro,

we demonstrated that DIM protected human intestinal epithelial cell-6 (HIEC-6)

against ionizing radiation, leading to increased cell vitality. Mechanistically,

the radioprotective effect of DIM was likely attributable to its anti-DNA damage

effects in irradiated HIEC-6 cells. Moreover, these changes were related to

reduction in reactive oxygen species (ROS) levels and increased the activities of

antioxidant enzymatic in irradiated HIEC-6 cells. Additionally, the DIM

radioprotective effects on the intestine resulted in the restoration of the

WAI-shifted gut bacteria composition in mice. Collectively, our findings

demonstrate that the beneficial properties of DIM mitigate intestinal radiation

injury, which provides a novel strategy for improving the therapeutic effects of

irradiation-induced intestinal injury.

 

Indian J Clin Biochem. 2018 Oct;33(4):397-405. doi: 10.1007/s12291-017-0701-2.

Epub 2017 Sep 23.

 

3,3'-Diindolylmethane Encapsulated Chitosan Nanoparticles Accelerates

Inflammatory Markers, ER/PR, Glycoprotein and Mast Cells Population During

Chemical Carcinogen Induced Mammary Cancer in Rats.

 

Isabella S(1), Mirunalini S(1), Pandiyan K(2).

 

The present study aimed to investigate the effect of 3,3'-diindolylmethane (DIM)

on inflammatory markers, estrogen receptors (ER), progesterone receptors (PR),

level of glycoprotein and the mast cell population in 7,12-dimethylbenz (a)

anthracene (DMBA) 25 mg/kg b.wt. induced rat mammary carcinogenesis. After 8

weeks of tumor formation, rats had access to an oral administrated with DIM

10 mg/kg b.wt. and DIM@CS-NP 0.5 mg/kg body weight respectively for 8 weeks. The

oral administration of DIM@CS-NP 0.5 mg/kg b.wt. suppressed the Cox-2, NF-κB and

TNF-α protein expression on DMBA induced rats compared to DIM 10 mg/kg b.wt. The

ER/PR levels were increased on DMBA induced rats, treated with DIM@CS-NP

0.5 mg/kg b.wt. reduced ER/PR level as well as glycoprotein and mast cell

population than DIM 10 mg/kg b.wt. The result shows that, DIM@CS-NP 0.5 mg/kg

b.wt. has the potentially inhibit abnormal levels of inflammatory markers, ER,

PR, levels of glycoprotein and mast cell population compared to DIM 10 mg/kg

b.wt.

 

Pharm Biol. 2018 Dec;56(1):407-414. doi: 10.1080/13880209.2018.1495747.

 

3,3'-Diindolylmethane enhances apoptosis in docetaxel-treated breast cancer cells

by generation of reactive oxygen species.

 

Lanza-Jacoby S(1), Cheng G(1).

 

CONTEXT: A major problem in the treatment of cancer is the development of toxic

side effects and resistance to chemotherapy. The use of plant compounds to

overcome resistance and prevent toxicity is a potential strategy for treatment.

OBJECTIVE: We evaluated whether 3,3'-diindolylmethane (DIM) enhanced the

sensitivity of breast cancer cells to docetaxel (DOC).

MATERIALS AND METHODS: MDA-MB231 and Sk-BR-3 cells were treated with and without

25 or 50 µM of DIM and 1 nM of DOC for 48 and 72 h, respectively. MTT assay was

used to measure cell survival. Apoptosis and intracellular reactive oxygen

species (ROS) were determined by flow cytometry. The expression of proteins

regulating ROS production and apoptosis was evaluated by immunoblotting

technique.

RESULTS: Combining 25 µM of DIM with 1 nM DOC decreased cell survival by 42% in

MDA-MB231 cells and 59% in Sk-BR-3 cells compared to control, DIM, or DOC

(p ≤ 0.05). The combination treatment increased apoptosis over 20% (p ≤ 0.01) in

both cell lines, which was associated with decreased Bcl-2, increased Bax,

cleaved PARP and activated JNK (p ≤ 0.01). ROS production increased by 46.5% in

the MDA-MB231 and 29.3% in Sk-BR-3 cells with the combination compared to DIM or

DOC alone. Pretreating cells with N-acetyl-cysteine or Tiron abrogated the

anti-survival effect of the combination. The increase in ROS was associated with

a 54% decrease in MnSOD and 47% increase in NOX2 protein compared to the other

groups.

CONCLUSIONS: Our findings indicated that DIM enhances the sensitivity of breast

cancer cells to DOC treatment by increasing ROS, which led to decreased cell

survival and apoptosis.

 

J Altern Complement Med. 2018 Sep/Oct;24(9-10):902-909. doi:

10.1089/acm.2018.0150.

 

Advising Women Undergoing Treatment for Breast Cancer: A Narrative Review.

 

Lemanne D(1)(2)(3), Maizes V(1).

 

A majority of women undergoing conventional treatment for breast cancer also

undertake complementary and integrative approaches. Practitioners knowledgeable

about the evidence base behind common integrative approaches can help patients

attain improved quality of life, and at times, improved survival. Evidence-based

recommendations include the following: a plant-based diet for general health

after diagnosis, and carbohydrate restriction for patients with estrogen

receptor-positive postmenopausal breast cancer may be prudent. Other dietary

recommendations include a 13-h daily overnight fast. Carefully selected patients

may choose to fast the day before and the day of chemotherapy to decrease side

effects. Specific food recommendations include avoidance or limitation of

alcohol, and liberal culinary use of cruciferous vegetables, coffee, green tea,

soy, and flaxseed. Promising supplements include diindolylmethane and melatonin.

Omega 3 fatty acids may help with bone density in patients on aromatase

inhibitors, but may increase chemotherapy resistance. Findings regarding the

usefulness of multivitamins, vitamin D, vitamin C, and vitamin E are weak and/or

mixed different exercise modalities may have different effects and thus play

different roles in breast cancer therapy. Aerobic and resistance training

combined during breast cancer chemotherapy may confer a survival benefit, while

yoga may improve outcome in lymphedema patients. Current evidence suggests that

meditation, yoga, breathing, music therapy, guided imagery, and hypnosis may

improve mood and quality of life during breast cancer treatment. Acupuncture is

useful for treating side effects of breast cancer therapies, including hot

flushes, aromatase inhibitor-induced joint pain, chemotherapy-induced peripheral

neuropathy, and vulvodynia. Vaginal moisturizers and vaginal rings supplying

low-dose estrogen can be useful in the treatment of symptoms of

estrogen-deprivation states caused by breast cancer treatments; such symptoms

include vaginal dryness, dyspareunia, and sexual dysfunction. Carbon dioxide

laser technology can rejuvenate atrophied vaginal mucosa and relieve dyspareunia,

allowing avoidance of estrogen therapy. Tertiary sexual health centers are

available for referral.

 

Toxicol Sci. 2019 Feb 1;167(2):375-384. doi: 10.1093/toxsci/kfy245.

 

Employing Dietary Comparators to Perform Risk Assessments for Anti-Androgens

Without Using Animal Data.

 

Dent MP(1), Li H(1), Carmichael PL(1), Martin FL(2).

 

This study investigated the use of androgen receptor (AR) reporter gene assay

data in a non-animal exposure-led risk assessment in which in vitro

anti-androgenic activity and exposure data were put into context using a

naturally occurring comparator substance with a history of dietary consumption.

First, several dietary components were screened to identify which selectively

interfered with AR signaling in vitro, using the AR CALUX® test. The IC50 values

from these dose-response data together with measured or predicted human exposure

levels were used to calculate exposure: activity ratios (EARs) for the dietary

components and a number of other well-known anti-androgenic substances. Both

diindolylmethane (DIM) and resveratrol are specifically acting dietary

anti-androgens. The EARs for several anti-androgens were therefore expressed

relative to the EAR of DIM, and how this 'dietary comparator ratio' (DCR)

approach may be used to make safety decisions was assessed using an exposure-led

case study for an anti-androgenic botanical ingredient. This highlights a

pragmatic approach which allows novel chemical exposures to be put into context

against dietary exposures to natural anti-androgenic substances. The DCR approach

may have utility for other modes of action where appropriate comparators can be

identified.

 

Medchemcomm. 2018 Apr 11;9(7):1114-1130. doi: 10.1039/c8md00055g. eCollection

2018 Jul 1.

 

Triazole-diindolylmethane conjugates as new antitubercular agents: synthesis,

bioevaluation, and molecular docking.

 

Danne AB(1), Choudhari AS(2), Chakraborty S(2), Sarkar D(2), Khedkar VM(3),

Shingate BB(1).

 

Author information:

(1)Department of Chemistry , Dr. Babasaheb Ambedkar Marathwada University ,

Aurangabad 431 004 , India . Email: [email protected] ; ; Tel: +(91) 240

2403312.

(2)Combi-Chem Bio-Resource Center , Organic Chemistry Division , CSIR-National

Chemical Laboratory , Pune 411 008 , India.

(3)Department of Pharmaceutical Chemistry , Shri Vile Parle Kelavani Mandal's

Institute of Pharmacy , Dhule , Maharashtra 424 001 , India.

 

We describe the synthesis of novel triazole-incorporated diindolylmethanes (DIMs)

using a molecular hybridization approach. The in vitro antitubercular activity of

the DIMs against Mycobacterium tuberculosis H37Ra (ATCC 25177) was tested in the

active and dormant state. Among all the synthesized conjugates, the compounds 6b,

6f, 6l, 6n, 6q, 6r, and 6s displayed good antitubercular activity against both

the active and dormant Mtb H37Ra strain. The compound 6l exhibited good

antitubercular activity against dormant Mtb H37Ra with an IC50 value of 1 μg mL-1

and IC90 (MIC) value of 3 μg mL-1. The compounds 6b, 6l, and 6r displayed good

antitubercular activity against active Mtb H37Ra with IC50 values of 2.19, 1.52,

and 0.22 μg mL-1, respectively. The compounds 6b, 6h, 6l, and 6s displayed more

than 70% inhibition against the Gram-positive Bacillus subtilus strain at 3 μg

mL-1. The molecular docking study showed the binding modes of the titled

compounds in the active site of the DprE1 enzyme and assisted with elucidating a

structural basis for the inhibition of Mycobacteria.

 

Ren Fail. 2018 Nov;40(1):447-454. doi: 10.1080/0886022X.2018.1490322.

 

3,3'-Diindolylmethane ameliorates renal fibrosis through the inhibition of renal

fibroblast activation in vivo and in vitro.

 

Xia ZE(1), Xi JL(2), Shi L(3)

 

3,3'-Diindolylmethane (DIM), a natural acid condensation extracted from

cruciferous plants, exhibits anti-fibrotic effects in hepatic and cardiac

fibrosis models. The effects of DIM on renal fibrosis, however, are unclear. This

study aimed to explore the protective effects of DIM on renal fibrosis.

Unilateral ureteral obstruction (UUO) and transforming growth factor

(TGF)-β1-stimulated normal rat kidney (NRK)-49F fibroblast cell mouse models were

established. The models were then treated with DIM for the assessment of its

anti-fibrotic effects and mechanisms. Results of HE and Masson staining showed

that DIM reduced kidney injury and production of interstitial collagens fibrosis.

CTS also inhibited expression of fibronectin, collagen-1 but retain E-cadherin in

the UUO model. Furthermore, DIM suppressed local fibroblast activation, as

evidenced by the suppressed expression of the myofibroblast markers α-SMA and

vimentin in vivo and in vitro. In addition, DIM significantly inhibited the

TGF-β1-induced proliferation of NRK49F cells in a time- and dose-dependent

manner. DIM decreased Smad2/3 phosphorylation but increased Smad7 expression.

Results suggested that DIM inhibits TGF-β/Smad2/3 signaling to attenuate renal

interstitial fibrosis via inhibiting local fibroblast activation. This mechanism

is likely related to Smad7 induction.

 

Biochem Pharmacol. 2018 Sep;155:419-424. doi: 10.1016/j.bcp.2018.07.032. Epub

2018 Jul 26.

 

From TCDD-mediated toxicity to searches of physiologic AHR functions.

 

Bock KW(1).

 

TCDD-mediated toxicity of human individuals together with animal studies led to

identification of the aryl hydrocarbon receptor (AHR). It was characterized as

multifunctional ligand-activated transcription factor and environmental sensor.

Comparison of human toxic responses and animal models provide hints to

physiologic AHR functions including chemical and microbial defense, homeostasis

of stem/progenitor cells and modulation of the immune system in barrier organs

such as skin and the gastrointestinal tract. Extrapolation from animals to humans

is difficult due to marked species differences and dependence of AHR function on

the cellular context. Nevertheless, therapeutic possibilities of AHR agonists and

antagonists are in development. The AHR remains challenging and fascinating.

 

Arch Biochem Biophys. 2018 Jul 21. pii: S0003-9861(18)30087-0. doi:

10.1016/j.abb.2018.07.002. [Epub ahead of print]

 

3,3'-Diindolylmethane suppresses ovarian cancer cell viability and metastasis and

enhances chemotherapy sensitivity via STAT3 and Akt signaling in vitro and in

vivo.

 

Zou M(1), Xu C(2), Li H(3), Zhang X(4), Fan W(5).

 

Signal transducer and activator of transcription-3 (STAT3) protein is

constitutively activated in ovarian cancer. The purpose of this study was to

investigate the effects of 3,3'-diindolylmethane (DIM) on the regulation of STAT3

signaling and ovarian cancer cell viability, invasion, and sensitivity to

chemotherapy. Ovarian cancer SKOV3 and A2780 cell lines were treated with various

concentrations of DIM for different periods of time for assessment of cell

viability as well as gene expression before and after knockdown of STAT3

expression using STAT3 shRNA. DIM treatment potently suppressed the viabilities

of ovarian cancer cells. Consequently, DIM inhibited xenograft growth in nude

mice. In addition, at the gene level, DIM inhibited phosphorylation of STAT3 and

AKT proteins and expression of their downstream proteins. Moreover, knockdown of

STAT3 expression significantly enhanced DIM antitumor activity and cisplatin

sensitivity. Their combination suppressed the protein expression of survivin,

Bcl-2, Mcl-1, HIF-1α, VEGF, and MMPs, but activated caspase-3. Taken together,

the antitumor activity of DIM is via inhibition of the STAT3 and Akt signaling

pathways. The combination of STAT3 knockdown with DIM treatment could be further

evaluated as a therapeutic strategy for the treatment of advanced ovarian cancer.

 

J Org Chem. 2018 Sep 7;83(17):9902-9913. doi: 10.1021/acs.joc.8b01349. Epub 2018

Jul 31.

 

General Synthesis of Unsymmetrical 3,3'-(Aza)diindolylmethane Derivatives.

 

Pillaiyar T(1), Gorska E(1), Schnakenburg G(2), Müller CE(1).

 

Diindolylmethane (DIM) and its derivatives have recently been in the focus of

interest due to their significant biological activities, specifically in cancer

prevention and therapy. Molecular targets of DIM have been identified, e.g., the

immunostimulatory G protein-coupled receptor GPR84. However, most of the reported

and investigated DIM derivatives are symmetrical because general methods for

obtaining unsymmetrical DIMs have been lacking. To optimize the interaction of

DIM derivatives with their protein targets, unsymmetrical substitution is

required. In the present study we developed a new, mild and efficient access to

unsymmetrically substituted 3,3'-DIMs by reaction of

(3-indolylmethyl)trimethylammonium iodides with a wide range of substituted

indole derivatives. 7-Azaindole also led to the 3,3'-connected DIM analogue,

while 4- and 5-azaindoles reacted at the N1-nitrogen atom as confirmed by X-ray

crystallography. The reactions were performed in water without the requirement of

a catalyst or other additives. Wide substrate scope, operational simplicity,

environmentally benign workup, and high yields are further advantages of the new

method. The synthetic protocol proved to be suitable for upscaling to yield gram

amounts for pharmacological studies. This procedure will allow the preparation of

a broad range of novel, unsymmetrical DIM derivatives to exploit their potential

as novel drugs.

 

Arch Biochem Biophys. 2018 Sep 15;654:47-54. doi: 10.1016/j.abb.2018.07.010. Epub

2018 Jul 18.

 

Aryl hydrocarbon receptor enhances the expression of miR-150-5p to suppress in

prostate cancer progression by regulating MAP3K12.

 

Yu J(1), Feng Y(1), Wang Y(1), An R(2).

 

It has been reported that mircoRNAs (miRNAs) can act as tumor inhibitors in

multiple malignant tumors. As a tumor suppressor, miR-150-5p has been reported in

some cancers. However, the biological impacts of miR-150-5p in prostate cancer is

not fully elaborated. This study aims to explore the biological role and

mechanism of miR-150-5p in prostate cancer. The expression level of miR-150-5p

was examined with Quantitative real time polymerase chain reaction (qRT-PCR).

Moreover, Kaplan Meier analysis revealed that downregulation of miR-150-5p

predicted unfavorable prognosis for patients with prostate cancer. To identify

the inhibitory effects of miR-150-5p on the cellular processes of prostate

cancer, gain-of function assay was conducted. Next, the inhibitory effects of

Tetrachlorodibenzo-p-dioxin (TCDD) and 3,3'-Diindolylmethane (DIM) on the

proliferation and invasion of prostate cancer cells were demonstrated. Knockdown

of Ahr reversed the TCDD/DIM-mediated proliferation and invasion. The expression

level of CYP1A1 also was measured to confirm that Ahr was activated by TCDD or

DIM in prostate cancer cells. Mechanism experiments revealed that MAP3K12 is a

target mRNA of miR-150-5p in prostate cancer cells. In conclusion, Aryl

hydrocarbon receptor enhances the expression of miR-150-5p to suppress cell

proliferation and invasion in prostate cancer by regulating MAP3K12.

 

Reprod Biol. 2018 Sep;18(3):252-258. doi: 10.1016/j.repbio.2018.07.002. Epub 2018

Jul 9.

 

Comparison of dienogest effects upon 3,3'-diindolylmethane supplementation in

models of endometriosis and clinical cases.

 

Morales-Prieto DM(1), Herrmann J(2), Osterwald H(3), Kochhar PS(3), Schleussner

E(1), Markert UR(4), Oettel M(5).

 

Dienogest (DNG) administration is a well-established treatment for endometriosis

but bleeding irregularities remain its main disadvantage. Changes in diet, mainly

to vegetable consumption, are beneficial in the treatment of estrogen-related

pathologies but their use for endometriosis has been poorly studied. In this

study, addition of the phytochemical 3,3'-diindolylmethane (DIM) to DNG therapy

has been investigated in in vitro and ex vivo models for endometriosis and in a

small cohort of women with endometriosis. Endometrial Ishikawa cells were treated

with DNG or DIM at dosages from 10-10 M to 10-5 M for up to 72 h. Cell

proliferation was measured by assessing BrdU incorporation. Endometrial tissue

from women with endometriosis and controls was incubated with DNG or a

combination of DNG and DIM. Tissue viability was determined using a modified

colorimetric MTS assay. 17β-estradiol secretion was quantified by an

electro-chemiluminescence immunoassay. Finally, DNG as monotherapy or in

combination with DIM was randomly administered to women with endometriosis

(n = 8) over 3 months. Bleeding patterns and associated pelvic pain were assessed

by Visual Analogue Scale (VAS). DNG and DIM significantly reduced cell

proliferation in Ishikawa cells. Ex vivo, DIM reduced viability and estradiol

secretion specifically in endometriotic but not in normal endometrial tissue.

This effect was enhanced by combination with DNG. Endometriosis associated pelvic

pain was significantly reduced in patients taking the DNG-DIM combination therapy

compared to those taking DNG alone. Bleeding pattern (number and duration of

episodes) was significantly improved by addition of DIM to the DNG treatment. In

conclusion, addition of DIM enhances effects of DNG ex vivo and may ameliorate

bleeding patterns in endometriosis patients.

 

Mol Immunol. 2018 Sep;101:46-54. doi: 10.1016/j.molimm.2018.05.024. Epub 2018 Jun

2.

 

A novel diindolylmethane analog, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane,

inhibits the tumor necrosis factor-induced inflammatory response in primary

murine synovial fibroblasts through a Nurr1-dependent mechanism.

 

Afzali MF(1), Popichak KA(2), Burton LH(1), Klochak AL(1), Wilson WJ(1), Safe

S(3), Tjalkens RB(1), Legare ME(4).

 

The progression of rheumatoid arthritis involves the thickening of the synovial

lining due to the proliferation of fibroblast-like synoviocytes (FLS) and

infiltration by inflammatory cells. Tumor necrosis factor alpha (TNFα) is a

pro-inflammatory cytokine involved in progression of the disease. Under

rheumatoid conditions, FLS express the tumor necrosis factor (TNF)-recognition

complex (TNFR1, TNFR2, VCAM-1 and ICAM-1), which induces local macrophage

activation and leads to downstream nuclear factor κB (NF-κB) signaling. The

NF-κB-regulated inflammatory gene, cyclooxygenase (COX), increases synthesis of

prostaglandins that contribute to the propagation of inflammatory damage within

the joint. Because the nuclear orphan receptor, NR4A2 (Nurr1), can negatively

regulate NF-κB-dependent inflammatory gene expression in macrophages, we

postulated that activation of this receptor by the Nurr1 ligand

1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) would modulate

inflammatory gene expression in synovial fibroblasts by inhibiting NF-κB.

Treatment with C-DIM12 suppressed TNFα-induced expression of adhesion molecules

and NF-κB regulated genes in primary synovial fibroblasts including vascular

adhesion molecule 1 (VCAM-1), PGE2 and COX-2. Immunofluorescence studies

indicated that C-DIM12 did not prevent translocation of p65 and stabilized

nuclear localization of Nurr1 in synovial fibroblasts. Knockdown of Nurr1

expression by RNA interference prevented the inhibitory effects of C-DIM12 on

inflammatory gene expression, indicating that the anti-inflammatory effects of

this compound are Nurr1-dependent. Collectively, these data suggest that this

receptor may be a viable therapeutic target in RA.

Int J Mol Sci. 2018 May 7;19(5). pii: E1388. doi: 10.3390/ijms19051388.

 

Towards Resolving the Pro- and Anti-Tumor Effects of the Aryl Hydrocarbon

Receptor.

 

Narasimhan S(1), Stanford Zulick E(2), Novikov O(3), Parks AJ(4), Schlezinger

JJ(5), Wang Z(6), Laroche F(7), Feng H(8), Mulas F(9), Monti S(10), Sherr DH(11).

 

We have postulated that the aryl hydrocarbon receptor (AHR) drives the later,

more lethal stages of some cancers when chronically activated by endogenous

ligands. However, other studies have suggested that, under some circumstances,

the AHR can oppose tumor aggression. Resolving this apparent contradiction is

critical to the design of AHR-targeted cancer therapeutics. Molecular (siRNA,

shRNA, AHR repressor, CRISPR-Cas9) and pharmacological (AHR inhibitors)

approaches were used to confirm the hypothesis that AHR inhibition reduces human

cancer cell invasion (irregular colony growth in 3D Matrigel cultures and Boyden

chambers), migration (scratch wound assay) and metastasis (human cancer cell

xenografts in zebrafish). Furthermore, these assays were used for a head-to-head

comparison between AHR antagonists and agonists. AHR inhibition or

knockdown/knockout consistently reduced human ER&minus;/PR&minus;/Her2&minus; and

inflammatory breast cancer cell invasion, migration, and metastasis. This was

associated with a decrease in invasion-associated genes (e.g., Fibronectin,

VCAM1, Thrombospondin, MMP1) and an increase in CDH1/E-cadherin, previously

associated with decreased tumor aggression. Paradoxically, AHR agonists

(2,3,7,8-tetrachlorodibenzo-p-dioxin and/or 3,3&prime;-diindolylmethane)

similarly inhibited irregular colony formation in Matrigel and blocked metastasis

in vivo but accelerated migration. These data demonstrate the complexity of

modulating AHR activity in cancer while suggesting that AHR inhibitors, and,

under some circumstances, AHR agonists, may be useful as cancer therapeutics.

 

Obesity (Silver Spring). 2018 Jul;26(7):1153-1160. doi: 10.1002/oby.22145. Epub

2018 May 3.

 

3,3'-Diindolylmethane Enhances Glucose Uptake Through Activation of Insulin

Signaling in 3T3-L1 Adipocytes.

 

Choi KM(1), Yoo HS(1).

 

Author information:

(1)College of Pharmacy, Chungbuk National University, Cheongju, Republic of

Korea.

 

OBJECTIVE: Indole-3-carbinol (I3C), a naturally occurring compound found in

cruciferous vegetables, and its metabolite 3,3'-diindolylmethane (DIM) reduce

body mass and serum glucose levels in high-fat-diet-induced obese mice. This

study aimed to determine whether I3C or DIM could increase glucose uptake via

enhanced insulin sensitivity in 3T3-L1 adipocytes, as well as the mechanism

involved.

METHODS: 3T3-L1 preadipocytes were differentiated by using a mixture of

adipogenic inducers, including a suboptimal concentration of insulin.

RESULTS: DIM, but not I3C, increased adipocyte differentiation through

upregulation of peroxisome proliferator-activated receptor γ and

CCAAT/enhancer-binding protein α. DIM also enhanced glucose uptake by increasing

expression of glucose transporter 4 in adipocytes. This was associated with

DIM-enhanced phosphorylation of the signaling intermediates Akt, insulin receptor

substrate-1, and insulin receptor early in differentiation.

CONCLUSIONS: Our findings suggest that DIM may improve insulin sensitivity

through the activation of the insulin signaling pathway, leading to enhanced

glucose uptake.

 

Mol Carcinog. 2018 Sep;57(9):1102-1115. doi: 10.1002/mc.22828. Epub 2018 May 15.

 

Dual role of Par-4 in abrogation of EMT and switching on Mesenchymal to

Epithelial Transition (MET) in metastatic pancreatic cancer cells.

 

Katoch A(1)(2), Suklabaidya S(3), Chakraborty S(1)(2), Nayak D(1)(2), Rasool

RU(1)(2), Sharma D(4), Mukherjee D(4), Faheem MM(2), Kumar A(5), Sharma PR(1)(2),

Senapati S(3), Kumar LD(5), Goswami A(1)(2).

 

Epithelial-mesenchymal transition (EMT) is a critical event that occurs during

the invasion and metastatic spread of cancer cells. Here, we conceive a dual

mechanism of Par-4-mediated inhibition of EMT and induction of MET in metastatic

pancreatic cancer cells. First, we demonstrate that

1,1'-β-D-glucopyranosyl-3,3'-bis(5-bromoindolyl)-octyl methane (NGD16), an

N-glycosylated derivative of medicinally important phytochemical

3,3'-diindolylmethane (DIM) abrogates EMT by inducing pro-apoptotic protein

Par-4. Induction of Par-4 (by NGD16 or ectopic overexpression) strongly impedes

invasion with inhibition of major mesenchymal markers viz. Vimentin and Twist-1

epithelial marker- E-cadherin. Further, NGD16 triggers MET phenotypes in

pancreatic cancer cells by augmenting ALK2/Smad4 signaling in a Par-4-dependent

manner. Conversely, siRNA-mediated silencing of endogenous Par-4 unveil reversal

of MET with diminished E-cadherin expression and invasive phenotypes.

Additionally, we demonstrate that intact Smad4 is essential for Par-4-mediated

maintenance of E-cadherin level in MET induced cells. Notably, we imply that

Par-4 induction regulates E-cadherin levels in the pancreatic cancer cells via

modulating Twist-1 promoter activity. Finally, in vivo studies with syngenic

mouse metastatic pancreatic cancer model reveal that NGD16 strongly suppresses

metastatic burden, ascites formation, and prolongs the overall survival of

animals effectively.

 

Int J Radiat Biol. 2018 Jun;94(6):558-568. doi: 10.1080/09553002.2018.1467063.

Epub 2018 May 3.

 

A combination of resveratrol and 3,3'-diindolylmethane, a potent radioprotector.

 

Thekkekkara D(1), Basavan D(1), Chandna S(2), Nanjan MJ(3).

 

PURPOSE: Exposure to ionizing radiation causes damage to the genomic integrity

and stability of the cell. Though a large number of molecules have been studied

for their radioprotective capability, no single agent is available today that

meets all the requirements of a good radiprotector. In this study, we have

investigated a combination of Resveratrol (RSV) and 3,3'-Diindolyl methane (DIM)

for its efficacy for radioprotection. It is our hypothesis that this combination

that possesses less toxicity than synthetic compounds, free radical scavenging

potential, and the capacity to interfere with the several of the signaling

cascades that trigger damage to cell by ionizing radiation may possess good

radioprotective capability.

MATERIALS AND METHODS: Mice were pre-treated with a combination of RSV and DIM

and the 30-day mortality assay, endogenous antioxidant levels in intestinal

mucosa, metaphase chromosomal aberrations, and micronuclei formation were

assessed after exposed to ionizing radiation.

RESULTS: The dose modifying factor (DRF) obtained for RSV, DIM, and the

combination is 1.15, 1.17, and 1.3, respectively. Pre-treatment of mice with the

combination results in significant (***p = .001) protection of the endogenous

antioxidant levels, chromosomal aberrations, micronuclei formation, after

exposure to ionizing radiation.

CONCLUSIONS: Our findings suggest that pre-treatment with the combination of RSV

and DIM protects effectively from the ionizing radiation-induced damage at the

molecular, cellular, and tissue levels by counteracting both the direct and

indirect effects.

 

J Am Podiatr Med Assoc. 2018 Mar;108(2):189-193. doi: 10.7547/16-115.

 

The Ring Verruca Plantaris in Cantharidin Use A Case Report.

 

Hood CR Jr, Miller JR.

 

Verrucae (warts) are the most common viral infections of the skin, affecting 7%

to 10% of the general population. Typically caused by human papillomavirus type

1, plantar warts manifest as benign proliferation of the epithelial cells on the

feet. It has been cited that up to one-third of nongenital warts become

recalcitrant, and biopsy is often required to confirm diagnosis and direct

appropriate treatment. These treatments can vary from various types of oral

medications, acids, ablative modalities, and injections. In this article, we

present a case of a recalcitrant plantar wart that appeared to circumferentially

spread from the initial site after first-line treatment and presumed resolution

with the product cantharidin. The development of ring warts is a known

complication associated with cantharidin use, with little described rationale to

the presentation.

 

Invest New Drugs. 2018 Aug;36(4):718-725. doi: 10.1007/s10637-018-0595-8. Epub

2018 Apr 2.

 

Autophagy inhibition improves the chemotherapeutic efficacy of cruciferous

vegetable-derived diindolymethane in a murine prostate cancer xenograft model.

 

Draz H(1)(2), Goldberg AA(1), Tomlinson Guns ES(3), Fazli L(3), Safe S(4),

Sanderson JT(5).

 

Prostate cancer is the second leading cause of cancer-related deaths in men in

North America and there is an urgent need for development of more effective

therapeutic treatments against this disease. We have recently shown that

diindolylmethane (DIM) and several of its halogenated derivatives (ring-DIMs)

induce death and protective autophagy in human prostate cancer cells. However,

the in vivo efficacy of ring-DIMs and the use of autophagy inhibitors as adjuvant

therapy have not yet been studied in vivo. The objective of this study was to

determine these effects on tumor growth in nude CD-1 mice bearing bioluminescent

androgen-independent PC-3 human prostate cancer cells. We found that chloroquine

(CQ) significantly sensitized PC-3 cells to death in the presence of sub-toxic

concentrations of DIM or 4,4'-Br2DIM in vitro. Moreover, a combination of DIM

(10 mg/kg) and CQ (60 mg/kg), 3× per week, significantly decreased PC-3 tumor

growth in vivo after 3 and 4 weeks of treatment. Furthermore, 4,4'-Br2DIM at

10 mg/kg (3× per week) significantly inhibited tumour growth after 4 weeks of

treatment. Tissues microarray analysis showed that DIM alone or combined with CQ

induced apoptosis marker TUNEL; the combination also significantly inhibited the

cell proliferation marker Ki67. In conclusion, we have confirmed that DIM and

4,4'-Br2DIM are effective agents against prostate cancer in vivo and shown that

inhibition of autophagy with CQ enhances the anticancer efficacy of DIM. Our

results suggest that including selective autophagy inhibitors as adjuvants may

improve the efficacy of existing and novel drug therapies against prostate

cancer.

 

Mini Rev Med Chem. 2018;18(11):962-968. doi: 10.2174/1389557518666180313100144.

 

Targeting the Microcirculation by Indole-3-carbinol and Its Main Derivate

3,3,'-diindolylmethane: Effects on Angiogenesis, Thrombosis and Inflammation.

 

Ampofo E(1), Schmitt BM(1), Menger MD(1), Laschke MW(1).

 

Author information:

(1)Institute for Clinical & Experimental Surgery, Saarland University, 66421

Homburg/Saar, Germany.

 

The pharmacological targeting of microcirculatory dysregulations is a therapeutic

strategy for the treatment of numerous pathological conditions, such as cancer,

thrombosis and inflammation. A promising candidate for this purpose is

indole-3-carbinol (I3C), a phytochemical compound of cruciferous vegetables, and

its main derivate 3,3,'-diindolylmethane (DIM). As summarized in this review, I3C

and DIM affect multiple molecular and cellular processes within the

microcirculation due to their pleiotropic action profile. These include

angiogenesis, leukocyte-endothelial cell interaction, cytokine and reactive

oxygen species (ROS) production, thrombus formation and microvascular leakage.

Hence, I3C may serve as a lead compound for the future chemical synthesis of

novel drugs that exert comparable beneficial effects while exhibiting an improved

bioavailability.

 

Int J Biol Macromol. 2018 Jul 1;113:515-525. doi: 10.1016/j.ijbiomac.2018.02.153.

Epub 2018 Feb 27.

 

Natural polymer functionalized graphene oxide for co-delivery of anticancer

drugs: In-vitro and in-vivo.

 

Deb A(1), Andrews NG(2), Raghavan V(3).

 

The present study focuses on the development of a chitosan functionalized

nanobiocomposite for the co-delivery of two anti-cancer drugs camptothecin (CPT)

and 3,3'‑Diindolylmethane (DIM). The difference in the mechanism of action of the

two drugs makes them a promising candidate to produce a synergistic effect

against breast cancer. Herein a nanobiocomposite was developed by functionalizing

a natural polymer chitosan to graphene oxide nanoparticles and decorated with

folic acid. The nanobiocomposite thus synthesized was loaded with camptothecin

and 3,3'‑Diindolylmethane and characterized by X-ray diffractometer (XRD),

Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy

(SEM), transmission electron microscopy (TEM), UV-visible spectroscopy (UV) and

atomic force microscopy (AFM).Biocompatibility was assayed by hemolysis and

anti-inflammatory assay. The cellular toxicity was measured by

3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑Diphenyltetrazolium Bromide (MTT),

Sulforhodamine B (SRB) and cell death assay against MCF-7 cell lines. Further in

vivo studies were carried out to analyze the biodistribution of the drug, blood

biochemical analysis and bioavailability of the drug. The data revealed a

significant increase in anticancer activity after co-loading of CPT and DIM to

the nanocarrier. Also in-vivo studies revealed that DIM successfully masked the

toxic effects produced by CPT.

 

J Pharm Bioallied Sci. 2017 Oct-Dec;9(4):259-265. doi: 10.4103/jpbs.JPBS_266_16.

 

Molecular Simulation Studies of 3,3'-Diindolylmethane as a Potent MicroRNA-21

Antagonist.

 

Junaid M(1)(2), Dash R(3)(2), Islam N(1), Chowdhury J(1), Alam MJ(1), Nath SD(4),

Shakil MAS(1), Azam A(1), Quader SM(1), Zahid Hosen SM(2).

 

Objective: In recent decades, the overexpression of microRNA-21 (miR-21) is found

to be progressively linked with many diseases such as different types of cancers,

cardiovascular diseases, and inflammation. Thereby, it has become an attractive

target for pharmacological and genetic modulation in various diseases, and also

for overcoming the resistance to chemotherapy in several cancers. Here, in this

study, the role of molecular therapeutics of 3,3'-diindolylmethane (DIM) has been

investigated for its ability to bind with the precursor miRNA as a target of

miR-21 (hsa-mir-21), which may alter the catalyzation process of dicer, a RNase

III enzyme, involved in miRNA transcription.

Methods: In this context, the present study describes the potential binding and

the structure alteration properties of DIM to precursor miR-21 (pre-miR-21)

through Molecular Docking and Molecular Dynamics simulation techniques.

Results: As a corollary, DIM formed both non-bonded and covalent interactions

with the bases of pre-miR, while covalent interaction with guanine in the 6th

position was found to be consensus in molecular dynamics simulation. Furthermore,

the stability of both DIM and pre-miR-21 was found to be inversely correlated to

each other in binding condition.

Conclusion: This result indicates that DIM can be used in target-based therapy

and also as a lead for further development of potent small molecule miRNA

antagonist.

 

Phytomedicine. 2018 Jan 15;39:100-110. doi: 10.1016/j.phymed.2017.12.006. Epub

2017 Dec 7.

 

Phytochemical-induced reactive oxygen species and endoplasmic reticulum

stress-mediated apoptosis and differentiation in malignant melanoma cells.

 

Heo JR(1), Lee GA(1), Kim GS(1), Hwang KA(1), Choi KC(2).

 

BACKGROUND: Phytochemicals are derived from plants, vegetables and daily products

and exert chemopreventive effects. Malignant melanoma is highly metastatic, and

melanoma patients can develop chemotherapeutic resistance against conventional

melanoma therapies.

METHODS: In the present study, we investigated the anti-cancer effect of the

phytochemicals kaempferol (Kaem), genistein (Gen), and 3'3-diindolylmethane (DIM)

on melanoma cell viability. We also evaluated the altered expression of cell

cycle-related genes. We verified the production of intracellular reactive oxygen

species (ROS) and endoplasmic reticulum (ER) stress at both the protein and

cellular level using a western blot, TUNEL assay, and Dihydrodichlorofluorescein

diacetate (DCF-DA) assay.

RESULTS: Treatment of A375SM melanoma cells with phytochemicals resulted in

inhibition of cell growth. Treatment with phytochemicals increased the gene

expression of p21 and decreased the gene expression of cyclin E and/or cyclin B.

The three phytochemicals activated the ROS-p38-p53 apoptotic pathway by

increasing the level of phosphorylated p38 MAPK and p53, and they activated the

ER stress-mediated apoptotic pathway by increasing the level of phosphorylated

eIF2α and C/EBP homologous protein (CHOP). Both the ROS-p38-p53 and ER

stress-mediated pathway induced the mitochondrial apoptotic pathway by

attenuating Bcl-2 expression and upregulating BAX. Detection of morphological

changes demonstrated that Kaem and Gen can induce differentiation in A375SM cell

line.

CONCLUSION: These results indicate that phytochemicals are potentially useful in

treatments for melanoma due to their ability to inhibit melanoma cell growth and

division via the ROS and ER stress pathway.

 

Int J Mol Sci. 2018 Jan 24;19(2). pii: E339. doi: 10.3390/ijms19020339.

 

Elucidating the Role of CD84 and AHR in Modulation of LPS-Induced Cytokines

Production by Cruciferous Vegetable-Derived Compounds Indole-3-Carbinol and

3,3'-Diindolylmethane.

 

Wang TTY(1), Pham Q(2), Kim YS(3).

 

Modulation of the immune system by cancer protective food bioactives has

preventive and therapeutic importance in prostate cancer, but the mechanisms

remain largely unclear. The current study tests the hypothesis that the

diet-derived cancer protective compounds, indole-3-carbinol (I3C) and

3,3'-diindolylmethane (DIM), affect the tumor microenvironment by regulation of

inflammatory responses in monocytes and macrophages. We also ask whether I3C and

DIM act through the aryl hydrocarbon (AHR)-dependent pathway or the signaling

lymphocyte activation molecule (SLAM) family protein CD84-mediated pathway. The

effect of I3C and DIM was examined using the human THP-1 monocytic cell in its

un-differentiated (monocyte) and differentiated (macrophage) state. We observed

that I3C and DIM inhibited lipopolysaccharide (LPS) induction of IL-1β mRNA and

protein in the monocyte form but not the macrophage form of THP-1. Interestingly,

CD84 mRNA but not protein was inhibited by I3C and DIM. AHR siRNA knockdown

experiments confirmed that the inhibitory effects of I3C and DIM on IL-1β as well

as CD84 mRNA are regulated through AHR-mediated pathways. Additionally, the AHR

ligand appeared to differentially regulate other LPS-induced cytokines

expression. Hence, cross-talk between AHR and inflammation-mediated pathways, but

not CD84-mediated pathways, in monocytes but not macrophages may contribute to

the modulation of tumor environments by I3C and DIM in prostate cancer.

 

Oncol Lett. 2017 Dec;14(6):8100-8105. doi: 10.3892/ol.2017.7185. Epub 2017 Oct

16.

 

Inhibitory effect of the low-toxic exogenous aryl hydrocarbon receptor modulator

3'3-diindolylmethane on gastric cancer in mice.

 

Su M(1), Qian C(1), Hu Y(2), Lu W(2), Huang R(3), Chen M(1), Chen J(1).

 

3'3-Diindolylmethane (DIM) has been proved to exhibit anticancer properties in

many solid tumors. In our previous study, we demonstrated that DIM inhibited

SGC7901 cell proliferation by inducing apoptosis and delaying cell cycle

progression. Herein, we further explored the anti-tumor effect of DIM on SGC-7901

tumor bearing mice. Tumors were excised, weighed, and tested by western blot and

TdT-UTP nick-end labeling (TUNEL) assay. Blood samples were collected for

biochemical analysis. The expression levels of AhR and cytochrome P450, family 1,

subfamily A, polypeptide 1 (CYP1A1) protein were evaluated by western-blot assay.

Our data show that with the increase of DIM dose (0, 5, 10, 20 mg/kg/day), AhR

protein gradually decreased as CYP1A1 protein increased. The weight of the tumors

found in the treated animals was significantly lower than that of the control

group (0.845±0.096 vs. 1.275±0.236 g, 0.768±0.161 vs. 1.275±0.236 g, 0.607±0.106

vs. 1.275±0.236 g, P<0.05). TUNEL test showed that DIM induced increased

apoptosis in the treatment groups in a dose-dependent manner. Blood tests also

indicated that DIM showed no toxic effect on animal weight or liver and kidney

function. These results indicated that DIM agent could be a safe and potent drug

in therapy of gastric cancer.

 

Int J Mol Sci. 2018 Jan 8;19(1). pii: E189. doi: 10.3390/ijms19010189.

 

3,3'-Diindolylmethane Suppressed Cyprodinil-Induced Epithelial-Mesenchymal

Transition and Metastatic-Related Behaviors of Human Endometrial Ishikawa Cells

via an Estrogen Receptor-Dependent Pathway.

 

Cyprodinil (CYP) is a pyrimidine amine fungicide that has been extensively used

in agricultural areas. 3,3'-Diindolylmethane (DIM) is a derivative of the dietary

phytoestrogen, indole-3-carbinol (I3C), which is derived from cruciferous

vegetables and considered to be a cancer-preventive phytonutrient agent. In this

study, the effects of CYP and DIM were examined on the cell viability, invasion,

and metastasis of human endometrial cancer cells, Ishikawa, via epithelial

mesenchymal transition (EMT). CYP increased the level of cell viability of

Ishikawa cells compared to DMSO as a control, as did E2. Ishikawa cells lost

cell-to-cell contact and obtained a spindle-shaped or fibroblast-like morphology

in response to the application of E2 or CYP by the cell morphology assay. In the

cell migration and invasion assay, CYP enhanced the ability of migration and

invasion of Ishikawa cells, as did E2. E2 and CYP increased the expressions of

N-cadherin and Snail proteins, while decreasing the expression of E-cadherin

protein as EMT-related markers. In addition, E2 and CYP increased the protein

expressions of cathepsin D and MMP-9, metastasis-related markers. Conversely,

CYP-induced EMT, cell migration, and invasion were reversed by fulvestrant (ICI

182,780) as an estrogen receptor (ER) antagonist, indicating that CYP exerts

estrogenic activity by mediating these processes via an ER-dependent pathway.

Similar to ICI 182,780, DIM significantly suppressed E2 and CYP-induced

proliferation, EMT, migration, and invasion of Ishikawa cancer cells. Overall,

the present study revealed that DIM has an antiestrogenic chemopreventive effect

to withdraw the cancer-enhancing effect of E2 and CYP, while CYP has the capacity

to enhance the metastatic potential of estrogen-responsive endometrial cancer.

Biomol Ther (Seoul). 2018 Sep 1;26(5):503-511. doi: 10.4062/biomolther.2017.160.

Treatment with Phytoestrogens Reversed Triclosan and Bisphenol A-Induced

Anti-Apoptosis in Breast Cancer Cells.

 

Lee GA(1), Choi KC(1), Hwang KA(1).

 

Triclosan (TCS) and bisphenol A (BPA) are endocrine-disrupting chemicals that

interfere with the hormone or endocrine system and may cause cancer. Kaempferol

(Kaem) and 3,3'-diindolylmethane (DIM) are phytoestrogens that play

chemopreventive roles in the inhibition of carcinogenesis and cancer progression.

In this study, the influence of TCS, BPA, Kaem, and DIM on proliferation and

apoptotic abilities of VM7Luc4E2 breast cancer cells were examined. MTT assay

revealed that TCS (0.1-10 µM), BPA (0.1-10 µM) and E2 (0.01-0.0001 µM) induced

significant cell proliferation of VM7Luc4E2 cells, which was restored to the

control (0.1% DMSO) by co-treatment with Kaem (30 µM) or DIM (15 µM). Reactive

oxygen species (ROS) production assays showed that TCS and BPA inhibited ROS

production of VM7Luc4E2 cells similar to E2, but that co-treatment with Kaem or

DIM on VM7Luc4E2 cells induced increased ROS production. Based on these results,

the effects of TCS, BPA, Kaem, and DIM on protein expression of apoptosis and ROS

production-related markers such as Bax and Bcl-xl, as well as endoplasmic

reticulum (ER) stress-related markers such as eIF2α and CHOP were investigated by

Western blot assay. The results revealed that TCS, and BPA induced anti-apoptosis

by reducing ROS production and ER stress. However, Kaem and DIM effectively

inhibited TCS and BPA-induced anti-apoptotic processes in VM7Luc4E2 cells.

Overall, TCS and BPA were revealed to be distinct xenoestrogens that enhanced

proliferation and anti-apoptosis, while Kaem and DIM were identified as natural

chemopreventive compounds that effectively inhibited breast cancer cell

proliferation and increased anti-apoptosis induced by TCS and BPA.

 

Sci Rep. 2017 Dec 20;7(1):17953. doi: 10.1038/s41598-017-18159-3.

 

Three classes of ligands each bind to distinct sites on the orphan G

protein-coupled receptor GPR84.

 

Mahmud ZA(1), Jenkins L(1), Ulven T(2), Labéguère F(3)(4), Gosmini R(3), De Vos

S(5), Hudson BD(1), Tikhonova IG(6), Milligan G(7).

 

Medium chain fatty acids can activate the pro-inflammatory receptor GPR84 but so

also can molecules related to 3,3'-diindolylmethane. 3,3'-Diindolylmethane and

decanoic acid acted as strong positive allosteric modulators of the function of

each other and analysis showed the affinity of 3,3'-diindolylmethane to be at

least 100 fold higher. Methyl decanoate was not an agonist at GPR84. This implies

a key role in binding for the carboxylic acid of the fatty acid. Via homology

modelling we predicted and confirmed an integral role of arginine172, located in

the 2nd extracellular loop, in the action of decanoic acid but not of

3,3'-diindolylmethane. Exemplars from a patented series of GPR84 antagonists were

able to block agonist actions of both decanoic acid and 3,3'-diindolylmethane at

GPR84. However, although a radiolabelled form of a related antagonist, [3H]G9543,

was able to bind with high affinity to GPR84, this was not competed for by

increasing concentrations of either decanoic acid or 3,3'-diindolylmethane and

was not affected adversely by mutation of arginine172. These studies identify

three separable ligand binding sites within GPR84 and suggest that if medium

chain fatty acids are true endogenous regulators then co-binding with a positive

allosteric modulator would greatly enhance their function in physiological

settings.

 

J BUON. 2017 Sep-Oct;22(5):1115-1121.

 

Remarkably higher efficacy and a wider safety window for nonfrontline over

first-line drug combinations in the adenocarcinoma Colo 320DM cell line.

 

Garza-Trevino EN(1), Rodriguez-Gonzalez MS, Delgado Gonzalez P, Alonso-Cruz YG,

Alonso-Cruz YG, Soto-Dominguez A, Gonzalez Guerrero JF, Castro-Govea Y, Michel

Sanchez E, Said Fernandez S, Martinez-Rodriguez HG.

 

Author information:

(1)Autonomous University of Nuevo León (UANL), Cellular Therapy Laboratory,

Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Mexico.

 

PURPOSE: To determine in vitro, the efficacy and safety window of not-front-line

and first-line anti-colorectal (CRC) drug combinations.

METHODS: The adenocarcinoma cell line Colo 320DM and normal human mesenchymal

stem cells derived from adipose tissue were used respectively to determine the

anti-CRC efficacy (% of Colo 320DM cell death [CD]) and safety window [SW] - %

Colo 320DM percent cancer death (PCD)/% of mesenchymal stem cell's death) of drug

combinations, using the adenosine triphosphate-based chemotherapy response assay

(ATP-CRA).

RESULTS: First-line anti-CRC drug combinations (5-fluorouracil [5FU]/oxaliplatin

[oxa] and 5-FU/Oxa /leucovorin [Leuco]) produced 57.7% and 52.4% CD, and 1.38 and

2.44 SW, respectively. Combinations of 5-FU/Oxa and 1 to 3 non-front line drugs

led to 56.3-99.8% CD and to 0.96-2.2 SW. The highest safety window corresponded

to 5FU/Oxa/ carboplatin [Carbo] (93% CD and 1.4 SW) and to 5-FU/ Oxa/cisplatin

[Cispl] (93.5% CD and 1.4 SW). In contrast, non-front line drugs led to

89.8-97.4% CD and to 1.1-78.2 SW. Outstandingly, those combinations containing

Carbo/ Cispl/3,3'-diindolylmethane (DIM), aspirin (Asp), or 3,3'- DIM/ Asp showed

a very high CD (91.9-96.9% [39.2-39.5 times higher than first-line-combined

drugs]) and very wide SW (57.8-81.56 [66.6-40 times higher than the first-line

drug combinations]).

CONCLUSIONS: Human mesenchymal stem cells could be an excellent alternative to

laboratory animals, when testing the safety profiles of drugs. The most promising

combinations of non-frontline drugs to treat CRC are Carbo/Cispl/ Asp and

Carbo/Cispl/DIM.

 

Mol Biol (Mosk). 2017 Sep-Oct;51(5):841-848. doi: 10.7868/S0026898417050123.

 

[Treatment with anti-cancer agents results in profound changes in lncRNA

expression in colon cancer cells].

 

[Article in Russian]

 

Zinovieva OL(1), Grineva EN(1), Prokofjeva MM(1), Karpov DS(1), Krasnov GS(1),

Prassolov VS(1), Mashkova TD(1), Lisitsyn NA(1)(2).

 

Author information:

(1)Engelhardt Institute of Molecular Biology, Russian Academy of Sciences,

Moscow, 119991 Russia.

(2)[email protected].

 

Using real-time RT-PCR in combination with bioinformatics, we have shown for the

first time that the treatment of HCT-116 and HT-29 colon cancer cells with two

anti-cancer agents (doxycycline or 3,3'-diindolylmethane) results in profound

changes in the intracellular content of several lncRNAs (by up to 100 times).

Since many of these RNAs are secreted by tumors into the bloodstream, the

obtained results provide a basis for developing more sensitive protocols for

serological monitoring of tumor relapse and metastasis, as well as for search of

new anti-cancer drugs.

 

Microb Pathog. 2017 Dec;113:330-334. doi: 10.1016/j.micpath.2017.10.045. Epub

2017 Oct 24.

 

Design and synthesis of indolopyridone hybrids as new antituberculosis agents.

 

Rather MA(1), Rasool F(2), Bhat ZS(3), Dar HU(4), Maqbool M(5), Amin S(6), Yousuf

SK(7), Ahmad Z(8).

 

Tuberculosis continues to be the most dangerous infectious disease globally and

need for development of new therapies is of utmost importance. In this study we

describe the rationale design for synthesis using molecular hybridization and

subsequent in-vitro antimycobacterial activity of various indolo-pyridone hybrid

molecules against Mycobacterium tuberculosis H37Rv. A total of 16 indolo-pyridone

hybrid molecules were synthesized with 85-90% yields and characterized by various

spectroscopic techniques. Four compounds were ineffective with MIC >256 μg/ml

(highest concentration tested), six exhibited poor activity with MIC > 100 μg/ml,

four showed moderate activity with MIC > 50 μg/ml and two had notable anti-TB

activity with MIC values 32 μg/ml. Interestingly the last two compounds were

observed equally effective against drug susceptible and various drug resistant

strains including multidrug-resistant (MDR) strains, thereby clearly

demonstrating their potential against MDR-TB. Our results showed that

un-substituted aryl rings posses better antituberculosis activity than those

having any kind of substitution and derivatives with small sized electron

withdrawing groups in aryl ring exhibited activity while bigger groups lead to

considerable loss in activity. The results of this study open up a new door for

further SAR guided synthesis on one hand and on the other hand provide a

promising opportunity that may lead to the discovery of a new class of

antituberculosis agents.

 

Molecules. 2017 Oct 17;22(10). pii: E1744. doi: 10.3390/molecules22101744.

 

A DFT Study of the Geometrical, Spectroscopical and Reactivity Properties of

Diindolylmethane-Phenylboronic Acid Hybrids.

 

Fragoso-Medina AJ(1), Escobedo-González RG(2), Nicolás-Vázquez MI(3), Arroyo-Razo

GA(4), Noguez-Córdova MO(5), Miranda-Ruvalcaba R(6).

 

The structure of the ortho-, meta- and para- hybrid

diindolylmethane-phenylboronic acids and their interactions were optimized with

by a quantum chemical method, using density functional theory at the (DFT) level.

Thus, infrared bands were assigned based on the scaled theoretical wavenumbers by

correlating the respective experimental data of the molecules. In addition, the

corresponding ¹H-/13C-/11B-NMR experimental and theoretical chemical shifts were

correlated. The target molecules showed a poor treatment of the OH shifts in the

GIAO method due to the absence of explicit solvent effects in these calculations;

therefore, they were explicitly considered with acetone molecules. Moreover, the

electron density at the hydrogen bond critical point increased, generating

stabilization energy, from weak to moderate or weak to strong, serving as an

indicator of the strength of the hydrogen bond between the different

intermolecular interactions. Finally, some properties related to the reactive

behavior of the target molecules associated with their cytotoxic effects and

metabolic pathways were also calculated.

 

Mol Med Rep. 2017 Dec;16(6):9553-9560. doi: 10.3892/mmr.2017.7788. Epub 2017 Oct

13.

 

3,3'‑Diindolylmethane attenuates cardiomyocyte hypoxia by modulating autophagy in

H9c2 cells.

 

Liang K(1), Qian WH(1), Zong J(1).

 

Author information:

(1)Institute of Cardiovascular Disease Research, Xuzhou Medical University,

Xuzhou, Jiangsu 221002, P.R. China.

 

Autophagy is activated in the ischemic heart and is a process that is essential

for survival, differentiation, development and homeostasis. 3,3'‑Diindolylmethane

(DIM) is a natural product of the acid‑catalyzed condensation of

indole‑3‑carbinol in cruciferous vegetables. Numerous studies have suggested that

DIM has various pharmacological effects, including antioxidant, antitumor,

anti‑angiogenic and anti‑apoptotic properties. However, the function of DIM on

hypoxia‑induced cardiac injury remains to be elucidated. In the present study,

H9c2 cells were pretreated with DIM (1, 5 and 10 µM) for 12 h and exposed to

hypoxia for 12 h. It was demonstrated that DIM markedly attenuated the increased

transcription of interleukin (IL)‑1β, IL‑6 and tumor necrosis factor‑α induced by

hypoxia. In addition, the transcription of autophagy associated genes increased

in the DIM pretreated group, compared with the hypoxia group. DIM additionally

attenuated the increased apoptosis, as determined by terminal deoxynucleotidyl

transferase dUTP nick end labeling staining, and regulated the relative protein

expression level of B cell lymphoma (Bcl) 2 associated X protein, Bcl‑xL and

cleaved caspase 3. Furthermore, the phosphorylation of the 5' AMP‑activated

protein kinase a (AMPKa) was activated and the phosphorylation of c‑Jun

N‑terminal kinase (JNK) was inhibited. The effect of DIM on hypoxia‑induced

apoptosis was abolished following pretreatment with JNK activator (anisomycin,

40 ng/ml). The effect of DIM on autophagy was reversed following pretreatment

with AMPKa inhibitor (CpC, 20 µM) following stimulation with hypoxia. The results

demonstrated that DIM prevented hypoxia‑induced inflammation and apoptosis and

activated cardiomyocyte autophagy, which may be mediated by activation of AMPKa

and inhibition of JNK pathways.

 

Oncotarget. 2017 Jun 27;8(39):65339-65358. doi: 10.18632/oncotarget.18689.

eCollection 2017 Sep 12.

 

Anti-SSTR2 peptide based targeted delivery of potent PLGA encapsulated

3,3'-diindolylmethane nanoparticles through blood brain barrier prevents glioma

progression.

 

Bhowmik A(1), Chakravarti S(1), Ghosh A(2), Shaw R(1), Bhandary S(2),

Bhattacharyya S(3), Sen PC(2), Ghosh MK(1).

 

Current therapy for Glioblastoma is insufficient because of the presence of blood

brain barrier. It limits the transport of essential drugs to the tumor sites. To

overcome this limitation we strategized the delivery of an anticancer compound

3,3'-diindolylmethane by encapsulation in poly (lactic-co-glycolic acid)

nanoparticles. These nanoparticles were tagged with a novel peptide against

somatostatin receptor 2 (SSTR2), a potential target in glioma. The

nanoformulation (27-87nm) had loading and encapsulation efficiency of 7.2% and

70% respectively. It was successfully internalized inside the glioma cells

resulting in apoptosis. Furthermore, an in vivo bio-distribution study revealed

the selective accumulation of the nanoformulation into rat brain tumor sites by

crossing the blood brain barrier. This resulted in abrogation of epidermal growth

factor receptor pathway activation in glioma cells. Our novel nanopreparation

therefore shows great promise to serve as a template for targeted delivery of

other therapeutics in treating GBM.

 

Int Immunopharmacol. 2017 Nov;52:342-351. doi: 10.1016/j.intimp.2017.09.015. Epub

2017 Oct 7.

 

Enhancing miR-132 expression by aryl hydrocarbon receptor attenuates

tumorigenesis associated with chronic colitis.

 

Alzahrani AM(1), Hanieh H(2), Ibrahim HM(2), Mohafez O(3), Shehata T(4), Bani

Ismail M(2), Alfwuaires M(2).

 

BACKGROUND: Chronic inflammation in ulcerative colitis (UC) patients is the major

risk factor for colitis-associated colon cancer (CAC). Recent evidences have

shown that microRNAs (miRNAs) are implicated in CAC pathogenesis. However, the

interaction of miRNAs with the transcription factors that alleviate CAC has not

been reported.

METHODS: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3,3'-diindolylmethane

(DIM) were used to activate aryl hydrocarbon receptor (Ahr) in azoxymethane

(AOM)/dextran sodium sulfate (DSS)-induced CAC in mice. Real-time PCR was used to

quantify the mRNAs of miRNA and coding genes while western blot and ELISA were

used to quantify protein levels. Silencing miRNA was carried out by means of

electroporation and locked nucleic acid (LNA)-miRNA.

RESULTS: Inducing CAC in mice upregulated miR-132 expression in the colon, spleen

and lymph nodes at all stages of disease development. Activation of Ahr by TCDD

or DIM boosted miR-132 expression and alleviated CAC severity by suppression of

macrophage infiltration and pro-inflammatory cytokines. Interestingly, TCDD, but

not DIM, augmented a cholinergic anti-inflammation by inducing

acetylcholinesterase (AChE)-targeting miR-132. This anti-inflammation was

manifested by suppressed production of TNF-α, IL-1β and IL-6. Silencing miR-132

in vivo in TCDD-treated mice abrogated the cholinergic anti-inflammation and

exacerbated CAC. In addition, inhibition of miR-132 in vitro in CD4+ cells and

macrophages mitigated the inhibitory effect of TCDD on AChE catalytic activity.

CONCLUSION: Our findings identify miR-132 as a new molecule implicated in CAC

pathogenesis, and reveal that miR-132 mediates the ameliorating effects of TCDD

on CAC, suggesting miR-132 as a promising therapeutic candidate to control

autoimmune inflammation and tumorigenesis in CAC patients.

 

Eur J Pharm Sci. 2018 Jan 1;111:133-141. doi: 10.1016/j.ejps.2017.09.050. Epub

2017 Sep 28.

 

Nanocapsules improve indole-3-carbinol photostability and prolong its

antinociceptive action in acute pain animal models.

 

Gehrcke M(1), Sari MHM(2), Ferreira LM(1), Barbieri AV(1), Giuliani LM(1), Prado

VC(2), Nadal JM(3), Farago PV(3), Nogueira CW(2), Cruz L(4).

 

This study aimed the development of nanocapsules (NCs) for oral indole-3-carbinol

(I3C) administration and evaluation of antinociceptive potential of this compound

in its two forms, free and nanoencapsulated, using acute pain models. NCs showed

adequate physicochemical characteristics and protected the I3C against UVC

radiation exposure. It was observed no chemical bond between I3C and polymer by

FTIR. Besides, X-ray and DSC analysis suggested that I3C was molecularly

dispersed in NCs. The dialysis bag technique showed that almost 100% of the

compound was released from NCs at 360min. Mathematical modeling demonstrated that

this release occurred in two rates, with an initial burst effect followed by a

slower release of I3C. Regarding the in vivo analysis, time-response curve showed

that both forms of I3C caused an inhibition in inflammatory phase of nociception

induced by formalin and increased the latency response in hot plate test.

Interestingly, NCs were able to prolong the I3C effect in both tests.

Furthermore, in dose-response curve, only I3C in its nanoencapsulated form

presented effect on inflammatory phase of the formalin test. In conclusion, NCs

to I3C incorporation presented adequate nanometric characteristics and prolonged

its antinociceptive action in acute pain models tested.

Med Chem. 2017 Sep 21. doi: 10.2174/1573406413666170922095011. [Epub ahead of
print]

IRS-1 pY612 and Akt-1/PKB pT308 Phosphorylation and Antiinflammatory Effect of
Diindolylmethane in Adipocytes Cocultured with Macrophages.

Alfonso LV(1), Javier GBJ(2), Sofía GGA(1), Ernesto ULP(1), Susana DTA(1), Ruth
BTM(1), Sergio SE(3), Francisco MVJ(4), Felipe JSL(5), Socorro ABJ(2), Estela
BRB(1).

BACKGROUND: 3,3'-Diindolylmethane (DIM) is a condensation product of
indole-3-carbinol, a glucosinolate naturally occurring in Brassica genus
vegetables. The antiinflammatory properties of DIM through the inhibition of
NF-κB, as well as its ameliorating effects on glucose tolerance and hyperglicemic
states, have been described. A subclinical proinflammatory profile resultant from
the interaction of adipocytes and macrophages have been reported in obesity,
affecting the insulin signaling pathway, contributing to insulin resistance.
OBJECTIVE: The aim of this study was to evaluate the effect of DIM on
proinflammatory cytokines and phosphorylation of IRS-1 pY612 and Akt-1/PKB pT308
in an obesity-induced inflammation model.
METHOD: Differentiated 3T3-L1 adipocytes were cocultured with RAW 264.7
macrophages and exposed to 20 μM, 40 μM and 60 μM DIM for 24 h followed by 100 nM
insulin for 20 min. MCP-1, IL-6 and TNFα were quantified in the supernatant
through individual ELISAs. Adipocyte lysates were used to determine the relative
expression of the proinflammatory mediators by qPCR, and the phosphorylation of
IRS-1 pY612 and Akt-1/PKB pT308 proteins by western blot analysis.
RESULTS: DIM significantly (p<0.05) reduced the production and mRNA expression of
MCP-1, IL-6, and TNFα in a DIM concentration dependant manner, concomitantly
increasing the abundance of IRS-1 pY612 and Akt-1/PKB pT308.
CONCLUSION: Our results suggest that DIM influences the insulin transduction
pathway by exerting an antiinflammatory effect. The potential therapeutic
benefits of DIM in the treatment of glucose metabolic disorders deserves further
studies.

DOI: 10.2174/1573406413666170922095011
PMID: 28934926

Cell Signal. 2017 Sep 18;40:172-182. doi: 10.1016/j.cellsig.2017.09.006. [Epub
ahead of print]

Diindolylmethane and its halogenated derivatives induce protective autophagy in
human prostate cancer cells via induction of the oncogenic protein AEG-1 and
activation of AMP-activated protein kinase (AMPK).

Draz H(1), Goldberg AA(2), Titorenko VI(3), Tomlinson Guns ES(4), Safe SH(5),
Sanderson JT(6).

3,3'-Diindolylmethane (DIM) and its synthetic halogenated derivatives 4,4'-Br2-
and 7,7'-Cl2DIM (ring-DIMs) have recently been shown to induce protective
autophagy in human prostate cancer cells. The mechanisms by which DIM and
ring-DIMs induce autophagy have not been elucidated. As DIM is a mitochondrial
ATP-synthase inhibitor, we hypothesized that DIM and ring-DIMs induce autophagy
via alteration of intracellular AMP/ATP ratios and activation of AMP-activated
protein kinase (AMPK) signaling in prostate cancer cells. We found that DIM and
ring-DIMs induced autophagy was accompanied by increased autophagic vacuole
formation and conversion of LC3BI to LC3BII in LNCaP and C42B human prostate
cancer cells. DIM and ring-DIMs also induced AMPK, ULK-1 (unc-51-like autophagy
activating kinase 1; Atg1) and acetyl-CoA carboxylase (ACC) phosphorylation in a
time-dependent manner. DIM and the ring-DIMs time-dependently induced the
oncogenic protein astrocyte-elevated gene 1 (AEG-1) in LNCaP and C42B cells.
Downregulation of AEG-1 or AMPK inhibited DIM- and ring-DIM-induced autophagy.
Pretreatment with ULK1 inhibitor MRT 67307 or siRNAs targeting either AEG-1 or
AMPK potentiated the cytotoxicity of DIM and ring-DIMs. Interestingly,
downregulation of AEG-1 induced senescence in cells treated with overtly
cytotoxic concentrations of DIM or ring-DIMs and inhibited the onset of apoptosis
in response to these compounds. In summary, we have identified a novel mechanism
for DIM- and ring-DIM-induced protective autophagy, via induction of AEG-1 and
subsequent activation of AMPK. Our findings could facilitate the development of
novel drug therapies for prostate cancer that include selective autophagy
inhibitors as adjuvants.

DOI: 10.1016/j.cellsig.2017.09.006
PMID: 28923415

Free Radic Res. 2017 Sep 18:1-43. doi: 10.1080/10715762.2017.1381694. [Epub ahead
of print]

Attenuation of doxorubicin-induced cardiotoxicity and genotoxicity by an indole
based natural compound 3,3'-diindolylmethane (DIM) through activation of Nrf2/ARE
signaling pathways and inhibiting apoptosis.

Hajra S(1), Basu A(1), Singha Roy S(1)(2), Patra AR(1), Bhattacharya S(1).

Author information:
(1)a Department of Cancer Chemoprevention, Chittaranjan National Cancer
Institute, Kolkata, India.
(2)b Centre of Biomedical Research, SGPGIMS Campus, Lucknow, India.

The most crucial complication related to doxorubicin (DOX) therapy is nonspecific
cytotoxic effect on healthy normal cells. The clinical use of this broad-spectrum
chemotherapeutic agent is restricted due to development of severe form of
cardiotoxicity, myelosuppression and genotoxicity which interfere with
therapeutic schedule, compromise treatment outcome and may lead to secondary
malignancy. 3,3'-diindolylmethane (DIM) is a naturally occurring plant alkaloid
formed by the hydrolysis of indolylmethyl glucosinolate (glucobrassicin).
Therefore, the present study was undertaken to investigate the protective role of
DIM against DOX-induced toxicity in mice. DOX was administered (5 mg/kg b.W,
i.p.) and DIM was administered (25 mg/kg b.W p.o.) in concomitant and 15-day
pretreatment schedule. Results showed that DIM significantly attenuated
DOX-induced oxidative stress in the cardiac tissues by reducing the levels of
free radicals and lipid peroxidation, and by enhancing the level of glutathione
(reduced) and the activity of antioxidant enzymes. The chemoprotective potential
of DIM was confirmed by histopathological evaluation of heart and bone marrow
niche. Moreover, DIM considerably mitigated DOX-induced clastogenicity, DNA
damage, apoptosis and myeloid hyperplasia in bone marrow niche. In addition, oral
administration of DIM significantly (p<0.05) stimulated the Nrf2-mediated
activation of antioxidant response element (ARE) pathway and promoted expression
of ARE-driven cytoprotective proteins, HO-1, NQO1 and glutathione-S-transferase
(GST). In connection with that, DIM significantly attenuated DOX-induced
apoptosis by upregulation of Bcl-2 expression and downregulation of Bax and
caspase-3 expression. Thus, this study suggests that DIM has promising
chemoprotective efficacy against DOX-induced toxicity and indicates its future
use as an adjuvant in chemotherapy.

DOI: 10.1080/10715762.2017.1381694
PMID: 28922986

Food Res Int. 2017 Oct;100(Pt 1):497-503. doi: 10.1016/j.foodres.2017.07.049.
Epub 2017 Jul 21.

Bioavailability and new biomarkers of cruciferous sprouts consumption.

Baenas N(1), Suárez-Martínez C(2), García-Viguera C(3), Moreno DA(4).

The evaluation of the bioavailability of bioactive compounds from cruciferous
foods is one challenge in the design of clinical trials for studying their
functionality. Currently, studies of bioavailability are mainly based of the
analysis of total isothiocyanates and indoles, and sulforaphane metabolites after
broccoli consumption. However, as far as we are aware, there are not any
biomarkers studied or established for the intake of radish sprouts. In this work,
a 7-days-cross-over study with fourteen women was undertaken to compare the
bioavailability of glucosinolates from broccoli and radish sprouts. The urinary
excretion of isothiocyanates, indoles and their metabolites was analysed by
UHPLC-QqQ-MS/MS. For the first time, sulforaphene,
sulforaphane-N-acetyl-l-cysteine (SFN-NAC) and 3,3'-diindolylmethane (DIM), were
studied as biomarkers of dietary exposure to radish. The SFN-NAC and DIM were
already considered biomarkers of broccoli consumption. Higher excretion of
conjugated isothiocyanates and constant excretion of indoles were found during
the first 12h after ingestion. Metabolites were excreted homogeneously during the
study, suggesting no accumulation. The different urinary biomarker profiles
provided new information to distinguish between the consumption of broccoli or
radish sprouts. The results provide valuable information to better understand the
bioavailability of cruciferous bioactives.

DOI: 10.1016/j.foodres.2017.07.049
PMID: 28873713

Food Chem Toxicol. 2017 Aug 24;109(Pt 1):284-295. doi: 10.1016/j.fct.2017.08.037.
[Epub ahead of print]

Inhibitory effects of 3,3'-diindolylmethane on epithelial-mesenchymal transition
induced by endocrine disrupting chemicals in cellular and xenograft mouse models
of breast cancer.

Lee GA(1), Hwang KA(2), Choi KC(3).

As a phytoestrogen, 3,3'-diindolylmethane (DIM) plays a chemopreventive role by
inhibiting cancer progression. In this study, we examined the effects of
17β-estradiol (E2), two endocrine disrupting chemicals (EDCs), triclosan (TCS)
and bisphenol A (BPA), and DIM on epithelial-mesenchymal transition (EMT) and
metastatic behaviors of estrogen receptor (ER)-positive MCF-7 breast cancer
cells. An in vitro assay revealed that E2 (10(-9) M), TCS (10(-5)-10(-7) M), and
BPA (10(-5)-10(-7) M) induced MCF-7 cell proliferation compared to a control
through the ER pathway. In addition, E2, TCS, and BPA changed the cell morphology
from the epithelial to the mesenchymal phenotype and increased the migration and
invasion capacity of MCF-7 cells via ER; however, co-treatment with DIM (20 μM)
effectively suppressed E2, TCS, and BPA-induced cell proliferation, EMT,
migration, and invasion of MCF-7 cells. Western blot assay revealed that DIM
regulated the protein expression of EMT- and metastasis-related genes toward the
inhibition of these processes. Moreover, E2, TCS, and BPA increased the protein
expression of CXCR4, which is a receptor of chemokine CXCL12 that is positively
involved in breast cancer metastasis via an ER-dependent pathway. Conversely, DIM
and a CXCR4 antagonist (AMD3100) decreased CXCR4 protein expression, which led to
inhibition of the EMT process, indicating that DIM may suppress E2, TCS or
BPA-induced EMT, migration, and invasion of MCF-7 breast cancer cells by
suppressing CXCR4 protein expression. These in vitro effects of E2, TCS, BPA, and
DIM were also identified in a xenografted mouse model transplanted with MCF-7
breast cancer cells. Taken together, DIM is a potent chemopreventive compound for
preventing metastatic behaviors of breast cancer cells induced by EDCs with
cancer-related toxicity.

Biomed Pharmacother. 2017 Oct;94:1197-1224. doi: 10.1016/j.biopha.2017.07.075.
Epub 2017 Aug 23.

Regulation of microRNA using promising dietary phytochemicals: Possible
preventive and treatment option of malignant mesothelioma.

Sayeed MA(1), Bracci M(2), Lucarini G(2), Lazzarini R(2), Di Primio R(2),
Santarelli L(2).

Malignant mesothelioma (MM) is a very aggressive, lethal cancer, and its
incidence is increasing worldwide. Development of multi-drug resistance, therapy
related side-effects, and disease recurrence after therapy are the major problems
for the successful treatment of MM. Emerging evidence indicates that dietary
phytochemicals can exert anti-cancer activities by regulating microRNA
expression. Until now, only one dietary phytochemical (ursolic acid) has been
reported to have MM microRNA regulatory ability. A large number of dietary
phytochemicals still remain to be tested. In this paper, we have introduced some
dietary phytochemicals (curcumin, epigallocatechin gallate, quercetin, genistein,
pterostilbene, resveratrol, capsaicin, ellagic acid, benzyl isothiocyanate,
phenethyl isothiocyanate, sulforaphane, indole-3-carbinol, 3,3'-diindolylmethane,
diallyl disulphide, betulinic acid, and oleanolic acid) which have shown microRNA
regulatory activities in various cancers and could regulate MM microRNAs. In
addition to microRNA regulatory activities, curcumin, epigallocatechin gallate,
quercetin, genistein, resveratrol, phenethyl isothiocyanate, and sulforaphane
have anti-mesothelioma potentials, and pterostilbene, capsaicin, ellagic acid,
benzyl isothiocyanate, indole-3-carbinol, 3,3'-diindolylmethane, diallyl
disulphide, betulinic acid, and oleanolic acid have potentials to inhibit cancer
by regulating the expression of various genes which are also known to be aberrant
in MM.

DOI: 10.1016/j.biopha.2017.07.075
PMID: 28841784Org Biomol Chem. 2017 Aug 23;15(33):6997-7007. doi: 10.1039/c7ob01701d.

A one-pot synthesis of 2,2'-disubstituted diindolylmethanes (DIMs) via a
sequential Sonogashira coupling and cycloisomerization/C3-functionalization of
2-iodoanilines.

Kayet A(1), Singh VK.

Author information:
(1)Department of Chemistry, Indian Institute of Science Education and Research, Bhopal, Bhopal-462 066, India.

A Pd(ii)-Ag(i) catalyzed highly efficient synthesis of diindolylmethane has been
developed. This transformation consists of a one-pot sequential Sonogashira
coupling (and desilylation) followed by cycloisomerization/C3-functionalization
of 2-iodoanilines. Six new bonds (four C-C and two C-N) are formed in a one-pot
fashion. A variety of diindolylmethanes were obtained in excellent yields (up to
94%) under mild reaction conditions and this strategy is amenable to gram scale
synthesis also. The products were transformed into various synthetically useful
compounds.

DOI: 10.1039/c7ob01701d
PMID: 28792550

Talanta. 2017 Nov 1;174:314-319. doi: 10.1016/j.talanta.2017.06.019. Epub 2017
Jun 8.

Development of a solid-phase extraction method with simple MEKC-UV analysis for
simultaneous detection of indole metabolites in human urine after administration
of indole dietary supplement.

Phonchai A(1), Wilairat P(2), Chantiwas R(3).

This work presents the development of a solid phase extraction method with simple
MEKC-UV analysis for the simultaneous determination of indole-3-carbinol (I3C)
and its metabolites (3, 3'-diindolylmethane (DIM), indole-3-carboxaldehyde
(I3CAL), indole-3-acetonitrile (I3A)) in human urine after oral administration of
an indole dietary supplement. Solid phase extraction (SPE) method was applied for
the first time for simultaneous analysis of these indole metabolites. The MEKC
separation method was developed in a previous work. Three commercial SPE
cartridges, each with different sorbent materials, were investigated: Sep-Pak(®)
C18, Oasis(®) HLB and Oasis(®) WCX. The Sep-Pak(®) C18 material provided the
highest extraction recovery of 88-113% (n = 9), for the four target indole
metabolites (I3C, DIM, I3CAL and I3A). The optimal washing and elution solutions
were 40% methanol/water (v/v) and 100% methanol, respectively, and optimal
elution volume was 2.0mL. The specificity of the proposed SPE method was
evaluated with negative control urine samples (n = 10) from healthy volunteers
who had not taken the dietary supplement or vegetables known to contain indole
compounds. Linear calibration curves were in the range of 0.2-25μgmL(-1) (r(2) >
0.998) using diphenylamine (DPA) as the internal standard. Intra-day and
inter-day precisions were 3.5-12.3%RSD and 2.7-14.1%RSD, respectively. Limits of
detection and quantification were 0.05-0.10μgmL(-1) and 0.10-0.50μgmL(-1),
respectively. The four target indole compounds were separated within only 5min by
MEKC-UV analysis. Urine from 5 subjects who had taken a dietary supplement
containing I3C and DIM were found to contain only the DIM metabolite at
concentrations ranging from 0.10 to 0.35µgmL(-1). Accuracy of the proposed method
based on the percentage recovery of spiked urine samples were 70-108%, 82-116%,
82-132% and 80-100% for I3C, I3CAL, I3A and DIM, respectively. The Sep-Pak(®)C18
cartridge was highly effective in extraction and sample cleanup for the
downstream simultaneous detection of urinary indole metabolites by MEKC-UV
method.

Int J Mol Sci. 2017 Jul 1;18(7). pii: E1409. doi: 10.3390/ijms18071409.

Dose-Dependent Responses of I3C and DIM on T-Cell Activation in the Human T
Lymphocyte Jurkat Cell Line.

Liu M(1), Yasmeen R(2), Fukagawa NK(3), Yu L(4), Kim YS(5), Wang TTY(6).

Indole-3-carbinol (I3C) and its dimer diindolylmethane (DIM) are bioactive
metabolites of a glucosinolate, glucobrassicin, found in cruciferous vegetables.
Both I3C and DIM have been reported to possess pro-apoptotic, anti-proliferative
and anti-carcinogenic properties via modulation of immune pathways. However,
results from these studies remain inconclusive since they lack thorough
evaluation of these bioactives' physiological versus pharmacological effects. In
the present study, we investigated I3C and DIM's dose-dependent effects on
cytokines production in human T lymphocytes Jurkat cell line (Clone E6-1). The
results showed that I3C and DIM pretreatment, at higher concentrations of 50 and
10 μM, respectively, significantly increased PMA/ionomycin-induced interleukin-2
(IL-2), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) production,
measured by real time polymerase chain reaction (RT-PCR) and enzyme linked
immunosorbent assay (ELISA). As a plausible mechanism underlying such pronounced
cytokine release, we found robust increase in downstream nuclear factor κB
(NF-κB) and nuclear factor of activated T-cells 1 (NFAT1) signaling with I3C
pretreatment, whereas DIM pretreatment only significantly induced NF-κB
activation, but not NFAT1. We hypothesize that I3C/DIM pretreatment primes the T
cells to become hyperresponsive upon PMA/ionomycin stimulation which in turn
differentially induces two major downstream Ca(2+)-dependent inflammatory
pathways, NF-κB and NFAT1. Our data show novel insights into the mechanisms
underlying induction of pro-inflammatory cytokine release by pharmacological
concentrations of I3C and DIM, an effect negligible under physiological
conditions.

PLoS One. 2017 Jun 30;12(6):e0180321. doi: 10.1371/journal.pone.0180321.
eCollection 2017.

Indole-3-carbinol, a plant nutrient and AhR-Ligand precursor, supports oral
tolerance against OVA and improves peanut allergy symptoms in mice.

Hammerschmidt-Kamper C(1), Biljes D(1), Merches K(1), Steiner I(2), Daldrup T(2),
Bol-Schoenmakers M(3), Pieters RHH(3), Esser C(1).

In general, dietary antigens are tolerated by the gut associated immune system.
Impairment of this so-called oral tolerance is a serious health risk. We have
previously shown that activation of the ligand-dependent transcription factor
aryl hydrocarbon receptor (AhR) by the environmental pollutant
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects both oral tolerance and food
allergy. In this study, we determine whether a common plant-derived, dietary
AhR-ligand modulates oral tolerance as well. We therefore fed mice with
indole-3-carbinole (I3C), an AhR ligand that is abundant in cruciferous plants.
We show that several I3C metabolites were detectable in the serum after feeding,
including the high-affinity ligand 3,3´-diindolylmethane (DIM). I3C feeding
robustly induced the AhR-target gene CYP4501A1 in the intestine; I3C feeding also
induced the aldh1 gene, whose product catalyzes the formation of retinoic acid
(RA), an inducer of regulatory T cells. We then measured parameters indicating
oral tolerance and severity of peanut-induced food allergy. In contrast to the
tolerance-breaking effect of TCDD, feeding mice with chow containing 2 g/kg I3C
lowered the serum anti-ovalbumin IgG1 response in an experimental oral tolerance
protocol. Moreover, I3C feeding attenuated symptoms of peanut allergy. In
conclusion, the dietary compound I3C can positively influence a vital immune
function of the gut.

Clin Cancer Res. 2017 Sep 15;23(18):5585-5597. doi:
10.1158/1078-0432.CCR-17-0387. Epub 2017 Jun 28.

DNA-Methyltransferase 1 Induces Dedifferentiation of Pancreatic Cancer Cells
through Silencing of Krüppel-Like Factor 4 Expression.

Xie VK(1), Li Z(2), Yan Y(2), Jia Z(2), Zuo X(3), Ju Z(4), Wang J(4), Du J(5),
Xie D(5), Xie K(6), Wei D(6).

Purpose: The dismal prognosis of pancreatic cancer has been linked to poor tumor
differentiation. However, molecular basis of pancreatic cancer differentiation
and potential therapeutic value of the underlying molecules remain unknown. We
investigated the mechanistic underexpression of Krüppel-like factor 4 (KLF4) in
pancreatic cancer and defined a novel epigenetic pathway of its activation for
pancreatic cancer differentiation and treatment.Experimental Design: Expressions
of KLF4 and DNMT1 in pancreatic cancer tissues were determined by IHC and the
genetic and epigenetic alterations of KLF4 in and KLF4's impact on
differentiation of pancreatic cancer were examined using molecular biology
techniques. The function of dietary 3,3'-diindolylmethane (DIM) on
miR-152/DNMT1/KLF4 signaling in pancreatic cancer was evaluated using both cell
culture and animal models.Results: Overexpression of DNMT1 and promoter
hypermethylation contributed to decreased KLF4 expression in and associated with
poor differentiation of pancreatic cancer. Manipulation of KLF4 expression
significantly affected differentiation marker expressions in pancreatic cancer
cells. DIM treatment significantly induced miR-152 expression, which blocked
DNMT1 protein expression and its binding to KLF4 promoter region, and
consequently reduced promoter DNA methylation and activated KLF4 expression in
pancreatic cancer cells. In addition, DIM treatment caused significant inhibition
of cell growth in vitro and tumorigenesis in animal models of pancreatic
cancer. Conclusions: This is the first demonstration that dysregulated KLF4
expression associates with poor differentiation of pancreatic cancer. Epigenetic
activation of miR-152/DNMT1/KLF4 signaling pathway by dietary DIM causes
differentiation and significant growth inhibition of pancreatic cancer cells,
highlighting its translational implications for pancreatic and other cancers.
Clin Cancer Res; 23(18); 5585-97.

Semin Cancer Biol. 2017 Jun 12. pii: S1044-579X(17)30153-0. doi:
10.1016/j.semcancer.2017.06.002. [Epub ahead of print]

Two likely targets for the anti-cancer effect of indole derivatives from
cruciferous vegetables: PI3K/Akt/mTOR signalling pathway and the aryl hydrocarbon
receptor.

Popolo A(1), Pinto A(1), Daglia M(2), Nabavi SF(3), Farooqi AA(4), Rastrelli
L(5).

Diets containing high quantities of plant foods are linked with a decreased
likelihood of incidence of cancer. Several common plant-based dietary components
exert effects on DNA methylation levels, and can positively influence genome
stability and the transcription of tumor suppressors and oncogenes.
Indole-3-carbinol (I3C) is a substance present in vegetables of the Brassicaeae
family, especially broccoli, white cabbage, Brussels sprouts and cauliflower. The
in vivo biological effects of I3C are ascribed to a series of oligomeric products
(including 3,3'-diindolylmethane), developed under acidic conditions. I3C is one
of the many natural products and bioactive compounds found in foods which have
recently received much attention for its potential effects in cancer prevention
and treatment. In vitro studies report that I3C suppresses the proliferation of
different tumor cells, including those isolated from breast, prostate,
endometrium, and colon cancers. I3C resulted to be a potent in vivo
chemopreventive agent for certain hormone-dependent cancers, including breast and
cervical cancer. However, the mechanisms underlying these effects are not well
defined. In this review, we have analysed recent literature on the use of indole
derivatives against various forms of cancer, and have identified the main
signalling pathways involved in their anti-cancer effect as PI3K/Akt/mTOR and the
aryl hydrocarbon receptor.

Front Plant Sci. 2017 May 23;8:845. doi: 10.3389/fpls.2017.00845. eCollection
2017.

Date Palm Tree (Phoenix dactylifera L.): Natural Products and Therapeutic
Options.

Al-Alawi RA(1), Al-Mashiqri JH(1), Al-Nadabi JSM(1), Al-Shihi BI(1), Baqi Y(1).

Author information:
(1)Department of Chemistry, Faculty of Science, Sultan Qaboos UniversityMuscat,
Oman.

Many plants, including some of the commonly consumed herbs and spices in our
daily food, can be safely and effectively used to prevent and/or treat some
health concerns. For example, caffeine the active ingredient found in coffee
beans (Coffea), shows biological activity in the treatment of the central nervous
system (CNS) disorders, indole-3-carbinol, and 3,3'-diindolylmethane are both
broccoli (Brassica oleracea) derived phytochemicals with potential anti-cancer
activity, and resveratrol, isolated from grape (Vitis vinifera), is reported to
extend lifespan and provide cardio-neuro-protective, anti-diabetic, and
anti-cancer effects. Date palm fruits possess high nutritional and therapeutic
value with significant antioxidant, antibacterial, antifungal, and
anti-proliferative properties. This review focuses on the date fruit extracts and
their benefits in individual health promoting conditions and highlights their
applications as useful to the pharmaceutical and nutraceutical industries in the
development of natural compound-based industrial products.

Mol Nutr Food Res. 2017 Jun 6. doi: 10.1002/mnfr.201700119. [Epub ahead of print]

3,3'-Diindolylmethane suppresses high-fat diet-induced obesity through inhibiting
adipogenesis of pre-adipocytes by targeting USP2 activity.

Yang H(1), Seo SG(1), Shin SH(1), Min S(1), Kang MJ(1), Yoo R(1), Kwon JY(2), Yue
S(3), Kim KH(2), Cheng JX(3)(4), Kim JR(5), Park JS(6), Kim JH(7), Park
JHY(7)(8), Lee HJ(1), Lee KW(1)(7)(8).

SCOPE: Indole-3-carbinol (I3C), a derivative abundant in cruciferous vegetables
such as cabbage, is well known for its various health benefits such as
chemo-preventive and anti-obesity effects. I3C is easily metabolized to
3,3'-diindolylmethane (DIM), a more stable form, in acidic conditions of the
stomach. However, the anti-obesity effect of DIM has not been investigated
clearly. We sought to investigate the effect of DIM on diet-induced obesity and
to elucidate its underlying mechanisms.
METHODS AND RESULTS: High-fat diet (HFD)-fed obese mouse and MDI-induced 3T3-L1
adipogenesis models were used to study the effect of DIM. We observed that the
administration of DIM (50 mg/kg BW) significantly suppressed HFD-induced obesity,
associated with a decrease in adipose tissue. Additionally, we observed that DIM
treatment (40 and 60 μM), but not I3C treatment, significantly inhibited
MDI-induced adipogenesis by reducing the levels of several adipogenic proteins
such as PPAR-γ and C/EBPα. DIM, but not I3C, suppressed cell cycle progression in
the G1 phase, which occurred in the early stage of adipogenesis, inducing
post-translational degradation of cyclin D1 by inhibiting ubiquitin specific
peptidase 2 (USP2) activities.
CONCLUSION: Our findings indicate that cruciferous vegetables, which can produce
DIM as a metabolite, have the potential to prevent or treat chronic obesity

Oncol Rep. 2017 Jul;38(1):569-574. doi: 10.3892/or.2017.5693. Epub 2017 Jun 1.

3,3'-diindolylmethane downregulates cyclin D1 through triggering endoplasmic
reticulum stress in colorectal cancer cells.

Zhang X(1), Sukamporn P(2), Zhang S(3), Min KW(4), Baek SJ(5).

As a major in vivo condensation product of indole-3-carbinol, which is mostly
present in cruciferous vegetables, 3,3'-diindolylmethane (DIM) has been
previously reported with anti-proliferative action in different types of cancer
by our group and others. To further elucidate these underlying mechanisms, we
examined the effect of DIM on cyclin D1, which was aberrantly overexpressed in
various cancer cells and tumors. Herein, we found that DIM downregulated
cyclin D1 expression in colorectal cancer cells (CRC), which was independent of
PPARγ expression and protease activity. Furthermore, DIM did not affect cyclin D1
mRNA expression, suggesting DIM modulated cyclin D1 expression at the
translational level. Subsequently, blocking eIF2α phosphorylation resulted from
endoplasmic reticulum (ER) stress restored cyclin D1 in the presence of DIM.
Thus, the present study demonstrates that DIM downregulates cyclin D1 through
triggering ER stress in human colorectal cancer cells.

J Nutr Biochem. 2017 Sep;47:113-119. doi: 10.1016/j.jnutbio.2017.05.005. Epub
2017 May 25.

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA
damage repair genes through repressive chromatin modifications and is associated
with DNA damage in prostate cancer cells.

Palomera-Sanchez Z(1), Watson GW(1), Wong CP(2), Beaver LM(3), Williams DE(4),
Dashwood RH(5), Ho E(6).

Author information:
(1)Biological and Population Health Sciences, Oregon State University, Corvallis,
OR.
(2)Biological and Population Health Sciences, Oregon State University, Corvallis,
OR; Moore Family Center, Oregon State University, Corvallis, OR.
(3)Biological and Population Health Sciences, Oregon State University, Corvallis,
OR; Moore Family Center, Oregon State University, Corvallis, OR; Linus Pauling
Institute, Oregon State University, Corvallis, OR.
(4)Linus Pauling Institute, Oregon State University, Corvallis, OR; Environmental
and Molecular Toxicology, Oregon State University, Corvallis, OR.
(5)Center for Epigenetics and Disease Prevention, Institute of Biosciences and
Technology, Texas A&M Science Center, Houston, TX; Department of Nutrition & Food
Science, Texas A&M University, College Station, TX; Department of Clinical Cancer
Prevention, MD Anderson Cancer Center, Houston, TX; Department of Molecular &
Cellular Medicine, Texas A&M University College of Medicine, College Station, TX.
(6)Biological and Population Health Sciences, Oregon State University, Corvallis,
OR; Moore Family Center, Oregon State University, Corvallis, OR; Linus Pauling

Institute, Oregon State University, Corvallis, OR.

Androgen receptor (AR) is a transcription factor involved in normal prostate
physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is
a promising phytochemical agent against PCa that affects AR activity and
epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via
epigenetic regulation of AR target genes is unknown. We assessed epigenetic
regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment
led to epigenetic suppression of AR target genes involved in DNA repair (PARP1,
MRE11, DNA-PK). Decreased expression of these genes was accompanied by an
increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment
to their regulatory regions. Decreased DNA repair gene expression was associated
with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat
elements LINE1 and α-satellite. Our results suggest that DIM suppresses
AR-dependent gene transcription through epigenetic modulation, leading to DNA
damage and genome instability in PCa cells.

DOI: 10.1016/j.jnutbio.2017.05.005
PMCID: PMC5583029 [Available on 2018-09-01]
PMID: 28582660

Breast Cancer Res Treat. 2017 Aug;165(1):97-107. doi: 10.1007/s10549-017-4292-7.
Epub 2017 May 30.

A randomized, placebo-controlled trial of diindolylmethane for breast cancer
biomarker modulation in patients taking tamoxifen.

Thomson CA(1)(2), Chow HHS(3), Wertheim BC(3), Roe DJ(3)(4), Stopeck A(5),
Maskarinec G(6), Altbach M(7), Chalasani P(3), Huang C(8), Strom MB(9), Galons
JP(3)(7), Thompson PA(5)(10).

Author information:
(1)Department of Health Promotion Sciences, Mel and Enid Zuckerman College of
Public Health, University of Arizona, 3950 S. Country Club, Suite 3210, Tucson,
AZ, 85714, USA. [email protected].
(2)University of Arizona Cancer Center, Tucson, AZ, USA.
[email protected].
(3)University of Arizona Cancer Center, Tucson, AZ, USA.
(4)Department of Epidemiology and Biostatistics, Mel and Enid Zuckerman College
of Public Health, University of Arizona, Tucson, AZ, USA.
(5)Stony Brook Cancer Center, Stony Brook University, Stony Brook, NY, USA.
(6)Cancer Epidemiology Program, University of Hawaii Cancer Center, Honolulu, HI,
USA.
(7)Department of Medical Imaging, College of Medicine, University of Arizona,
Tucson, AZ, USA.
(8)Departments of Radiology, Psychiatry, School of Medicine, Stony Brook
University, Stony Brook, NY, USA.
(9)Department of Nutritional Sciences, University of Arizona, Tucson, AZ, USA.
(10)Department of Pathology, School of Medicine, Stony Brook University, Stony
Brook, NY, USA.

PURPOSE: Diindolylmethane (DIM), a bioactive metabolite of indole-3-carbinol
found in cruciferous vegetables, has proposed cancer chemoprevention activity in
the breast. There is limited evidence of clinically relevant activity of DIM or
long-term safety data of its regular use. A randomized, double-blind,
placebo-controlled trial was conducted to determine the activity and safety of
combined use of DIM with tamoxifen.
METHODS: Women prescribed tamoxifen (n = 130) were randomly assigned oral DIM
at 150 mg twice daily or placebo, for 12 months. The primary study endpoint was
change in urinary 2/16α-hydroxyestrone (2/16α-OHE1) ratio. Changes in
4-hydroxyestrone (4-OHE1), serum estrogens, sex hormone-binding globulin (SHBG),
breast density, and tamoxifen metabolites were assessed.
RESULTS: Ninety-eight women (51 placebo, 47 DIM) completed intervention;
compliance with treatment was >91%. DIM increased the 2/16α-OHE1 ratio (+3.2
[0.8, 8.4]) compared to placebo (-0.7 [-1.7, 0.8], P < 0.001). Serum SHBG
increased with DIM compared to placebo (+25 ± 22 and +1.1 ± 19 nmol/L,
respectively). No change in breast density measured by mammography or by MRI was
observed. Plasma tamoxifen metabolites (endoxifen, 4-OH tamoxifen, and
N-desmethyl-tamoxifen) were reduced in women receiving DIM versus placebo
(P < 0.001). Minimal adverse events were reported and did not differ by treatment
arm.
CONCLUSION: In patients taking tamoxifen for breast cancer, daily DIM promoted
favorable changes in estrogen metabolism and circulating levels of SHBG. Further
research is warranted to determine whether DIM associated decreases in
tamoxifen metabolites, including effects on endoxifen levels, attenuates the
clinical benefit of tamoxifen.
TRIAL REGISTRATION: ClinicalTrials.gov NCT01391689.

DOI: 10.1007/s10549-017-4292-7
PMCID: PMC5571834 [Available on 2018-08-01]
PMID: 28560655

Theranostics. 2017 Apr 10;7(6):1674-1688. doi: 10.7150/thno.18082. eCollection
2017.

3,3'-Diindolylmethane stimulates exosomal Wnt11 autocrine signaling in human
umbilical cord mesenchymal stem cells to enhance wound healing.

Shi H(1), Xu X(1), Zhang B(1), Xu J(1), Pan Z(1), Gong A(1), Zhang X(1), Li R(1),
Sun Y(1), Yan Y(1), Mao F(1), Qian H(1), Xu W(1).

Author information:
(1)Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of
Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, China.

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) are suggested as a
promising therapeutic tool in regenerative medicine, however, their efficacy
requires improvement. Small molecules and drugs come up to be a convenient
strategy in regulating stem cells fate and function. Here, we evaluated
3,3'-diindolylmethane (DIM), a natural small-molecule compound involved in the
repairing effects of hucMSCs on a deep second-degree burn injury rat model.
HucMSCs primed with 50 μM of DIM exhibited desirable repairing effects compared
with untreated hucMSCs. DIM enhanced the stemness of hucMSCs, which was related
to the activation of Wnt/β-catenin signaling. β-catenin inhibition impaired the
healing effects of DIM-primed hucMSCs (DIM-hucMSCs) in vivo. Moreover, we
demonstrated that DIM upregulated Wnt11 expression in hucMSC-derived exosomes.
Wnt11 knockdown inhibited β-catenin activation and stemness induction in
DIM-hucMSCs and abrogated their therapeutic effects in vivo. Thus, our findings
indicate that DIM promotes the stemness of hucMSCs through increased exosomal
Wnt11 autocrine signaling, which provides a novel strategy for improving the
therapeutic effects of hucMSCs on wound healing.

DOI: 10.7150/thno.18082
PMCID: PMC5436520
PMID: 28529644

J Med Food. 2017 Jul;20(7):646-652. doi: 10.1089/jmf.2016.0165. Epub 2017 May 1.

3,3'-Diindolylmethane Suppresses Adipogenesis Using AMPKα-Dependent Mechanism in
3T3-L1 Adipocytes and Caenorhabditis elegans.

Lee J(1), Yue Y(2), Park Y(2), Lee SH(1).

Author information:
(1)1 Department of Nutrition and Food Science, College of Agriculture and Natural
Resources, University of Maryland , College Park, Maryland, USA .
(2)2 Department of Food Science, University of Massachusetts , Amherst,
Massachusetts, USA.

3,3'-diindolylmethane is a major in vivo metabolite of indole-3-carbinol, a
bioactive compound found in cruciferous vegetables. Although
3,3'-diindolylmethane has been implicated to possess antitumorigenic and
anti-inflammatory properties, the effect of 3,3'-diindolylmethane on adipogenesis
has not been explored previously. Thus, the present study was conducted to
determine if 3,3'-diindolylmethane affects adipogenesis using 3T3-L1 adipocytes
and Caenorhabditis elegans. Treatment of 3,3'-diindolylmethane significantly
reduced fat accumulation without affecting viability in 3T3-L1 adipocytes.
3,3'-diindolylmethane suppressed expression of peroxisome proliferator-activated
receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), fatty acid binding
protein 4 (FABP4), and perilipin. In addition, 3,3'-diindolylmethane activated
AMP-activated protein kinase α (AMPKα), which subsequently inactivated acetyl CoA
carboxylase (ACC), resulting in reduced fat accumulation. These observations were
further confirmed in C. elegans as treatment with 3,3'-diindolylmethane
significantly reduced body fat accumulation, which was partly associated with
aak-1, but not aak-2, orthologs of AMPKα catalytic subunits α1 and α2,
respectively. The current results demonstrate that 3,3'-diindolylmethane, a
biologically active metabolite of indole-3-carbinol, may prevent adipogenesis
through the AMPKα-dependent pathway.

DOI: 10.1089/jmf.2016.0165
PMID: 28459610  [Indexed for MEDLINE]

Curr Opin Toxicol. 2017 Feb;2:24-29. doi: 10.1016/j.cotox.2017.01.012. Epub 2017
Feb 1.

The Aryl Hydrocarbon Receptor (AhR) as a Drug Target for Cancer Chemotherapy.

Safe S(1), Cheng Y(1), Jin UH(1).

Author information:
(1)Department of Veterinary Physiology & Pharmacology, Texas A&M University,
College Station, TX 77843.

The aryl hydrocarbon receptor (AhR) is overexpressed in some patients with
different tumor types, and the receptor can be a negative or positive prognostic
factor. There is also evidence from both in vivo and in vitro cell culture models
that the AhR can exhibit tumor-specific pro-oncogenic and tumor suppressor-like
functions and therefore can be treated with AhR antagonists or agonists,
respectively. Successful clinical applications of AhR ligands will require the
synthesis and development of selective AhR modulators (SAhRMs) with
tumor-specific AhR agonist or antagonist activity, and some currently available
compounds such as indole-3-carbinol and diindolylmethane-(DIM) and synthetic AhR
antagonists are potential drug candidates. There is also evidence that some
AhR-active pharmaceuticals, including tranilast, flutamide, hydroxytamoxifen and
omeprazole or their derivatives, may be effective AhR-dependent anticancer agents
for single or combination cancer chemotherapies for treatment of breast and
pancreatic cancers.

DOI: 10.1016/j.cotox.2017.01.012
PMCID: PMC5407490 [Available on 2018-02-01]
PMID: 28459113

Comb Chem High Throughput Screen. 2017 Apr 25. doi:
10.2174/1386207320666170425123248. [Epub ahead of print]

Fe3O4@SiO2@KIT-6 as an efficient and reusable catalyst for the synthesis of novel
derivatives of 3,3'-((Aryl-1-phenyl-1H-pyrazol-4-yl)methylene)bis(1H-indole).

Nikpassand M(1), Fekri LZ(2), Nabatzadeh M(1).

Author information:
(1)Department of Chemistry, Rasht Branch, Islamic Azad University, Rasht. Iran.
(2)Department of Chemistry, Payame Noor University, PO Box 19395-3697 Tehran.
Iran.

Korea advanced institute of science and technology cubic ordered mesoporous
silica (KIT-6 mesoporous) silica coated magnetite nanoparticles, is an effective,
eco-benign and recyclable catalyst for the electrophilic substitution reactions
of indoles with various synthetized aldehydes to afford the corresponding novel
diindolylmethanes in high yields and short reaction times. The catalyst can be
recovered and reused without loss of activity. The work-up of the reaction
consists of a simple separation, followed by concentration of the crude product
and purification. The present methodology offers several advantages such as
aqueous media, excellent yields, simple procedure, mild conditions and reduced
environmental consequences. All of synthesized compounds are new and were
characterized by IR, NMR and elemental analyses.

DOI: 10.2174/1386207320666170425123248
PMID: 28443502

J Med Chem. 2017 May 11;60(9):3636-3655. doi: 10.1021/acs.jmedchem.6b01593. Epub
2017 May 1.

Diindolylmethane Derivatives: Potent Agonists of the Immunostimulatory Orphan G
Protein-Coupled Receptor GPR84.

Pillaiyar T(1), Köse M(1), Sylvester K(1), Weighardt H(2), Thimm D(1), Borges
G(1), Förster I(2), von Kügelgen I(3), Müller CE(1).

Author information:
(1)PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I,
University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
(2)Life and Medical Sciences (LIMES) Institute, Immunology and Environment,
University of Bonn, Carl-Troll-Straße 31, 53115 Bonn, Germany.
(3)Department of Pharmacology and Toxicology, University of Bonn , 53105 Bonn,
Germany.

The Gi protein-coupled receptor GPR84, which is activated by (hydroxy)fatty
acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was
identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad
range of diindolylmethane derivatives by condensation of indoles with
formaldehyde in water under microwave irradiation. The products were evaluated at
the human GPR84 in cAMP and β-arrestin assays. Structure-activity relationships
(SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at
the 5- and/or 7-position of the indole rings displayed the highest activity in
cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane
(38, PSB-15160, EC50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57,
PSB-16671, EC50 41.3 nM). In β-arrestin assays, SARs were different, indicating
biased agonism. The new compounds were selective versus related fatty acid
receptors and the arylhydrocarbon receptor. Selected compounds were further
investigated and found to display an ago-allosteric mechanism of action and
increased stability in comparison to the lead structure.

DOI: 10.1021/acs.jmedchem.6b01593
PMID: 28406627  [Indexed for MEDLINE]

Bone Rep. 2017 Feb 16;6:51-59. doi: 10.1016/j.bonr.2017.02.003. eCollection 2017
Jun.

Mechanistic insight into the effects of Aryl Hydrocarbon Receptor activation on
osteogenic differentiation.

Yun C(1), Weiner JA(1), Chun DS(1), Yun J(1), Cook RW(1), Schallmo MS(1), Kannan
AS(1), Mitchell SM(1), Freshman RD(1), Park C(1), Hsu WK(1), Hsu EL(1).

Author information:
(1)Northwestern University Department of Orthopaedic Surgery, Chicago, IL, USA.

While inhibition of bone healing and increased rates of pseudarthrosis are known
adverse outcomes associated with cigarette smoking, the underlying mechanisms by
which this occurs are not well understood. Recent work has implicated the Aryl
Hydrocarbon Receptor (Ahr) as one mediator of the anti-osteogenic effects of
cigarette smoke (CS), which contains numerous toxic ligands for the Ahr.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a high-affinity Ahr ligand
frequently used to evaluate Ahr pathway activation. The purpose of this study was
to elucidate the downstream mechanisms of dioxin action on bone regeneration and
investigate Ahr antagonism as a potential therapeutic approach to mitigate the
effects of dioxin on bone. Markers of osteogenic activity and differentiation
were assessed in primary rat bone marrow stromal cells (BMSC) after exposure to
dioxin, Ahr antagonists, or antagonist + dioxin. Four Ahr antagonists were
evaluated: α-Naphthoflavone (ANF), resveratrol (Res), 3,3'-Diindolylmethane
(DIM), and luteolin (Lut). Our results demonstrate that dioxin inhibited ALP
activity, migratory capacity, and matrix mineralization, whereas co-treatment
with each of the antagonists mitigated these effects. Dioxin also inhibited BMSC
chemotaxis, while co-treatment with several antagonists partially rescued this
effect. RNA and protein expression studies found that dioxin down-regulated
numerous pro-osteogenic targets, whereas co-treatment with Ahr antagonists
prevented these dioxin-induced expression changes to varying degrees. Our results
suggest that dioxin adversely affects bone regeneration in a myriad of ways, many
of which appear to be mediated by the Ahr. Our work suggests that the Ahr should
be investigated as a therapeutic target to combat the adverse effects of CS on
bone healing.

DOI: 10.1016/j.bonr.2017.02.003
PMCID: PMC5365310
PMID: 28377982

Bioorg Med Chem Lett. 2017 Apr 1;27(7):1561-1565. doi:
10.1016/j.bmcl.2017.02.033. Epub 2017 Feb 17.

Preliminary SAR on indole-3-carbinol and related fragments reveals a novel
anticancer lead compound against resistant glioblastoma cells.

Sherer C(1), Tolaymat I(2), Rowther F(3), Warr T(3), Snape TJ(4).

Author information:
(1)Molecular Preventive Medicine, Institute of Prevention and Cancer
Epidemiology, University Medical Center Freiburg, Freiburg, Germany.
(2)Institute of Environmental Health Sciences, University Medical Center
Freiburg, Freiburg, Germany.
(3)Department of Obstetrics & Gynecology, University Medical Center Freiburg,
Freiburg, Germany.
(4)Department of Gynecology and Obstetrics, University Medical Center Regensburg,
Regensburg, Germany.

The prognosis for glioblastoma patients is, at best, poor, with the median time
of survival after diagnosis measured in months. As such, there is much need for
the rapid development of potent and novel treatments. Herein, we report our
preliminary findings on the SAR of a series of indole-3-carbinol and related
fragments and reveal a potent lead with low micromolar activity against a
particularly resistant glioblastoma cell culture, providing a new platform for
future development of a new therapy in this area.

DOI: 10.1016/j.bmcl.2017.02.033
PMID: 28256372  [Indexed for MEDLINE]

Cell Death Discov. 2017 Feb 27;3:17013. doi: 10.1038/cddiscovery.2017.13.
eCollection 2017.

MPP+ induces necrostatin-1- and ferrostatin-1-sensitive necrotic death of
neuronal SH-SY5Y cells.

Ito K(1), Eguchi Y(2), Imagawa Y(3), Akai S(4), Mochizuki H(5), Tsujimoto Y(3).

Author information:
(1)Laboratory of Molecular Genetics, Department of Medical Genetics, Osaka
University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871,
Japan; Department of Molecular and Cellular Biology, Research Institute of Osaka
Medical Center for Cancer and Cardiovascular Diseases, 1-3-2 Nakamichi,
Higashinari-ku, Osaka 537-8511, Japan; Department of Neurology, Osaka University
Graduate School of Medicine, Suita, Japan.
(2)Laboratory of Molecular Genetics, Department of Medical Genetics, Osaka
University Graduate School of Medicine , 2-2 Yamadaoka, Suita, Osaka 565-0871,
Japan.
(3)Department of Molecular and Cellular Biology, Research Institute of Osaka
Medical Center for Cancer and Cardiovascular Diseases , 1-3-2 Nakamichi,
Higashinari-ku, Osaka 537-8511, Japan.
(4)Laboratory of Synthetic Medicinal Chemistry, Osaka University Graduate School
of Pharmaceutical Sciences , 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
(5)Department of Neurology, Osaka University Graduate School of Medicine , Suita,
Japan.

Regulation of cell death is potentially a powerful treatment modality for
intractable diseases such as neurodegenerative diseases. Although there have been
many reports about the possible involvement of various types of cell death in
neurodegenerative diseases, it is still unclear exactly how neurons die in
patients with these diseases, thus treatment strategies based on cell death
regulation have not been established yet. To obtain some insight into the
mechanisms of cell death involved in neurodegenerative diseases, we studied the
effect of 1-methyl-4-phenylpyridinium (MPP+) on the human neuroblastoma cell line
SH-SY5Y (a widely used model of Parkinson's disease). We found that MPP+
predominantly induced non-apoptotic death of neuronally differentiated SH-SY5Y
cells. This cell death was strongly inhibited by necrostatin-1 (Nec-1), a
necroptosis inhibitor, and by an indole-containing compound
(3,3'-diindolylmethane: DIM). However, it occurred independently of
receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3), indicating
that this form of cell death was not necroptosis. MPP+-induced cell death was
also inhibited by several inhibitors of ferroptosis, including ferrostatin-1
(Fer-1). Although MPP+-induced death and ferroptosis shared some features, such
as occurrence of lipid peroxidation and inhibition by Fer-1, MPP+-induced death
seemed to be distinct from ferroptosis because MPP+-induced death (but not
ferroptosis) was inhibited by Nec-1, was independent of p53, and was accompanied
by ATP depletion and mitochondrial swelling. Further investigation of
MPP+-induced non-apoptotic cell death may be useful for understanding the
mechanisms of neuronal loss and for treatment of neurodegenerative diseases such
as Parkinson's disease.

DOI: 10.1038/cddiscovery.2017.13
PMCID: PMC5327502
PMID: 28250973

BMC Complement Altern Med. 2017 Feb 16;17(1):115. doi: 10.1186/s12906-017-1621-7.

BreastDefend enhances effect of tamoxifen in estrogen receptor-positive human
breast cancer in vitro and in vivo.

Cheng S(1)(2), Castillo V(1), Welty M(1), Alvarado M(1), Eliaz I(3), Temm CJ(4),
Sandusky GE(4), Sliva D(5)(6)(7).

Author information:
(1)Cancer Research Laboratory, Methodist Research Institute, Indiana University
Health, Indianapolis, IN, 46202, USA.
(2)Present address: Department of Food Quality and Safety, School of Engineering,
China Pharmaceutical University, Nanjing, People's Republic of China.
(3)Amitabha Medical Clinic and Healing Center, Santa Rosa, CA, 95401, USA.
(4)Department of Pathology, Indiana University School of Medicine, Indianapolis,
IN, 46202, USA.
(5)Cancer Research Laboratory, Methodist Research Institute, Indiana University
Health, Indianapolis, IN, 46202, USA. [email protected].
(6)Department of Medicine, Indiana University School of Medicine, Indianapolis,
IN, 46202, USA. [email protected].
(7)DSTest Laboratories, Purdue Research Park, 5225 Exploration Drive,
Indianapolis, IN, 46241, USA. [email protected].

BACKGROUND: Tamoxifen (TAM) has been widely used for the treatment of estrogen
receptor (ER)-positive breast cancer and its combination with other therapies is
being actively investigated as a way to increase efficacy and decrease side
effects. Here, we evaluate the therapeutic potential of co-treatment with TAM and
BreastDefend (BD), a dietary supplement formula, in ER-positive human breast
cancer.
METHODS: Cell proliferation and apoptosis were determined in ER-positive human
breast cancer cells MCF-7 by MTT assay, quantitation of cytoplasmic
histone-associated DNA fragments and expression of cleaved PARP, respectively.
The molecular mechanism was identified using RNA microarray analysis and western
blotting. Tumor tissues from xenograft mouse model were analyzed by
immunohistochemistry.
RESULTS: Our data clearly demonstrate that a combination of 4-hydroxytamoxifen
(4-OHT) with BD lead to profound inhibition of cell proliferation and induction
of apoptosis in MCF-7 cells. This effect is consistent with the regulation of
apoptotic and TAM resistant genes at the transcription and translation levels.
Importantly, TAM and BD co-treatment significantly enhanced apoptosis, suppressed
tumor growth and reduced tumor weight in a xenograft model of human ER-positive
breast cancer.
CONCLUSION: BD sensitized ER-positive human breast cancer cells to 4-OHT/TAM
treatment in vitro and in vivo. BreastDefend can be used in an adjuvant therapy
to increase the therapeutic effect of tamoxifen in patients with ER-positive
breast cancer.

DOI: 10.1186/s12906-017-1621-7
PMCID: PMC5314617
PMID: 28209156  [Indexed for MEDLINE]

Hum Mol Genet. 2016 Nov 15;25(22):5006-5016. doi: 10.1093/hmg/ddw329.

AHR/CYP1A1 interplay triggers lymphatic barrier breaching in breast cancer
spheroids by inducing 12(S)-HETE synthesis.

Nguyen CH(1), Brenner S(2), Huttary N(1), Atanasov AG(3)(4), Dirsch VM(3),
Chatuphonprasert W(2)(5), Holzner S(1), Stadler S(1), Riha J(2), Krieger S(1), de
Martin R(6), Bago-Horvath Z(1), Krupitza G(1), Jäger W(2).

Author information:
(1)Clinical Institute of Pathology, Medical University of Vienna, Vienna,
Austria.
(2)Department of Clinical Pharmacy and Diagnostics, University of Vienna, Vienna,
Austria.
(3)Department of Pharmacognosy, University of Vienna, Vienna, Austria.
(4)Institute of Genetics and Animal Breeding of the Polish Academy of Sciences,
Jastrzebiec, Poland
(5)Faculty of Medicine, Mahasarakham University, Mahasarakham, Thailand
(6)Department of Vascular Biology and Thrombosis Research, Center of Biomolecular
Medicine and Pharmacology, Medical University of Vienna, Vienna, Austria.

A causal link between overexpression of aryl hydrocarbon receptor (AHR) and its
target cytochrome P450 1A1 (CYP1A1) and metastatic outgrowth of various cancer
entities has been established. Nevertheless, the mechanism how AHR/CYP1A1 support
metastasis formation is still little understood. In vitro we discovered a
potential mechanism facilitating tumour dissemination based on the production of
12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Utilising a three-dimensional
lymph endothelial cell (LEC) monolayer & MDA-MB231 breast cancer cell spheroid
co-culture model in combination with knock-down approach allowed elucidation of
the molecular/biochemical basis of AHR/CYP1A1-induced tumour breaching through
the LEC barrier. Enzyme immunoassay evidenced the potential of recombinant CYP1A1
to synthesise 12(S)-HETE in vitro and qPCR and Western blotting measured gene and
protein expression in specific experimental settings. In detail, AHR induced
CYP1A1 expression and 12(S)-HETE secretion in tumour spheroids, which caused LEC
junction retraction thereby forming large discontinuities allowing transmigration
of the tumour. This was enforced by the activating AHR ligand 6-formylindolo
(3,3-b)carbazole (FICZ), or inhibited by the AHR antagonist 3,3’-diindolylmethane
(DIM) as well as by siRNA against AHR and CYP1A1. AHR and NF-κB were negatively
cross talking and therefore, the inhibition of AHR (but not CYP1A1) induced RELA,
RELB, NFKB1, NFKB2 and the NF-κB target MMP1, which itself promotes tumour
intravasation by a mechanism that is different from 12(S)-HETE. Conversely, the
inhibition of NFKB2 induced AHR, CYP1A1 and 12(S)-HETE synthesis. The approved
clinical drugs guanfacine and vinpocetine, which inhibit CYP1A1 and NF-κB,
respectively, significantly inhibited LEC barrier breaching in vitro indicating
an option to reduce metastatic dissemination.

DOI: 10.1093/hmg/ddw329
PMID: 28171546

Curr Pharm Des. 2017;23(19):2697-2721. doi: 10.2174/1381612823666170120160832.

Food as Pharma? The Case of Glucosinolates.

Capuano E(1), Dekker M(2), Verkerk R(2), Oliviero T(2).

Author information:
(1)Food Quality Design, WU Agrotechnology & Food Sciences, Axis building 118,
Bornse Weilanden 9, 6708 WG Wageningen, Netherlands.
(2)Food Quality & Design Group, Wageningen University, Axis building, 6708WG,
Wageningen, Netherlands.

BACKGROUND: Glucosinolates (GLSs) are dietary plant secondary metabolites
occurring in the order Brassicales with potential health effects, in particular
as anti-carcinogenic compounds. GLSs are converted into a variety of breakdown
products (BPs) upon plant tissue damage and by the gut microbiota. GLS biological
activity is related to BPs rather than to GLSs themselves.
METHODS: we have reviewed the most recent scientific literature on the metabolic
fate and the biological effect of GLSs with particular emphasis on the
epidemiological evidence for health effect and evidence from clinical trials. An
overview of potential molecular mechanisms underlying GLS biological effect is
provided. The potential toxic or anti-nutritional effect has also been discussed.
RESULTS: Epidemiological and human in vivo evidence point towards a potential
anti-cancer effect for sulforaphane, indole-3-carbinol and 3,3-diindolylmethane.
A number of new human clinical trials are on-going and will likely shed further
light on GLS protective effect towards cancer as well as other diseases. BPs
biological effect is the results of a plurality of molecular mechanisms acting
simultaneously which include modulation of xenobiotic metabolism, modulation of
inflammation, regulation of apoptosis, cell cycle arrest, angiogenesis and
metastasis and regulation of epigenetic events. BPs have been extensively
investigated for their protective effect towards cancer but in recent years the
interest also includes other diseases.
CONCLUSION: It appears that certain BPs may protect against and may even
represent a therapeutic strategy against several forms of cancer. Whether this
latter effect can be achieved through diet or supplements should be investigated
more thoroughly.

DOI: 10.2174/1381612823666170120160832
PMID: 28117016

J Cell Biochem. 2017 Aug;118(8):1979-1983. doi: 10.1002/jcb.25903. Epub 2017 Apr
25.

Therapeutic Potential of Targeting Wnt/β-Catenin Pathway in Treatment of
Colorectal Cancer: Rational and Progress.

Bahrami A(1), Amerizadeh F(1), ShahidSales S(2), Khazaei M(3), Ghayour-Mobarhan
M(2), Sadeghnia HR(1), Maftouh M(4), Hassanian SM(5)(4), Avan A(4)(2).

Author information:
(1)Department of Modern Sciences and Technologies, Faculty of Medicine, Mashhad
University of Medical Sciences, Mashhad, Iran.
(2)Cancer Research Center, School of Medicine, Mashhad University of Medical
Sciences, Mashhad, Iran.
(3)Department of Physiology, Neurogenic Inflammatory Research Center, Mashhad
University of Medical Sciences, Mashhad, Iran.
(4)Metabolic Syndrome Research Center, School of Medicine, Mashhad University of
Medical Sciences, Mashhad, Iran.
(5)Department of Medical Biochemistry, School of Medicine, Mashhad University of
Medical Sciences, Mashhad, Iran.

Wnt/β-catenin pathway is one of the main/frequent dysregulated pathways in
several tumor types, including colon cancer. Aberrant activation of this pathway
is associated with cell proliferation, invasive behaviors, and cell resistance,
suggesting its potential value as a therapeutic target in treatment of CRC.
Several agents have been developed for targeting of this pathway (e.g, natural
agents: curcumin, 3,3-diindolylmethane, phytoestrogen; Synthetic/small Wnt
inhibitors: Rofecoxib; PRI-724, CWP232291; and monoclonal antibody against
frizzled receptors, Vanituctumab). This review summarizes the current knowledge
about the therapeutic potential of targeting Wnt pathway with particular emphasis
on preclinical/clinical studies in treatment of colorectal cancer. J. Cell.
Biochem. 118: 1979-1983, 2017.

DOI: 10.1002/jcb.25903
PMID: 28109136

Aquat Toxicol. 2017 Feb;183:135-143. doi: 10.1016/j.aquatox.2016.12.021. Epub
2016 Dec 30.

Linking genomic responses of gonads with reproductive impairment in marine medaka
(Oryzias melastigma) exposed chronically to the chemopreventive and antifouling
agent, 3,3'-diindolylmethane (DIM).

Chen L(1), Au DW(2), Hu C(3), Zhang W(1), Zhou B(4), Cai L(1), Giesy JP(5), Qian
PY(6).

Author information:
(1)Division of Life Science, Hong Kong University of Science and Technology,
Clear Water Bay, Hong Kong SAR, China.
(2)State Key Laboratory in Marine Pollution, Department of Biology and Chemistry,
City University of Hong Kong, Kowloon, Hong Kong SAR, China.
(3)School of Chemistry and Environmental Engineering, Wuhan Institute of
Technology, Wuhan 430072, China.
(4)State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of
Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
(5)Department of Veterinary Biomedical Sciences and Toxicology Centre, University
of Saskatchewan, 44 Campus Drive, Saskatoon, SK S7N 5B3, Canada; Department of
Zoology, and Center for Integrative Toxicology, Michigan State University, East
Lansing, MI, USA.
(6)Division of Life Science, Hong Kong University of Science and Technology,
Clear Water Bay, Hong Kong SAR, China. Electronic address: [email protected].

3,3'-Diindolylmethane (DIM) has been promoted as an effective chemopreventive and
antifouling additive. However, the concurrent risks or side effects of DIM are
not fully understood, especially on tissues responsive to estrogen. Therefore,
this study employed marine medaka (Oryzias melastigma) as a test model to
evaluate relative safety and explore mechanisms of toxic action of DIM on
development and function of gonad after chronic (28days) aqueous exposure to
relatively low doses (0μg/L or 8.5μg/L). Integration of comprehensive
toxicogenomic analysis at the transcriptome and proteome levels with apical
endpoints, such as production of eggs and swimming performance of larvae,
elucidated the molecular linkage in gonad from bottom up along the reproductive
adverse outcome pathway. A series of sequential changes at the transcript and
protein levels were linked to lesser fecundity and viability of larvae exposed to
DIM. Anomalous production of vitellogenin (VTG) and eggshell proteins in testis
confirmed the estrogenic potency of DIM. In the ovary, although storage of VTG
was greater, lesser expressions of cathepsin enzymes blocked cleavage and
incorporation of VTG into oocytes as yolk, which acted together with lower
eggshell proteins to inhibit maturation of primary oocyte and thus contributed to
impairment of fecundity. Overall, this study demonstrated that exposure to DIM
impaired reproductive fitness. Diverse molecular initiating changes in gonads
were linked to apical endpoints that could be used in assessment of risks posed
by DIM on gametogenesis. In combination with chemical stability and potent
endocrine disruption, the results of this study can inform decisions about the
use of DIM either as chemopreventive or antifouling agent.

DOI: 10.1016/j.aquatox.2016.12.021
PMID: 28063342  [Indexed for MEDLINE]

Free Radic Biol Med. 2016 Oct;99:463-471. doi:
10.1016/j.freeradbiomed.2016.09.007. Epub 2016 Sep 5.

3,3'-diindolylmethane mitigates total body irradiation-induced hematopoietic
injury in mice.

Lu L(1), Dong J(2), Li D(2), Zhang J(2), Fan S(3).

Author information:
(1)Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,
Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking
Union Medical College, Tianjin 300192, China. Electronic address:
[email protected].
(2)Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,
Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking
Union Medical College, Tianjin 300192, China.
(3)Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine,
Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking
Union Medical College, Tianjin 300192, China. Electronic address:
[email protected].

We have reported that hematopoietic system injury induced by total body
irradiation (TBI) leads to generation of intracellular reactive oxygen species
(ROS) and DNA damage, which are ameliorated by antioxidant agents. In the present
study, we reported that administration of DIM, a potent antioxidant agent, not
only protected mice against TBI-induced lethality, also ameliorated TBI-induced
hematopoietic injury. The latter effect was probably attributable to DIM's
inhibition of TBI-induced increases in ROS production in hematopoietic stem cells
(HSCs) and the phosphorylation of histone H2AX (γ-H2AX). In particular, DIM led
to significant improvements in bone marrow (BM) HSC frequency, hematopoietic
progenitor cell (HPC) clonogenic function, and multilineage engraftment after
transplantation. A downregulation of NADPH oxidase 4 (NOX4) and an upregulation
of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1)
expression were observed following DIM treatment. Notably, the anti-apoptotic
potential of DIM was correlated with increased expression of the anti-apoptotic
protein Bcl-2 and decreased expression of the pro-apoptotic protein Bax. These
findings suggest that DIM attenuates TBI-induced hematopoietic injury through the
inhibition of both oxidative stress in HSCs and hematopoietic cell apoptosis.
Furthermore, we demonstrated that DIM protected BM hematopoietic cells against
ionizing radiation and led to increased clonogenicity in vitro. Therefore, DIM
has the potential to be used as an effective radioprotectant to ameliorate
TBI-induced hematopoietic injury.

DOI: 10.1016/j.freeradbiomed.2016.09.007
PMID: 27609226

J Agric Food Chem. 2016 Oct 4. [Epub ahead of print]

Effect of Oral Administration of 3,3'-Diindolylmethane on Dextran Sodium
Sulfate-Induced Acute Colitis in Mice.

Jeon EJ(1), Davaatseren M(2), Hwang JT(1)(3), Park JH(1)(3), Hur HJ(1), Lee
AS(1), Sung MJ(1)(3).

Author information:
(1)Research Division Emerging Innovative Technology, Korea Food Research
Institute , Songnam, Keongki, Republic of Korea.
(2)Department of Food Science and Technology, Chung-ang University , Ansung,
Keongki, Republic of Korea.
(3)Food Biotechnology, University of Science and Technology , Daejeon, Republic
of Korea.

In patients with inflammatory bowel disease (IBD), inflammation is induced and
maintained by lymphangiogenesis and angiogenesis. 3,3'-Diindolylmethane (DIM) is
a natural product formed in acidic conditions from indole-3-carbinol in
cruciferous vegetables, and it is known for its chemotherapeutic activity. This
study evaluated DIM's effects on angiogenesis, lymphangiogenesis, and
inflammation in a mouse colitis model. Experimental colitis was induced in mice
by administering 3% dextran sulfate sodium (DSS) via drinking water. DIM
remarkably attenuated the clinical signs and histological characteristics in mice
with DSS-induced colitis. DIM suppressed neutrophil infiltration and
pro-inflammatory cytokines. Moreover, it significantly suppressed the expression
of vascular endothelial growth factor (VEGF)-A and VEGF receptor (VEGFR)-2,
indicating that the mechanism may be related to the repression of
pro-angiogenesis activity. DIM also remarkably suppressed the expression of
VEGF-C, VEGF-D, VEGFR-3, and angiopoietin-2; thus, the mechanism may also be
related to the suppression of lymphangiogenesis. Therefore, DIM is a possible
treatment option for inflammation of the intestine and associated angiogenesis
and lymphangiogenesis.

DOI: 10.1021/acs.jafc.6b02604
PMID: 27700072

Sci Rep. 2016 Sep 29;6:34529. doi: 10.1038/srep34529.

Regulation of PCGEM1 by p54/nrb in prostate cancer.

Ho TT(1)(2), Huang J(3)(4), Zhou N(3)(5), Zhang Z(6), Koirala P(3), Zhou X(7), Wu
F(5), Ding X(1)(8), Mo YY(1).

Author information:
(1)Department of Pharmacology and Toxicology, Cancer Institute, University of
Mississippi Medical Center, Jackson, MS, USA.
(2)Department of Radiation Oncology, University of Mississippi Medical Center,
Jackson, MS, USA.
(3)Department of Biochemistry, Cancer Institute, University of Mississippi
Medical Center, Jackson, MS, USA.
(4)Department of Radiation Oncology, Duke University Medical Center, Durham, NC,
USA.
(5)System Biosciences, Mountain View, CA, USA.
(6)Department of Pulmonary Medicine, Tongji Hospital, Tongji University,
Shanghai, China.
(7)Department of Pathology, University of Mississippi Medical Center, Jackson,
MS, USA.
(8)College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.

PCGEM1 is a long non-coding RNA (lncRNA) that is often upregulated in prostate
cancer. However, little is known how PCGEM1 is regulated. In the present study,
we show transcriptional regulation of PCGEM1 in response to androgen deprivation
by p54/nrb. While ectopic expression of p54/nrb increases, suppression of p54/nrb
by RNAi or knockout (KO) reduces PCGEM1. Moreover, rescue experiments indicate
that re-expression of p54/nrb in KO cells restores the ability to induce PCGEM1,
leading to upregulation of the androgen receptor splice variant AR3 which has
been shown to play a role in castration resistance. Finally,
3,3'-Diindolylmethane (DIM), a known chemoprevention agent, is capable of
suppressing PCGEM1 expression by preventing the interaction of p54/nrb with the
PCGEM1 promoter. In particular, DIM reduces tumor growth by suppression of PCGEM1
and promoting apoptosis in the castrated xenograft mouse model. Together, these
results demonstrate a novel mechanism of p54/nrb-mediated expression of PCGEM1
and AR3, contributing to castration resistance in prostate cancer.

DOI: 10.1038/srep34529
PMCID: PMC5041109
PMID: 27682980

Adv Exp Med Biol. 2016;928:131-154.

Indole-3-Carbinol and Its Role in Chronic Diseases.

Licznerska B(1), Baer-Dubowska W(2).

Author information:
(1)Department of Pharmaceutical Biochemistry, Poznan University of Medical
Sciences, Poznan, Poland.
(2)Department of Pharmaceutical Biochemistry, Poznan University of Medical
Sciences, Poznan, Poland. [email protected].

Indole-3-carbinol (I3C), a common phytochemical in cruciferous vegetables, and
its condensation product, 3,3'-diindolylmethane (DIM) exert several biological
activities on cellular and molecular levels, which contribute to their
well-recognized chemoprevention potential. Initially, these compounds were
classified as blocking agents that increase drug-metabolizing enzyme activity.
Now it is widely accepted that I3C and DIM affect multiple signaling pathways and
target molecules controlling cell division, apoptosis, or angiogenesis
deregulated in cancer cells. Although most of the current data support the role
of I3C and DIM in prevention of hormone-dependent cancers, it seems that their
application in prevention of the other cancer as well as cardiovascular disease,
obesity, and diabetes reduction is also possible. This chapter summarizes the
current experimental data on the I3C and DIM activity and the results of clinical
studies indicating their role in prevention of chronic diseases.

DOI: 10.1007/978-3-319-41334-1_6
PMID: 27671815  [Indexed for MEDLINE]

Eur J Pharm Biopharm. 2016 Nov;108:168-179. doi: 10.1016/j.ejpb.2016.08.006. Epub
2016 Aug 30.

Novel diindolylmethane derivatives based NLC formulations to improve the oral
bioavailability and anticancer effects in triple negative breast cancer.

Godugu C(1), Doddapaneni R(2), Safe SH(3), Singh M(4).

Author information:
(1)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University,
Tallahassee, FL, USA; Department of Regulatory Toxicology, National Institute of
Pharmaceutical Education and Research (NIPER), Hyderabad, Telangana 500037,
India.
(2)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University,
Tallahassee, FL, USA.
(3)Department of Veterinary Physiology and Pharmacology, Texas A&M University,
College Station, TX, USA.
(4)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University,
Tallahassee, FL, USA. Electronic address: [email protected].

The present study demonstrates the promising anticancer effects of novel
C-substituted diindolylmethane (DIM) derivatives DIM-10 and DIM-14 in aggressive
TNBC models. In vitro studies demonstrated that these compounds possess strong
anticancer effects. Caco-2 permeability studies resulted in poor permeability and
poor oral bioavailability was demonstrated by pharmacokinetic studies. Nano
structured lipid carrier (NLC) formulations were prepared to increase the
clinical acceptance of these compounds. Significant increase in oral
bioavailability was observed with NLC formulations. Compared to DIM-10, DIM-10
NLC formulation showed increase in Cmax and AUC values by 4.73 and 11.19-folds,
respectively. Similar pattern of increase was observed with DIM-14 NLC
formulations. In dogs DIM-10 NLC formulations showed an increase of 2.65 and
2.94-fold in Cmax and AUC, respectively. The anticancer studies in MDA-MB-231
orthotopic TNBC models demonstrated significant reduction in tumor volumes in
DIM-10 and DIM-14 NLC treated animals. Our studies suggest that NLC formulation
of both DIM-10 and 14 is effective in TNBC models.

DOI: 10.1016/j.ejpb.2016.08.006
PMID: 27586082  [Indexed for MEDLINE]

Cancer Prev Res (Phila). 2016 Oct;9(10):788-793. Epub 2016 Aug 18.

Harnessing the Power of Cruciferous Vegetables: Developing a Biomarker for
Brassica Vegetable Consumption Using Urinary 3,3'-Diindolylmethane.

Fujioka N(1), Ransom BW(2), Carmella SG(2), Upadhyaya P(2), Lindgren BR(2),
Roper-Batker A(3), Hatsukami DK(4), Fritz VA(5), Rohwer C(6), Hecht SS(2).

Author information:
(1)Division of Hematology, Oncology, and Transplantation, University of
Minnesota, Minneapolis, Minnesota. Masonic Cancer Center, University of
Minnesota, Minneapolis, Minnesota. [email protected].
(2)Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota.
(3)College of Medicine, University of Vermont, Burlington, Vermont.
(4)Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota.
Tobacco Research Programs, University of Minnesota, Minneapolis, Minnesota.
(5)Department of Horticultural Science, University of Minnesota, St. Paul,
Minnesota. Southern Research and Outreach Center, University of Minnesota,
Waseca, Minnesota.
(6)Southern Research and Outreach Center, University of Minnesota, Waseca,
Minnesota.

Glucobrassicin in Brassica vegetables gives rise to indole-3-carbinol (I3C), a
compound with potent anticancer effects in preclinical models. We previously
showed that the urinary metabolite 3,3'-diindolylmethane (DIM) could discriminate
between volunteers fed high and low doses of Brassica vegetables. However, the
quantitative relationship between glucobrassicin exposure and urinary DIM level
is unclear. We conducted a clinical trial to examine the hypotheses that a range
of glucobrassicin exposure from Brassica vegetables is reflected in urinary DIM
and that this effect plateaus. Forty-five subjects consumed vegetables, a mixture
of brussels sprouts and/or cabbage, at one of seven discrete dose levels of
glucobrassicin ranging from 25 to 500 μmol, once daily for 2 consecutive days.
All urine was collected for 24 hours after each vegetable-eating session. Urinary
DIM was measured using our published liquid chromatography-electrospray
ionization-tandem mass spectrometry-selected reaction monitoring
(LC/ESI-MS/MS-SRM) method. Urinary DIM excretion increased predictably with
increasing glucobrassicin dose and plateaued between 200 and 300 μmol of
glucobrassicin. The association between glucobrassicin dose and urinary DIM was
strong and positive (R(2) = 0.68). The majority of DIM was excreted in the first
12 hours after vegetable consumption. We conclude that urinary DIM is a reliable
biomarker of glucobrassicin exposure and I3C uptake and that feeding
glucobrassicin beyond 200 μmol did not consistently lead to more urinary DIM,
suggesting a plateau in potential chemopreventive benefit. Cancer Prev Res;
9(10); 788-93.

DOI: 10.1158/1940-6207.CAPR-16-0136
PMCID: PMC5220883 [Available on 2017-10-01]
PMID: 27538743

Int J Mol Sci. 2016 Jul 19;17(7). pii: E1155. doi: 10.3390/ijms17071155.

Cellular and Molecular Mechanisms of 3,3'-Diindolylmethane in Gastrointestinal
Cancer.

Kim SM(1).

Author information:
(1)Department of Physiology, Chonbuk National University Medical School, Jeonju
561-180, Korea. [email protected].

Studies in humans have shown that 3,3'-diindolylmethane (DIM), which is found in
cruciferous vegetables, such as cabbage and broccoli, is effective in the
attenuation of gastrointestinal cancers. This review presents the latest findings
on the use, targets, and modes of action of DIM for the treatment of human
gastrointestinal cancers. DIM acts upon several cellular and molecular processes
in gastrointestinal cancer cells, including apoptosis, autophagy, invasion, cell
cycle regulation, metastasis, angiogenesis, and endoplasmic reticulum (ER)
stress. In addition, DIM increases the efficacy of other drugs or therapeutic
chemicals when used in combinatorial treatment for gastrointestinal cancer. The
studies to date offer strong evidence to support the use of DIM as an anticancer
and therapeutic agent for gastrointestinal cancer. Therefore, this review
provides a comprehensive understanding of the preventive and therapeutic
properties of DIM in addition to its different perspective on the safety of DIM
in clinical applications for the treatment of gastrointestinal cancers.

DOI: 10.3390/ijms17071155
PMCID: PMC4964527
PMID: 27447608  [Indexed for MEDLINE]

Sci Rep. 2016 Jul 19;6:29878. doi: 10.1038/srep29878.

The autism-related gene SNRPN regulates cortical and spine development via
controlling nuclear receptor Nr4a1.

Li H(1), Zhao P(2), Xu Q(1), Shan S(2), Hu C(1), Qiu Z(2), Xu X(1).

Author information:
(1)Department of Child Health Care, Children's Hospital of Fudan University, 399
Wanyuan Road, Shanghai 201102, China.
(2)Institute of Neuroscience, Key Laboratory of Primate Neurobiology, CAS Center
for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

The small nuclear ribonucleoprotein polypeptide N (SNRPN) gene, encoding the
RNA-associated SmN protein, duplications or deletions of which are strongly
associated with neurodevelopmental disabilities. SNRPN-coding protein is highly
expressed in the brain. However, the role of SNRPN protein in neural development
remains largely unknown. Here we showed that the expression of SNRPN increased
markedly during postnatal brain development. Overexpression or knockdown of SNRPN
in cortical neurons impaired neurite outgrowth, neuron migration, and the
distribution of dendritic spines. We found that SNRPN regulated the expression
level of Nr4a1, a critical nuclear receptor during neural development, in
cultured primary cortical neurons. The abnormal spine development caused by SNRPN
overexpression could be fully rescued by Nr4a1 co-expression. Importantly, we
found that either knockdown of Nr4a1 or 3, 3'- Diindolylmethane (DIM), an Nr4a1
antagonist, were able to rescue the effects of SNRPN knockdown on neurite
outgrowth of embryonic cortical neurons, providing the potential therapeutic
methods for SNRPN deletion disorders. We thus concluded that maintaining the
proper level of SNRPN is critical in cortical neurodevelopment. Finally, Nr4a1
may serve as a potential drug target for SNRPN-related neurodevelopmental
disabilities, including Prader-Willi syndrome (PWS) and autism spectrum disorders
(ASDs).

DOI: 10.1038/srep29878
PMCID: PMC4949425
PMID: 27430727

Drug Deliv Transl Res. 2016 Oct;6(5):526-39. doi: 10.1007/s13346-016-0302-2.

Lipid-based oral delivery systems for skin deposition of a potential
chemopreventive DIM derivative: characterization and evaluation.

Boakye CH(1), Patel K(1), Patel AR(1), Faria HA(2), Zucolotto V(2), Safe S(3),
Singh M(4).

Author information:
(1)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, 1520
S Martin Luther King Jr. Blvd, Tallahassee, FL, 32307, USA.
(2)Nanomedicine and Nanotoxicology Group, Physics Institute of São Carlos, USP,
São Carlos, SP, 13566-590, Brazil.
(3)Department of Veterinary Physiology and Pharmacology, Texas A&M University,
College Station, TX, USA.
(4)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, 1520
S Martin Luther King Jr. Blvd, Tallahassee, FL, 32307, USA.

The objective of this study was to explore the oral route as a viable potential
for the skin deposition of a novel diindolylmethane derivative (DIM-D) for
chemoprevention activity. Various lipid-based oral delivery systems were
optimized and compared for enhancing DIM-D's oral bioavailability and skin
deposition. Preformulation studies were performed to evaluate the log P and
solubility of DIM-D. Microsomal metabolism, P-glycoprotein efflux, and caco-2
monolayer permeability of DIM-D were determined. Comparative evaluation of the
oral absorption and skin deposition of DIM-D-loaded various lipid-based
formulations was performed in rats. DIM-D showed pH-dependent solubility and a
high log P value. It was not a strong substrate of microsomal degradation and
P-glycoprotein. SMEDDs comprised of medium chain triglycerides, monoglycerides,
and kolliphor-HS15 (36.70 ± 0.42 nm). SNEDDs comprised of long chain
triglycerides, cremophor RH40, labrasol, and TPGS (84.00 ± 14.14 nm).
Nanostructured lipid carriers (NLC) consisted of compritol, miglyol, and
surfactants (116.50 ± 2.12 nm). The blank formulations all showed >70 % cell
viability in caco-2 cells. Differential Scanning Calorimetry confirmed the
amorphization of DIM-D within the lipid matrices while Atomic Force Microscopy
showed particle size distribution similar to the dynamic light scattering data.
DIM-D also showed reduced permeation across caco-2 monolayer that was enhanced
(p < 0.05) by SNEDDs in comparison to SMEDDs and NLC. Fabsolute for DIM-D SNEDDs,
SMEDDs, and NLC was 0.14, 0.04, and 0.007, respectively. SNEDDs caused 53.90,
11.32, and 15.08-fold more skin deposition of DIM-D than the free drug, SMEDDs,
and NLC, respectively, at 2 h following oral administration and shows a viable
potential for use in skin cancer chemoprevention. Graphical Abstract ᅟ.

DOI: 10.1007/s13346-016-0302-2
PMID: 27405772

Biochem Pharmacol. 2016 Sep 1;115:77-84. doi: 10.1016/j.bcp.2016.06.018. Epub
2016 Jun 29.

3,3'-Diindolylmethane induces anti-human gastric cancer cells by the miR-30e-ATG5
modulating autophagy.

Ye Y(1), Fang Y(2), Xu W(3), Wang Q(4), Zhou J(4), Lu R(5).

Author information:
(1)Department of Preventive Medicine and Public Health Laboratory Science, School
of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China.
(2)Department of Preventive Medicine and Public Health Laboratory Science, School
of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China; Department of
Digestive Diseases, Affiliated Kunshan Hospital, Jiangsu University, Kunshan,
Suzhou, Jiangsu, China.
(3)Labortaory of Cancer Biology, Key Laboratory of Biotherapy in Zhejiang, Sir
Runrun Shaw Hospital, Medical School of Zhejiang University, Zhejiang, China.
(4)Department of Molecular Cell Biology and Toxicology, Cancer Center, School of
Public Health, Nanjing Medical University, Nanjing, Jiangsu, China.
(5)Department of Preventive Medicine and Public Health Laboratory Science, School
of Medicine, Jiangsu University, Zhenjiang, Jiangsu, China; Center for
Experimental Research, Affiliated Kunshan Hospital, Jiangsu University, Kunshan,
Suzhou, Jiangsu, China. Electronic address: [email protected].

3,3'-Diindolylmethane (DIM), a class of relatively non-toxic indole derivatives
from cruciferous vegetables, has been reported as a promising anticancer
phytochemical, but the underlying molecular mechanism is not completely
elucidated. In the present study we report a novel regulation of autophagy by DIM
in human gastric cancer cells. We found that DIM dose-dependently inhibited the
growth of gastric cancer cells in vitro and in vivo. Moreover, ATG5 and LC3 were
activated by DIM in gastric cancer cells. Furthermore, miR-30e was down-regulated
by DIM and miR-30e targeted the 3'-UTR of ATG5 to inhibit its translation.
Overall, these results suggest that DIM may through the miR-30e-ATG5 modulating
autophagy inhibit the proliferation of gastric cancer cells.

DOI: 10.1016/j.bcp.2016.06.018
PMID: 27372603  [Indexed for MEDLINE]

Biochem Pharmacol. 2017 Jan 1;123:1-7. doi: 10.1016/j.bcp.2016.06.015. Epub 2016
Jun 25.

From dioxin toxicity to putative physiologic functions of the human Ah receptor
in homeostasis of stem/progenitor cells.

Bock KW(1).

Author information:
(1)Department of Toxicology, Institute of Experimental and Clinical Pharmacology
and Toxicology, Wilhelmstrasse 56, D-72074 Tübingen, Germany. Electronic address:
[email protected].

Despite decades of intensive research physiologic Ah receptor (AHR) functions are
not yet elucidated. Challenges include marked species differences and dependence
of AHR function on the cell type and cellular context. Hints to physiologic
functions may be derived (i) from feedback loops between endogenous ligands and
substrates of major target enzymes such as CYP1A1 and UGT1A1, and (ii) from
dioxin toxicity in human individuals. For example, dioxin-mediated chloracne is
probably due to dysregulated homeostasis of sebocyte stem/progenitor cells.
Dioxin-mediated inflammatory responses may be due to complex dysregulation of
hematopoiesis. Comparison of AHR functions with those of PXR and its target
enzyme CYP3A4 may be helpful to emphasize AHR functions in specialized cells: PXR
is known to be mainly involved in regulation of systemic metabolism of endo- and
xenobiotics. However, AHR may be mostly controlling local homeostasis of signals
in specialized cells such as stem/progenitor cells. Accumulating evidence
suggests that knowledge about physiologic AHR functions may stimulate drug
development.

DOI: 10.1016/j.bcp.2016.06.015
PMID: 27349986  [Indexed for MEDLINE]

Pharm Biol. 2016 Dec;54(12):3164-3168. Epub 2016 Jun 16.

Development of novel application of 3,3'-diindolylmethane: sensitizing multidrug
resistance human breast cancer cells to γ-irradiation.

Wang W(1), Lv M(1), Wang Y(1), Zhang J(1).

Author information:
(1)a Department of Blood Biopharmaceuticals and Viral Detection , Institute of
Transfusion Medicine, The Academy of Military Medical Sciences , Beijing , P.R.
China.

CONTEXT: Multidrug resistance (MDR) is known as a major obstacle to effective
cancer therapy. The effects of irradiation on MDR in cancer cells had rarely been
reported.
OBJECTIVE: The effect of 3,3'-diindolylmethane (DIM) sensitizing MDR human breast
carcinoma to γ-irradiation was investigated.
MATERIALS AND METHODS: MCF-7/ADR cells were exposed to different concentrations
of DIM (0-30 μM) for 48 or 2 h before IR (γ-Co(60), 10 Gy, room temperature) then
cultured for 48 h. Cell survival was determined by MTT assay. Intracellular
reactive oxygen spices (ROS) induced by DIM (20 and 30 μM, 2 h before
irradiation) was measured by flow cytometry. Propidium iodide staining assay was
used for cell cycle distribution studies; cell apoptosis was measured by flow
cytometry and confocal microscopy.
RESULTS: DIM (20 and 30 μM, 2 h before irradiation) sensitized MCF-7/ADR cells to
IR with survival rates decreased from 100% to 79% and 63%, respectively. DIM
combined with γ-radiation demonstrated that the activity of G2/M phase cell cycle
arresting with percentages enhanced from 9% to 49% and 52%. DIM can increase
intracellular ROS generation by 1.45- and 1.55-times compared to control group.
Significantly enhanced radiation-induced apoptosis by DIM was also observed.
DISCUSSION AND CONCLUSION: These data provide a rationale for the use of DIM as a
promising radio-sensitizer to MDR cancer cells.

DOI: 10.1080/13880209.2016.1192198
PMID: 27307186

Nutr Rev. 2016 Jul;74(7):432-43. doi: 10.1093/nutrit/nuw010. Epub 2016 May 31.

Chemopreventive properties of 3,3'-diindolylmethane in breast cancer: evidence
from experimental and human studies.

Thomson CA(1), Ho E(2), Strom MB(2).

Author information:
(1)Cynthia A. Thomson is with the Mel & Enid Zuckerman College of Public Health,
the University of Arizona Cancer Center, and the Department of Nutritional
Sciences, University of Arizona, Tucson, Arizona, USA. Emily Ho is with the Moore
Family Center for Whole Grain Foods, Nutrition and Preventive Health, College of
Public Health and Human Sciences, Oregon State University, Corvallis, Oregon, and
the Linus Pauling Institute, Oregon State University, Corvallis, Oregon, USA.
Meghan B. Strom is with the Department of Nutritional Sciences, University of
Arizona, Tucson, Arizona, USA. [email protected].
(2)Cynthia A. Thomson is with the Mel & Enid Zuckerman College of Public Health,
the University of Arizona Cancer Center, and the Department of Nutritional
Sciences, University of Arizona, Tucson, Arizona, USA. Emily Ho is with the Moore
Family Center for Whole Grain Foods, Nutrition and Preventive Health, College of
Public Health and Human Sciences, Oregon State University, Corvallis, Oregon, and
the Linus Pauling Institute, Oregon State University, Corvallis, Oregon, USA.
Meghan B. Strom is with the Department of Nutritional Sciences, University of
Arizona, Tucson, Arizona, USA.

Diet is a modifiable factor associated with the risk of several cancers, with
convincing evidence showing a link between diet and breast cancer. The role of
bioactive compounds of food origin, including those found in cruciferous
vegetables, is an active area of research in cancer chemoprevention. This review
focuses on 3,3'-diindolylmethane (DIM), the major bioactive indole in crucifers.
Research of the cancer-preventive activity of DIM has yielded basic mechanistic,
animal, and human trial data. Further, this body of evidence is largely supported
by observational studies. Bioactive DIM has demonstrated chemopreventive activity
in all stages of breast cancer carcinogenesis. This review describes current
evidence related to the metabolism and mechanisms of DIM involved in the
prevention of breast cancer. Importantly, this review also focuses on current
evidence from human observational and intervention trials that have contributed
to a greater understanding of exposure estimates that will inform recommendations
for DIM intake.

DOI: 10.1093/nutrit/nuw010
PMCID: PMC5059820
PMID: 27261275  [Indexed for MEDLINE]

Toxins (Basel). 2016 May 24;8(6). pii: E162. doi: 10.3390/toxins8060162.

Roles of Dietary Phytoestrogens on the Regulation of Epithelial-Mesenchymal
Transition in Diverse Cancer Metastasis.

Lee GA(1), Hwang KA(2), Choi KC(3).

Author information:
(1)Laboratory of Biochemistry and Immunology, College of Veterinary Medicine,
Chungbuk National University, Cheongju, Chungbuk 361-763, Korea.
[email protected].
(2)Laboratory of Biochemistry and Immunology, College of Veterinary Medicine,
Chungbuk National University, Cheongju, Chungbuk 361-763, Korea.
[email protected].
(3)Laboratory of Biochemistry and Immunology, College of Veterinary Medicine,
Chungbuk National University, Cheongju, Chungbuk 361-763, Korea. [email protected].

Epithelial-mesenchymal transition (EMT) plays a key role in tumor progression.
The cells undergoing EMT upregulate the expression of cell motility-related
proteins and show enhanced migration and invasion. The hallmarks of EMT in cancer
cells include changed cell morphology and increased metastatic capabilities in
cell migration and invasion. Therefore, prevention of EMT is an important tool
for the inhibition of tumor metastasis. A novel preventive therapy is needed,
such as treatment of natural dietary substances that are nontoxic to normal human
cells, but effective in inhibiting cancer cells. Phytoestrogens, such as
genistein, resveratrol, kaempferol and 3,3'-diindolylmethane (DIM), can be raised
as possible candidates. They are plant-derived dietary estrogens, which are found
in tea, vegetables and fruits, and are known to have various biological
efficacies, including chemopreventive activity against cancers. Specifically,
these phytoestrogens may induce not only anti-proliferation, apoptosis and cell
cycle arrest, but also anti-metastasis by inhibiting the EMT process in various
cancer cells. There have been several signaling pathways found to be associated
with the induction of the EMT process in cancer cells. Phytoestrogens were
demonstrated to have chemopreventive effects on cancer metastasis by inhibiting
EMT-associated pathways, such as Notch-1 and TGF-beta signaling. As a result,
phytoestrogens can inhibit or reverse the EMT process by upregulating the
expression of epithelial phenotypes, including E-cadherin, and downregulating the
expression of mesenchymal phenotypes, including N-cadherin, Snail, Slug, and
vimentin. In this review, we focused on the important roles of phytoestrogens in
inhibiting EMT in many types of cancer and suggested phytoestrogens as prominent
alternative compounds to chemotherapy.

DOI: 10.3390/toxins8060162
PMCID: PMC4926129
PMID: 27231938

J Org Chem. 2016 May 20;81(10):3994-4001. doi: 10.1021/acs.joc.6b00106. Epub 2016
May 11.

Access to Indole Derivatives from Diaryliodonium Salts and 2-Alkynylanilines.

Li P(1), Weng Y(1), Xu X(1), Cui X(1).

Author information:
(1)Key Laboratory of Xiamen Marine and Gene Drugs, Institutes of Molecular
Medicine and School of Biomedical Sciences, Huaqiao University & Engineering
Research Center of Molecular Medicine, Ministry of Education , Xiamen 361021,
China.

An efficient, environmentally friendly, and operationally simple procedure to
1,2-disubstituted indoles from 2-alkynylanilines and diaryliodonium salts has
been developed. This reaction proceeds smoothly under metal-free conditions. The
products obtained could be transferred into 3,3'-diindolylmethane with DMSO
catalyzed by palladium. The isotopic label experiments indicated that the
methylene group in 3,3'-diindolylmethane is derived from DMSO. The diverse
indoles were obtained in up to 90% yield for 28 examples.

DOI: 10.1021/acs.joc.6b00106
PMID: 27156581

J Plant Physiol. 2016 Jun 1;196-197:93-8. doi: 10.1016/j.jplph.2016.03.013. Epub
2016 Apr 13.

Exogenous 3,3'-diindolylmethane increases Brassica napus L. seedling shoot growth
through modulation of superoxide and hydrogen peroxide content.

Gokul A(1), Roode E(1), Klein A(2), Keyster M(3).

Author information:
(1)Environmental Biotechnology Laboratory, Department of Biotechnology,
University of the Western Cape, Private Bag X17, Bellville 7535, South Africa.
(2)Proteomics Research Unit, Department of Biotechnology, University of the
Western Cape, Private Bag X17, Bellville 7535, South Africa.
(3)Environmental Biotechnology Laboratory, Department of Biotechnology,
University of the Western Cape, Private Bag X17, Bellville 7535, South Africa.
Electronic address: [email protected].

Brassica napus L. (cv. AV Garnet) seeds were pre-treated with 15μM
3,3'-diindolylmethane (DIM) to investigate whether DIM could enhance seed
germination. Further treatment of seedlings with 15μM DIM for 14days explored the
effects on seedling shoot growth. Exogenous DIM led to improved germination
percentage, increased seedling shoot lengths, and increased fresh and dry
weights. Furthermore, DIM triggered induction of superoxide radical (O2(-)) and
hydrogen peroxide (H2O2) content however, no change in malondialdehyde (MDA)
content and cell death (assessed with Evans Blue assay) was detected for both the
control and DIM treated seedling shoots. We also observed increases in superoxide
dismutase (SOD) activity and ascorbate peroxidase (APX) activity in response to
exogenous DIM, two fundamental enzymes in the control of reactive oxygen species
(ROS) in plants. These results indicate that exogenous DIM treatment enhances
seed germination and improves seedling shoot growth through possible activation
of a reactive oxygen species signalling pathway involving O2(-) and H2O2 in B.
napus.

DOI: 10.1016/j.jplph.2016.03.013
PMID: 27100938  [Indexed for MEDLINE]

Pathol Oncol Res. 2016 Oct;22(4):747-54. doi: 10.1007/s12253-016-0054-9. Epub
2016 Apr 18.

Diindolylmethane and Lupeol Modulates Apoptosis and Cell Proliferation in
N-Butyl-N-(4-Hydroxybutyl) Nitrosamine Initiated and Dimethylarsinic Acid
Promoted rat Bladder Carcinogenesis.

Prabhu B(1), Sivakumar A(1), Sundaresan S(2).

Author information:
(1)Department of Medical Research, SRM Medical College Hospital Research Centre,
SRM University, Kattankulathur, 603203, Kanchipuram District, Tamilnadu, India.
(2)Department of Medical Research, SRM Medical College Hospital Research Centre,
SRM University, Kattankulathur, 603203, Kanchipuram District, Tamilnadu, India.
[email protected].

Bladder cancer has been shown to resist programmed cell death with altered
expression of both pro-apoptotic and anti-apoptotic proteins. To study is to
investigate the apoptotic properties of Diindolylmethane (DIM) and Lupeol on
N-Butyl-N-(4-hydroxybutyl) Nitrosamine (BBN) initiated and Dimethylarsinic Acid
(DMA) promoted urinary bladder cancer. Sixty male Wistar rats were divided into 6
groups. Group I: Control. Group II: Rats were experimentally developed bladder
carcinogenesis with BBN and DMA. Group III and IV: DIM and lupeol were
administered after BBN treatment for 28 weeks. Group V and VI: DIM and lupeol
alone treatment for 36 weeks. All the experimental rats were maintained and
euthanized after 36 weeks protocol. Urinary bladder tissues were collected and
processed for further investigations. Apoptotis and cell proliferative marker
such as Bax, Bcl-2, caspase-3, caspase-9 and PCNA were quantified using
immunohistochemical analysis. The Immunohistochemical expression of Bax, Bcl-2,
caspase-3, caspase-9 and PCNA were aberrant in BBN + DMA treated tumor group.
Administration of DIM and lupeol inhibited the progression of bladder cancer,
induced the expression of apoptotic Bax, caspase-3, caspase-9 and inhibited the
expression of anti-apoptotic Bcl-2, PCNA in the urinary bladder of rats.
Administration of diindolylmethane and lupeol treatment induces apoptosis and
cellular proliferation by its anti-carcinogenic properties. From our results DIM
and lupeol would be the agent or adjunct for the treatment of bladder
carcinogenesis.

DOI: 10.1007/s12253-016-0054-9
PMID: 27091758  [Indexed for MEDLINE]

Am J Transl Res. 2016 Jan 15;8(1):166-76. eCollection 2016.

Anti-androgenic activity of 3,3'-diindolylmethane in prostatectomy patients.

Hwang C(1), Sethi S(2), Heilbrun LK(3), Gupta NS(4), Chitale DA(4), Sakr WA(2),
Menon M(5), Peabody JO(5), Smith DW(3), Sarkar FH(6), Heath EI(3).

Author information:
(1)Department of Hematology/Oncology, Josephine Ford Cancer Institute, Henry Ford
Health System Detroit, MI, USA.
(2)Department of Pathology, Karmanos Cancer Institute, Wayne State University
School of Medicine Detroit, MI, USA.
(3)Department of Oncology, Karmanos Cancer Institute, Wayne State University
School of Medicine Detroit, MI, USA.
(4)Department of Pathology, Josephine Ford Cancer Institute, Henry Ford Health
System Detroit, MI, USA.
(5)Department of Vattikuti Institute of Urology, Josephine Ford Cancer Institute,
Henry Ford Health System Detroit, MI, USA.
(6)Department of Pathology, Karmanos Cancer Institute, Wayne State University
School of MedicineDetroit, MI, USA; Department of Oncology, Karmanos Cancer
Institute, Wayne State University School of MedicineDetroit, MI, USA.

Consumption of cruciferous vegetables is associated with a decreased risk of developing prostate cancer. Antineoplastic effects of cruciferous vegetables are attributable to bioactive indoles, most prominently, 3, 3'-diindolylmethane (DIM). In addition to effects on proliferation and apoptosis, DIM acts as anantiandrogen in prostate cancer cell lines. This study characterized the effects of prostatic DIM on the androgen receptor (AR) in patients with prostate cancer. Men with localized prostate cancer were treated with a specially formulated DIM
at a dose of 225 mg orally twice daily for a minimum of 14 days. DIM levels and AR activity were assessed at the time of prostatectomy. Out of 28 evaluable patients, 26 (93%) had detectable prostatic DIM levels, with a mean concentration of 14.2 ng/gm. The mean DIM plasma level on DIM therapy was 9.0 ng/mL; levels were undetectable at
baseline and in follow-up samples. AR localization in the prostate was assessed with immunohistochemistry. After DIM therapy, 96% of patients exhibited exclusion of the AR from the cell nucleus. In contrast, in prostate biopsy
samples obtained prior to DIM therapy, no patient exhibited AR nuclear exclusion. Declines in PSA were observed in a majority of patients (71%). Compliance was excellent and toxicity was minimal. In summary, DIM treatment
resulted in reliable prostatic DIM levels and anti-androgenic biologic effects at well tolerated doses. These results support further investigation of DIM as a chemopreventive and therapeutic agent in prostate cancer.

PMCID: PMC4759426
PMID: 27069550

EPMA J. 2016 Apr 2;7:5. doi: 10.1186/s13167-016-0057-3. eCollection 2016.

First results of the double-blind randomized placebo-controlled multicenter
clinical trial of DIM-based therapy designed as personalized approach to reverse
prostatic intraepithelial neoplasia (PIN).

Paltsev M(1), Kiselev V(2), Drukh V(2), Muyzhnek E(3), Kuznetsov I(4), Andrianova
E(4), Baranovskiy P(1).

Author information:
(1)National Research Centre (NRC "Kurchatov Institute"), 1, Akademika Kurchatova
Pl., Moscow, 123182 Russia.
(2)Peoples' Friendship University of Russia, Miklukho-Maklaya St., 6, Moscow,
117198 Russia.
(3)MiraxBioPharma, Closed Joint Stock Company, 12 Kutuzovsky av., 121248 Moscow,
Russia.
(4)IlmixGroup, Closed Joint Stock Company, 12 Kutuzovsky av., 121248 Moscow,
Russia.

BACKGROUND: Targeted pharmacological correction is used extensively in medical
practice today. 3,3'-Diindolylmethane (DIM) is known as a substance with various
anticancer properties. An interim study of the efficacy of a new drug Infemin on
the basis of diindolylmethane (DIM) with improved bioavalability has been
conducted.
METHODS: The clinical trial had a multicenter, randomized, placebo-controlled,
double-blind design and was carried out in two parallel groups. The interim
analysis of data included 21 patients diagnosed with a high-grade prostatic
intraepithelial neoplasia (PIN). Group 1 (11 patients) received Infemin in a dose
of 900 mg of DIM a day, and group 2 (10 patients) received placebo. To assess the
efficacy of therapy, the analysis of morphological index (MI) changes based on
the results of histological examinations of prostate biopsy specimens was
performed, and a proportion of patients with persistent PIN in 12 months after
Infemin initiation was calculated. Researchers also evaluated prostate size,
urodynamic parameters (Qmax, Qave, Vres), IPSS, and QoL (quality of life) indices
and International Index of Erectile Function (IIEF) at 3, 6, 9, and 12 months
after the Infemin administration start.
RESULTS: After 12 months of treatment in the Infemin group, MI decreased from
0.50 to 0.08, while in the placebo group, it increased from 0.27 to 0.58; the
difference between the groups was statistically significant (p = 0.0003,
Mann-Whitney test). In 45.5 % of patients in the Infemin group, a complete
regression of PIN was also observed, while in the placebo group, PIN regression
was not observed in any patients (p = 0.053, Yates' corrected chi-square). Study
results in the Infemin group show improvement of maximal urinary flow rate Qmax
(53.3 % increase compared to the initial value); however, the statistical
significance was not reached (p = 0.180, Mann-Whitney test) due to the small
sample size. Evaluation of other urodynamic parameters, prostate volume, quality
of life, symptoms reflecting urination disorder, and erectile dysfunction
symptoms did not reveal significant differences between the Infemin and placebo
groups either which is probably due to the small sample size.
CONCLUSIONS: The intermediate results of the 21 patients in this multicenter,
randomized, placebo-controlled, double-blind study show that Infemin may be a
promising drug candidate in patients with  high-grade PIN.
TRIAL REGISTRATION: www.chictr.org.cnChiCTR-INR-15007496.

DOI: 10.1186/s13167-016-0057-3
PMCID: PMC4818865
PMID: 27042242

Colloids Surf B Biointerfaces. 2016 Jul 1;143:156-167. doi:
10.1016/j.colsurfb.2016.03.036. Epub 2016 Mar 15.

Ultra-flexible nanocarriers for enhanced topical delivery of a highly lipophilic
antioxidative molecule for skin cancer chemoprevention.

Boakye CHA(1), Patel K(1), Doddapaneni R(1), Bagde A(1), Behl G(2), Chowdhury
N(1), Safe S(3), Singh M(4).

Author information:
(1)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University,
Tallahassee, FL 32307, USA.
(2)Florida A&M College of Pharmacy, USA.
(3)Department of Veterinary Physiology and Pharmacology, Texas A&M University, TX
77843, USA.
(4)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University,
Tallahassee, FL 32307, USA. Electronic address: [email protected].

PURPOSE: In this study, we developed cationic ultra-flexible nanocarriers
(UltraFLEX-Nano) to surmount the skin barrier structure and to potentiate the
topical delivery of a highly lipophilic antioxidative diindolylmethane derivative
(DIM-D) for the inhibition of UV-induced DNA damage and skin carcinogenesis.
METHODS: UltraFLEX-Nano was prepared with
1,2-dipalmitoyl-sn-glycero-3-phosphocholine,
1,2-dioleoyl-3-trimethylammonium-propane, cholesterol and tween-80 by ethanolic
injection method; was characterized by Differential Scanning Calorimetric (DSC),
Fourier Transform Infrared (FT-IR) and Atomic Force Microscopic (phase-imaging)
analyses and permeation studies were performed in dermatomed human skin. The
efficacy of DIM-D-UltraFLEX-Nano for skin cancer chemoprevention was evaluated in
UVB-induced skin cancer model in vivo.
RESULTS: DIM-D-UltraFLEX-Nano formed a stable mono-dispersion (110.50±0.71nm)
with >90% encapsulation of DIM-D that was supported by HPLC, DSC, FT-IR and AFM
phase imaging. The blank formulation was non-toxic to human embryonic kidney
cells. UltraFLEX-Nano was vastly deformable and highly permeable across the
stratum corneum; there was significant (p<0.01) skin deposition of DIM-D for
UltraFLEX-Nano that was superior to PEG solution (13.83-fold).
DIM-D-UltraFLEX-Nano pretreatment delayed the onset of UVB-induced tumorigenesis
(2 weeks) and reduced (p<0.05) the number of tumors observed in SKH-1 mice
(3.33-fold), which was comparable to pretreatment with sunscreen (SPF30). Also,
DIM-D-UltraFLEX-Nano caused decrease (p<0.05) in UV-induced DNA damage
(8-hydroxydeoxyguanosine), skin inflammation (PCNA), epidermal hyperplasia
(c-myc, CyclinD1), immunosuppression (IL10), cell survival (AKT), metastasis
(Vimentin, MMP-9, TIMP1) but increase in apoptosis (p53 and p21).
CONCLUSION: UltraFLEX-Nano was efficient in enhancing the topical delivery of
DIM-D. DIM-D-UltraFLEX-Nano was efficacious in delaying skin tumor incidence and
multiplicity in SKH mice comparable to sunscreen (SPF30).

DOI: 10.1016/j.colsurfb.2016.03.036
PMCID: PMC5371508
PMID: 27003466  [Indexed for MEDLINE]

Food Chem. 2016 Jul 15;203:340-7. doi: 10.1016/j.foodchem.2016.02.079. Epub 2016
Feb 12.

Stability of glucosinolates and glucosinolate degradation products during storage
of boiled white cabbage.

Ciska E(1), Drabińska N(2), Narwojsz A(3), Honke J(2).

Author information:
(1)Department of Chemistry and Biodynamics of Food, Institute of Animal
Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10 Str.,
10-748 Olsztyn, Poland. Electronic address: [email protected].
(2)Department of Chemistry and Biodynamics of Food, Institute of Animal
Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10 Str.,
10-748 Olsztyn, Poland.
(3)Department of Human Nutrition, Faculty of Food Science, University of Warmia
and Mazury, Plac Cieszyński 1, 10-726 Olsztyn, Poland.

The aim of the study was to investigate the effect of storage on the contents of
glucosinolates (GLS) and their degradation products in a boiled white cabbage. A
24h storage at 4 °C resulted in a decrease in GLS content (20-40%, depending on
the cooking time applied) in the edible parts. The most significant losses were
observed for sinigrin (20-45%), and the least for glucobrassicin (12-32%).
Storage had a diversified effect on GLS breakdown products
(indole-3-acetonitrile, indole-3-carbinol, ascorbigen and 3,3'-diindolylmethane
released from glucobrassicin and 4-methylsulfinylbutanenitrile released from
glucoiberin) in the boiled cabbage. The increase in the content of
indole-3-acetonitrile, especially considerable within the first 24h of storage
(and a simultaneous decrease in glucobrassicin) clearly indicates that
degradation of GLS may occur during storage or cooling to 4 °C.

DOI: 10.1016/j.foodchem.2016.02.079
PMID: 26948623  [Indexed for MEDLINE]

Andrologia. 2016 Dec;48(10):1155-1165. doi: 10.1111/and.12554. Epub 2016 Feb 29.

3,3 diindolylmethane leads to apoptosis, decreases sperm quality, affects blood
estradiol 17 β and testosterone, oestrogen (α and β) and androgen receptor levels
in the reproductive system in male rats.

Aksu EH(1), Akman O(1), Ömür AD(1), Karakuş E(2), Can I(3), Kandemir FM(4),
Dorman E(5), Uçar Ö(1).

Author information:
(1)Faculty of Veterinary Medicine, Department of Reproduction and Artificial
Insemination, Atatürk University, Erzurum, Turkey.
(2)Faculty of Veterinary Medicine, Department of Pharmacology, Atatürk
University, Erzurum, Turkey.
(3)Faculty of Medicine, Department of Histology, Kafkas University, Kars, Turkey.
(4)Faculty of Veterinary Medicine, Department of Biochemistry, Atatürk
University, Erzurum, Turkey.
(5)Faculty of Medicine, Biochemistry Laboratories, Atatürk University, Erzurum,
Turkey.

3,3 Diindolylmethane (DIM) is a major digestive product of indole-3 carbinol,
obtained from Brassica family vegetables such as broccoli, cabbage and Brussels
sprouts. This study aimed to investigate the effects of DIM on sperm parameters,
histological structures of testicular tissues, blood testosterone (T) and
estradiol 17-β (E2) in male rats. Thirty-eight male Sprague Dawley rats were
used. Rats were divided into four groups: Group I: referred as Control group,
received corn oil only; Group II: as DIM-10, rats received 10 mg kg(-1) DIM;
Group III: as DIM-50, rats received 50 mg kg(-1) DIM; Group IV: as DIM-100,
received 100 mg kg(-1) DIM during 53 days. Spermatological parameters,
malondialdehyde (MDA) levels of testes and serum T and E2 levels were assayed.
Histopathological examinations of tests were done. DIM caused an increase in MDA
levels. It decreased motility and live sperm rates and increased degeneration of
testicular tissues. While DIM-10 did not affect abnormal sperm rate, higher
concentrations increased the abnormalities. Sperm density was higher in DIM-10
groups when compared to both other groups. Only DIM-50 had an anti-androgenic
effect among all groups. Only, DIM-10 showed anti-estrogenic activity as compared
to higher DIM groups. In conclusion, DIM (i) had side effect on some sperm
characteristics, (ii) increased the MDA levels and (iii) led to histological
degeneration of testicular tissues and apoptosis in a dose-dependent manner.

DOI: 10.1111/and.12554
PMID: 26926141  [Indexed for MEDLINE]

Oncotarget. 2016 Mar 29;7(13):16311-24. doi: 10.18632/oncotarget.7684.

Anti-cancer drug 3,3'-diindolylmethane activates Wnt4 signaling to enhance
gastric cancer cell stemness and tumorigenesis.

Zhu Y(1)(2), Zhang B(1), Gong A(1), Fu H(1), Zhang X(1), Shi H(1), Sun Y(1), Wu
L(1), Pan Z(1), Mao F(1), Zhu W(1), Qian H(1), Xu W(1).

Author information:
(1)Key Laboratory of Laboratory Medicine of Jiangsu Province, The Affiliated
Hospital, School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, P. R.
China.
(2)Department of Clinical Laboratory, Dali Bai Autonomous Prefecture People's
Hospital, Dali, Yunnan, P. R. China.

As a natural health supplement, 3,3'-diindolylmethane (DIM) is proposed as a
preventive and chemotherapeutic agent for cancer by inhibiting cell proliferation
and inducing cell apoptosis. However, we found that in contrary to high level of
DIM (30 μM), low level of DIM (1 μM and 10 μM) obviously promoted gastric cancer
cell growth and migration. In addition, we found that low level of DIM increased
the expression of stemness factors and enhanced the pluripotency of gastric
cancer cells. Low level of DIM promoted gastric cancer progression by inducing
the PORCN-dependent secretion of Wnt4 and the activation of β-catenin signaling.
Wnt4 knockdown reversed the effects of low level of DIM on gastric cancer cells.
The results of in vivo studies showed that gastric cancer cells treated with low
level of DIM (1 μM) grew faster and expressed higher level of Wnt4 than control
cells. Taken together, our findings indicate that low level of DIM activates
autocrine Wnt4 signaling to enhance the progression of gastric cancer, which may
suggest an adverse aspect of DIM in cancer therapy. Our findings will provide a
new aspect for the safety of DIM in its clinical application.

DOI: 10.18632/oncotarget.7684
PMCID: PMC4941316
PMID: 26918831

Arch Toxicol. 2017 Feb;91(2):967-982. doi: 10.1007/s00204-016-1672-4. Epub 2016
Feb 8.

The Brassica-derived phytochemical indolo[3,2-b]carbazole protects against
oxidative DNA damage by aryl hydrocarbon receptor activation.

Faust D(1), Nikolova T(1), Wätjen W(2), Kaina B(1), Dietrich C(3).

Author information:
(1)Institute of Toxicology, University Medical Center of the Johannes
Gutenberg-University Mainz, Obere Zahlbacherstr. 67, 55131, Mainz, Germany.
(2)Institute of Agricultural and Nutritional Sciences, Martin-Luther University
Halle-Wittenberg, Weinbergweg 22, 06120, Halle (Saale), Germany.
(3)Institute of Toxicology, University Medical Center of the Johannes
Gutenberg-University Mainz, Obere Zahlbacherstr. 67, 55131, Mainz, Germany.
[email protected].

Epidemiological studies suggest that a high intake of Brassica vegetables
protects against colon carcinogenesis. Brassica vegetables are rich in
glucosinolates which are hydrolysed during digestion to various products
including indole-3-carbinol. In animal studies, a protective effect of
indole-3-carbinol has been demonstrated in colon carcinogenesis.
Indole-3-carbinol is highly unstable and, therefore, the observed protection
likely results from condensation products of indole-3-carbinol, e.g.
diindolylmethane or indolo[3,2-b]carbazole (ICZ). Interestingly, ICZ is a potent
activator of the aryl hydrocarbon receptor (AhR), a transcription factor known to
mediate toxic effects of environmental pollutants, such as dioxin and polycyclic
aromatic hydrocarbons. Here, we show that ICZ protects against oxidative DNA
damage in various cell lines including the colon carcinoma cell line Caco-2. When
preincubated for 24 h, ICZ decreases DNA single-strand break (SSB) and 8-oxo-dG
formation induced by tertiary-butylhydroperoxide (t-BOOH), hydrogen peroxide or
benzo[a]pyrene. Simultaneous addition of ICZ does not protect against
t-BOOH-induced SSB formation, which disproves a direct radical scavenging effect.
The repair of SSBs was not enhanced, but the data indicate that ICZ attenuates
the ROS level following t-BOOH. The antioxidant response factor Nrf2 was not
activated following ICZ. Functional inhibition of the AhR and AhR-/ARNT-defective
cell lines demonstrate that the AhR/ARNT pathway is mandatory for the observed
ROS defence caused by ICZ, supporting the hypothesis that AhR-mediated regulation
of defence genes is involved. The data point to a hitherto unknown protective
function of ICZ and a novel role of the AhR in the defence against oxidative DNA
damage.

DOI: 10.1007/s00204-016-1672-4
PMID: 26856715  [Indexed for MEDLINE]

Mol Nutr Food Res. 2016 Jun;60(6):1228-38. doi: 10.1002/mnfr.201500889. Epub 2016
May 23.

Research on cruciferous vegetables, indole-3-carbinol, and cancer prevention: A
tribute to Lee W. Wattenberg.

Fujioka N(1), Fritz V(2), Upadhyaya P(1), Kassie F(1), Hecht SS(1).

Author information:
(1)Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
(2)Southern Research and Outreach Center, University of Minnesota, Minneapolis,
MN, USA.

Lee W. Wattenberg, who spent his entire career at the University of Minnesota,
was a true pioneer in the field of chemoprevention. This paper is a tribute to
his groundbreaking research which uncovered the cancer prevention properties of
many dietary compounds, including those discussed here in some
detail-indole-3-carbinol and diindolylmethane. These compounds occur as
glucosinolate conjugates in cruciferous vegetables and are released when one
chews or otherwise macerates the vegetable. They have numerous beneficial effects
including the ability to prevent cancer in laboratory animals treated with
carcinogens. We review some of the early work on indole-3-carbinol and
diindolylmethane which spurred subsequent studies on their efficacy and molecular
mechanisms of prevention. We also present unique data on field conditions that
affect levels of their glucosinolate precursors in vegetables and on the release
of diindolylmethane in people who consume cruciferous vegetables.

DOI: 10.1002/mnfr.201500889
PMID: 26840393

J Pharmacol Exp Ther. 2016 Apr;357(1):177-87.

3,39-Diindolylmethane Ameliorates Staphylococcal Enterotoxin B–Induced Acute Lung
Injury through Alterations in the Expression of MicroRNA that Target Apoptosis
and Cell-Cycle Arrest in Activated T Cells.

Elliott DM, Nagarkatti M, Nagarkatti PS.

3,39-Diindolylmethane (DIM), a natural indole found in cruciferous vegetables,
has significant anti-cancer and anti-inflammatory properties. In this current
study, we investigated the effects of DIM on acute lung injury (ALI) induced by
exposure to staphylococcal enterotoxin B (SEB). We found that pretreatment of
mice with DIM led to attenuation of SEB-induced inflammation in the lungs,
vascular leak, and IFN-g secretion. Additionally, DIM could induce cell-cycle
arrest and cell death in SEB-activated T cells in a concentration-dependent
manner. Interestingly, microRNA (miRNA) microarray analysis uncovered an altered
miRNA profile in lung-infiltrating mononuclear cells after DIM treatment of
SEB-exposed mice. Moreover, computational analysis of miRNA gene targets and
regulation networks indicated that DIM alters miRNA in the cell death and
cell-cycle progression pathways. Specifically, DIM treatment significantly
downregulated several miRNA and a correlative increase associated gene targets.
Furthermore, overexpression and inhibition studies demonstrated that DIM-induced
cell death, at least in part, used miR-222. Collectively, these studies
demonstrate for the first time that DIM treatment attenuates SEB-induced ALI and
may do so through the induction of microRNAs that promote apoptosis and
cell-cycle arrest in SEB-activated T cells.

DOI: 10.1124/jpet.115.226563
PMCID: PMC4809322
PMID: 26818958  [Indexed for MEDLINE]

Mol Nutr Food Res. 2016 Jun;60(6):1251-63. doi: 10.1002/mnfr.201500867. Epub 2016
Feb 16.

The bounty of nature for changing the cancer landscape.

Ahmad A(1), Li Y(1), Sarkar FH(1)(2).

Author information:
(1)Department of Pathology, Wayne State University School of Medicine and
Karmanos Cancer Institute, Detroit, MI, USA.
(2)Department of Oncology, Wayne State University School of Medicine and Karmanos
Cancer Institute, Detroit, MI, USA.

The landscape of cancer has changed considerably in past several years, due
mainly to aggressive screening, accumulation of data from basic and
epidemiological studies, and the advances in translational research. Natural
anticancer agents have always been a part and parcel of cancer research. The
initial focus on natural anticancer agents was in context of their cancer
chemopreventive properties but their ability to selectively target oncogenic
signaling pathways has also been recognized. In light of the rapid advancements
in our understanding of the role of microRNAs, cancer stem cells, and epigenetic
events in cancer initiation and progression, a number of natural anticancer
agents are showing promise in vitro, in vivo as well as in preclinical studies.
Moreover, parent structures of natural agents are being extensively modified with
the hope of improving efficacy, specificity, and bioavailability. In this
article, we focus on two natural agents, 3,3'-diindolylmethane and garcinol,
along with 3,4-difluorobenzo curcumin, a synthetic analog of natural agent
curcumin. We showcase how these anticancer agents are changing cancer landscape
by modulating novel microRNAs, epigenetic factors, and cancer stem cell markers.
These activities are relevant and being appreciated for overcoming drug
resistance and inhibition of metastases, the two overarching clinical challenges
in modern medicine.

DOI: 10.1002/mnfr.201500867
PMID: 26799714

Immune Netw. 2015 Dec;15(6):278-90. doi: 10.4110/in.2015.15.6.278. Epub 2015 Dec
24.

3,3'-Diindolylmethane Inhibits Flt3L/GM-CSF-induced-bone Marrow-derived CD103(+)
Dendritic Cell Differentiation Regulating Phosphorylation of STAT3 and STAT5.

Park JH(1), Choi AJ(1), Kim SJ(1), Jeong SY(1).

Author information:
(1)Department of Biology, Changwon National University, Changwon 51140, Korea.

The intestinal immune system maintains oral tolerance to harmless antigens or
nutrients. One mechanism of oral tolerance is mediated by regulatory T cell
(Treg)s, of which differentiation is regulated by a subset of dendritic cell
(DC)s, primarily CD103(+) DCs. The aryl hydrocarbon receptor (AhR), a
ligand-activated transcription factor, plays an important role in regulating
immunity. The intestines are exposed to various AhR ligands, including endogenous
metabolites and phytochemicals. It was previously reported that AhR activation
induced tolerogenic DCs in mice or in cultures of bone marrow-derived DCs.
However, given the variety of tolerogenic DCs, which type of tolerogenic DCs is
regulated by AhR remains unknown. In this study, we found that AhR ligand
3,3'-diindolylmethane (DIM) inhibited the development of CD103(+) DCs from mouse
bone marrow cells stimulated with Flt3L and GM-CSF. DIM interfered with
phosphorylation of STAT3 and STAT5 inhibiting the expression of genes, including
Id2, E2-2, IDO-1, and Aldh1a2, which are associated with DC differentiation and
functions. Finally, DIM suppressed the ability of CD103(+) DCs to induce Foxp3(+)
Tregs.

DOI: 10.4110/in.2015.15.6.278
PMCID: PMC4700404
PMID: 26770182

J Immunol. 2016 Feb 1;196(3):1108-22. doi: 10.4049/jimmunol.1501727. Epub 2015
Dec 28.

Dietary Indoles Suppress Delayed-Type Hypersensitivity by Inducing a Switch from
Proinflammatory Th17 Cells to Anti-Inflammatory Regulatory T Cells through
Regulation of MicroRNA.

Singh NP(1), Singh UP(1), Rouse M(1), Zhang J(2), Chatterjee S(3), Nagarkatti
PS(1), Nagarkatti M(4).

Author information:
(1)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, SC 29208;
(2)Department of Epidemiology and Biostatistics, University of South Carolina,
Columbia, SC 29208; and.
(3)Department of Environmental Health Sciences, University of South Carolina,
Columbia, SC 29208.
(4)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, SC 29208; [email protected].

Aryl hydrocarbon receptor (AhR) has been shown to have profound influence on T
cell differentiation, and use of distinct AhR ligands has shown that whereas some
ligands induce regulatory T cells (Tregs), others induce Th17 cells. In the
present study, we tested the ability of dietary AhR ligands (indole-3-carbinol
[I3C] and 3,3'-diindolylmethane [DIM]) and an endogenous AhR ligand,
6-formylindolo(3,2-b)carbazole (FICZ), on the differentiation and functions of
Tregs and Th17 cells. Treatment of C57BL/6 mice with indoles (I3C or DIM)
attenuated delayed-type hypersensitivity (DTH) response to methylated BSA and
generation of Th17 cells while promoting Tregs. In contrast, FICZ exacerbated the
DTH response and promoted Th17 cells. Indoles decreased the induction of IL-17
but promoted IL-10 and Foxp3 expression. Also, indoles caused reciprocal
induction of Tregs and Th17 cells only in wild-type (AhR(+/+)) but not in AhR
knockout (AhR(-/-)) mice. Upon analysis of microRNA (miR) profile in draining
lymph nodes of mice with DTH, treatment with I3C and DIM decreased the expression
of several miRs (miR-31, miR-219, and miR-490) that targeted Foxp3, whereas it
increased the expression of miR-495 and miR-1192 that were specific to IL-17.
Interestingly, treatment with FICZ had precisely the opposite effects on these
miRs. Transfection studies using mature miR mimics of miR-490 and miR-1192 that
target Foxp3 and IL-17, respectively, or scrambled miR (mock) or inhibitors
confirmed that these miRs specifically targeted Foxp3 and IL-17 genes. Our
studies demonstrate, to our knowledge for the first time, that the ability of AhR
ligands to regulate the differentiation of Tregs versus Th17 cells may depend on
miR signature profile.

DOI: 10.4049/jimmunol.1501727
PMCID: PMC4724476
PMID: 26712945  [Indexed for MEDLINE]

EPMA J. 2015 Dec 21;6:25. doi: 10.1186/s13167-015-0048-9. eCollection 2015.

Double-blind randomized placebo-controlled multicenter clinical trial (phase IIa)
on diindolylmethane's efficacy and safety in the treatment of CIN: implications
for cervical cancer prevention.

Ashrafian L(1), Sukhikh G(2), Kiselev V(3), Paltsev M(4), Drukh V(3), Kuznetsov
I(5), Muyzhnek E(6), Apolikhina I(2), Andrianova E(7).

Author information:
(1)Russian Scientific Center of Roentgenoradiology of the Ministry of Health of
the Russian Federation, Moscow, 86, Profsouznaya Str., Moscow, 117837 Russia.
(2)Federal State Budget Institution "Research Center for Obstetrics, Gynecology
and Perinatology" of the Ministry of Health of Russian Federation, 4, Akademika
Oparina Str., Moscow, 117198 Russia.
(3)Federal State Autonomous Educational Institution of Higher Education "Peoples'
Friendship University of Russia" (PFUR), 6, Mikluho-Maklaya Str., Moscow, 117198
Russia.
(4)Federal State Budget Institution «Russian Academy of Sciences», 14 Leninsky
av., Moscow, 119991 Russia.
(5)State Budgetary Educational Institution of Higher Professional Education
"Moscow State Medical Stomatological University named after A.I. Evdokimov"
(MSMSU) of the Ministry of Health of Russian Federation, 20 Delegatskaya Str.,
Build. 1, Moscow, 127473 Russia.
(6)CJSC "MiraxBioPharma", 12, Kutuzovsky av., Build. 2, Moscow, 121248 Russia.
(7)CJSC "IlmixGroup", 12, Kutuzovsky av., Build. 2, Moscow, 121248 Russia.

BACKGROUND: The article presents the results of a clinical trial on the efficacy
and safety of a novel pharmaceutical composition in the form of vaginal
suppositories containing diindolylmethane in the course of cervical
intraepithelial neoplasia (CIN) I-II conservative treatment. It offers an
attractive drug therapy for more personalized prevention of cervical cancer.
METHODS: A total of 78 women of reproductive age were included. This was a
multicenter, randomized, placebo-controlled, double-blind, parallel-group trial
with efficacy determined by histological evaluation of cervical biopsies. The
efficacy of active drug treatment (100 and 200 mg/day) in both treatment groups
was significantly higher in comparison with the placebo group, according to the
primary efficacy end point (proportion of patients with complete CIN regression
after 90-180 days of the study drug treatment).
RESULTS: The efficacies were 100.0 % (confidence interval (CI) 95 %:
82.35-100.00 %), 90.5 % (CI 95 %: 69.62-98.83 %), and 61.1 % (CI 95 %:
35.75-82.70 %), for the high dose, low does, and placebo, respectively. Adverse
events in the placebo group were reported in 22 % of patients (CI 95 %:
7.5-43.7 %); in the first treatment group (100 mg/day), adverse events were
reported in 40.0 % of patients (CI 95 %: 21.1-61.3 %); in the second treatment
group (200 mg/day), adverse events were reported in 42.0 % of patients (CI 95 %:
22.1-63.4 %). The differences in side effects between treatment groups treated
with the active drug and placebo were statistically significant. No serious
adverse events were reported in any of the groups.
CONCLUSIONS: Thus, the use of diindolylmethane in the form of intravaginal
suppositories can be effective in patients with CIN I-II and is not accompanied
by clinically significant side effects. This approach could be a better option
for young women with CIN I-II as it takes in attention their reproductive plans.
TRIAL REGISTRATION: ID: ChiCTR-INR-15007497 (2 December 2015).

DOI: 10.1186/s13167-015-0048-9
PMCID: PMC4685602
PMID: 26693258

Curr Drug Metab. 2016;17(4):401-9.

Indole-3-Carbinol (I3C) and its Major Derivatives: Their Pharmacokinetics and
Important Roles in Hepatic Protection.

Wang SQ, Cheng LS, Liu Y, Wang JY, Jiang W(1).

Author information:
(1)Department of Gastroenterology, Zhongshan Hospital, Fudan University, 180
Fenglin Road, Shanghai 200032, China. [email protected].

BACKGROUND: Indoles, including indole-3-carbinol (I3C) and its derivatives, are
the products of glucosinolate hydrolysis catalyzed by the enzyme myrosinase.
Under acidic conditions, I3C polymerizes into 3, 3- diindolylmethane (DIM),
[2-(indol-3-ylmethyl)-indol-3-yl]indol-3-ylmethane (LTr1), 1-(3-hydroxymethyl)-
indolyl-3-indolylmethane (HI-IM) and indolo[3,2b]carbazole (ICZ). Recently, I3C
and its dimer DIM have shown pleiotropic protective effects on chronic liver
injuries, including viral hepatitis, hepatic steatosis, hepatic cirrhosis,
hepatocellular carcinoma, and so on.
METHODS: We reviewed the published papers about the pharmacokinetics of I3C and
its derivatives in vitro and in vivo, and summarized their multiple protective
roles in the processes of chronic liver diseases.
RESULTS: Indoles not only regulate transcriptional factors and their respective
signaling pathways, but also relieve oxidative stress and inhibit the synthesis
of DNA to influence the activation, proliferation and apoptosis of target cells.
Moreover, indoles modulate the enzymes that are relevant to hepatitis viral
replication, lipogenesis, and the metabolism of ethanol and some hepatotoxic
substances to protect the liver. Currently, the immunomodulatory biofunction of
indoles contributes to improving non-alcoholic steatohepatitis. In addition,
indoles also function as the inhibitors of pro-inflammatory cytokines and
chemokines to reduce microbial-induced liver injures.
CONCLUSION: Indoles, especially I3C and DIM as phytochemicals, exert
anti-fibrosis, anti-tumor, anti-oxidant, immunomodulatory, detoxification and
anti-inflammation effects on hepatic protection through pleiotropic mechanism.

PMID: 26651978  [Indexed for MEDLINE]

Food Chem Toxicol. 2016 Jan;87:23-30. doi: 10.1016/j.fct.2015.11.015. Epub 2015
Nov 25.

The estrogenic effect of trigonelline and 3,3-diindolymethane on cell growth in
non-malignant colonocytes.

Yoo G(1), Allred CD(2).

Author information:
(1)Department of Nutrition and Food Science, Texas A&M University, College
Station, TX, United States.
(2)Department of Nutrition and Food Science, Texas A&M University, College
Station, TX, United States. Electronic address: [email protected].

Epidemiological and animal data have demonstrated the protective effects of
estrogen signaling on colon carcinogenesis. Nonetheless, studies have suggested
that estrogen replacement therapy is positively correlated to increased risk of
breast cancer. Therefore, there is considerable interest in investigating novel
phytoestrogens that mimic the protective actions of estrogen in the colon.
Trigonelline (Trig) and 3,3-diindolylmethane (DIM) have been reported as
phytoestrogens in spite of their distinct chemical structures from other
phytoestrogens. Both compounds elicit estrogenic responses without directly
interacting with the binding domain of the estrogen receptor (ER). We examined
the influence of Trig and DIM on non-malignant colonocytes. Both compounds
reduced cell growth of young adult mouse colonocytes (YAMCs). Trig and DIM
induced cell cycle arrest in the G0/G1 phase and enhanced apoptosis in YAMCs. The
inhibitory effect of Trig on cell growth was disrupted by co-treatment of ICI
182,780, an ER antagonist. DIM elevated ER mediated transcriptional activity.
Both compounds changed gene expression related to apoptosis and cell
proliferation in unique ways. In conclusion, Trig and DIM impact cell physiology
and gene expression in YAMCs via novel estrogenic actions and these data suggest
that intake of novel phytoestrogens may activate protective effects of estrogen
signaling in the colon.

DOI: 10.1016/j.fct.2015.11.015
PMID: 26593444  [Indexed for MEDLINE]

Biomed Res Int. 2015;2015:465105. doi: 10.1155/2015/465105. Epub 2015 Oct 22.

3,3'-Diindolylmethane: A Promising Sensitizer of γ-Irradiation.

Wang W(1), Lv M(1), Huangfu C(1), Wang F(1), Zhang J(1).

Author information:
(1)Department of Blood Biopharmaceuticals and Viral Detection, Institute of
Transfusion Medicine, The Academy of Military Medical Sciences, Beijing 100850,
China.

PURPOSE: Radiotherapy is an effective treatment modality in the clinical
treatment of breast cancer. The present work investigated the effect of
3,3'-diindolylmethane (DIM) on γ-irradiation sensitizing human breast carcinoma.
METHODS: Cell survival, intracellular ROS levels, cell cycle distribution, cell
apoptosis, and expression of proteins related to apoptosis were measured with MTT
assays, flow cytometry, and Western blot analysis, respectively.
RESULTS: In vitro DIM plus γ-irradiation arrested the activity of G2/M phase cell
cycle, increased intracellular ROS level, significantly suppressed PARP (poly
ADP-ribose polymerase), and enhanced γ-irradiation-induced apoptosis, thereby
inhibiting the proliferation of MCF-7 cells.
CONCLUSION: These data provide a rationale for the use of DIM as a promising
sensitizer of γ-irradiation.

DOI: 10.1155/2015/465105
PMCID: PMC4633530
PMID: 26579534  [Indexed for MEDLINE]

Oncol Rep. 2016 Feb;35(2):955-61. doi: 10.3892/or.2015.4428. Epub 2015 Nov 16.

ERK signaling mediates long-term low concentration 3,3'-diindolylmethane
inhibited nasopharyngeal carcinoma growth and metastasis: An in vitro and in vivo
study.

Li F(1), Chen C(1), Chen SM(1), Xiao BK(1), Tao ZZ(1).

Author information:
(1)Otolaryngology-Head and Neck Surgery Research Institute, Renmin Hospital of
Wuhan University, Wuhan, Hubei 430060, P.R. China.

It is well known that crucifers have antitumor effects and 3,3'-diindolylmethane
(DIM) is one of the major bioactive components, and the associated molecular
mechanisms in a short-term high-dose manner are widely discussed. However, the
antitumor effects of DIM in a long-term low-dose manner in nasopharyngeal
carcinoma (NPC) has not been reported yet, as to the potential mechanisms in the
human body. In the present study, NPC cells were induced by 20 µmol/l DIM for
over a month, and the proliferation, apoptosis, migration and in vivo metastasis
were investigated. The results showed that DIM significantly reduced the
proliferation and migration; however, changes in apoptosis were not observed. In
vivo study showed the metastasis was significantly reduced. Compared to the
short-term high-dose manner, incomplete similar qualities were observed; next we
explored the possible signal pathway revolved, the ERK signaling showed similar
changes, while the PI3K/Akt, NF-κB, P38, JNK pathways were significantly altered
in the short-term high-dose manner (our previous study) showed no obvious change,
indicating the ERK signaling may be the main effector of DIM.

DOI: 10.3892/or.2015.4428
PMID: 26574660  [Indexed for MEDLINE]

Cell Oncol (Dordr). 2016 Feb;39(1):47-57. doi: 10.1007/s13402-015-0251-7. Epub
2015 Oct 28.

IL6-induced metastasis modulators p-STAT3, MMP-2 and MMP-9 are targets of
3,3'-diindolylmethane in ovarian cancer cells.

Zou M(1), Zhang X(2), Xu C(3).

Author information:
(1)Department of Oncology, The Second Affiliated Hospital, Chongqing Medical
University, 74 Linjiang Road, Chongqing, 400010, China. [email protected].
(2)Department of Oncology, The Second Affiliated Hospital, Chongqing Medical
University, 74 Linjiang Road, Chongqing, 400010, China. [email protected].
(3)Department of Oncology, The Second Affiliated Hospital, Chongqing Medical
University, 74 Linjiang Road, Chongqing, 400010, China. [email protected].

PURPOSE: Ovarian cancer is a highly lethal gynecological malignancy for which the
overall prognosis has remained poor over the past few decades. Interleukin (IL6)
has been found to be a major contributor to the initiation and progression of
ovarian cancer. This cytokine exerts its activity through activation of several
signaling pathways, in particular the signal transducer and activator of
transcription (STAT3) pathway. Here, we aimed at investigating the capacity of a
natural dietary compound found in cruciferous vegetables, i.e.,
3,3'-diindolylmethane (DIM), to target the metastatic phenotype of ovarian cancer
cells through functional p-STAT3.
METHODS: The human ovarian carcinoma-derived cell lines SKOV3 and A2780 were
treated with IL6 and/or DIM and subjected to in vitro proliferation, adhesion,
migration and invasion assays to assess the anti-metastatic and anti-IL6 effects
of DIM, as well as to assess gene expression alterations before and after
shRNA-mediated STAT3 silencing.
RESULTS: We found that DIM inhibits IL6-mediated increases in ovarian cancer cell
adhesion, migration and invasion. These results were corroborated by
shRNA-mediated STAT3 silencing. Through Western blot and ELISA analyses direct
evidence was provided for the capacity of DIM to inhibit ovarian cancer cell
adhesion, migration and invasion, which was found to be associated with
down-regulation of the matrix metalloproteinases MMP-2 and MMP-9.
CONCLUSIONS: From our data we conclude that DIM exhibits an anti-IL6-like
activity by inhibiting p-STAT3 enhanced ovarian cancer cell proliferation and in
vitro metastasis-associated events, i.e., adhesion, migration and invasion. Most
significantly, MMP-2 and MMP-9, which are known to promote and enhance
metastasis, were found to act as targets of DIM. This anti-IL6-like property of
DIM may pave the way for the development of novel ovarian cancer preventive
and/or therapeutic strategies.

DOI: 10.1007/s13402-015-0251-7
PMID: 26510945  [Indexed for MEDLINE]

Med Princ Pract. 2016;25 Suppl 2:11-7. doi: 10.1159/000439307. Epub 2015 Oct 27.

Role of 3,3'-Diindolylmethane in the Treatment of Human Prostate
Cancer: Clinical Experience.

Li Y(1), Sarkar FH.

Author information:
(1)Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, Mich., USA.

Castration-resistant prostate cancer (CRPC) progression after androgen
deprivation therapy shows upregulated expression of androgen receptor (AR) splice
variants, induced epithelial-to-mesenchymal transition phenotypes and enhanced
stem cell characteristics, all of which are associated with resistance to
enzalutamide. Since there is no curative treatment for CRPC, innovative
treatments are urgently needed. In our recent study, we found that resistance to
enzalutamide was partly due to deregulated expression of microRNAs such as
miR-34a, miR-124, miR-27b, miR-320 and let-7, which play important roles in
regulating AR and stem cell marker gene expression that appears to be linked with
resistance to enzalutamide. Importantly, we found that 3,3'-diindolylmethane (DIM) treatment in vitro and in vivo causeddownregulation in the expression of wild-type AR. The AR splice variants, Lin28B
and EZH2, appear to be deregulated through the re-expression of let-7, miR-27b,
miR-320 and miR-34a in human prostate cancer (PCa). DIM administered in
clinical trials was well tolerated, and 93% of patients had detectable prostatic
DIM levels. The inhibitory effects of DIM on AR and AR target gene such as
prostate-specific antigen were also observed in the clinical trial. Our
preclinical and clinical studies provide the scientific basis for a
'proof-of-concept' clinical trial in CRPC patients treated with enzalutamide in
combination with DIM. This strategy could be expanded in future clinical
trials in patients with PCa to determine whether or not they could achieve a
better treatment outcome which could be partly mediated by delaying or preventing
the development of CRPC.

DOI: 10.1159/000439307
PMCID: PMC4848191
PMID: 26501150  [Indexed for MEDLINE]

Chin J Nat Med. 2015 Oct;13(10):730-42. doi: 10.1016/S1875-5364(15)30073-X.

Modulation of signal transduction pathways by natural compounds in cancer.

Ranjan A(1), Fofaria NM(1), Kim SH(2), Srivastava SK(3).

Author information:
(1)Department of Biomedical Sciences and Cancer Biology Center, Texas Tech
University Health Sciences Center, Amarillo, TX 79106, USA.
(2)Cancer Preventive Material Development Research Center, College of Korean
Medicine, Department of Pathology, Kyunghee University, Seoul 131-701, South
Korea. Electronic address: [email protected].
(3)Department of Biomedical Sciences and Cancer Biology Center, Texas Tech
University Health Sciences Center, Amarillo, TX 79106, USA; Cancer Preventive
Material Development Research Center, College of Korean Medicine, Department of
Pathology, Kyunghee University, Seoul 131-701, South Korea. Electronic address:
[email protected].

Cancer is generally regarded as the result of abnormal growth of cells. According
to World Health Organization, cancer is the leading cause of mortality worldwide.
Mother nature provides a large source of bioactive compounds with excellent
therapeutic efficacy. Numerous phytochemicals from nature have been investigated
for anticancer properties. In this review article, we discuss several natural
compounds, which have shown anti-cancer activity. Natural compounds induce cell
cycle arrest, activate intrinsic and extrinsic apoptosis pathways, generate
Reactive Oxygen Species (ROS), and down-regulate activated signaling pathways,
resulting in inhibition of cell proliferation, progression and metastasis of
cancer. Several preclinical studies have suggested that natural compounds can
also increase the sensitivity of resistant cancers to available chemotherapy
agents. Furthermore, combining FDA approved anti-cancer drugs with natural
compounds results in improved efficacy. On the basis of these exciting outcomes
of natural compounds against several cancer types, several agents have already
advanced to clinical trials. In conclusion, preclinical results and clinical
outcomes against cancer suggest promising anticancer efficacy of agents from
natural sources.

DOI: 10.1016/S1875-5364(15)30073-X
PMID: 26481373  [Indexed for MEDLINE]

Mol Neurobiol. 2016 Oct;53(8):5591-606. doi: 10.1007/s12035-015-9471-0. Epub 2015
Oct 17.

Selective Aryl Hydrocarbon Receptor Modulator 3,3'-Diindolylmethane Impairs AhR
and ARNT Signaling and Protects Mouse Neuronal Cells Against Hypoxia.

Rzemieniec J(1), Litwa E(1), Wnuk A(1), Lason W(1), Krzeptowski W(2), Kajta M(3).

Author information:
(1)Department of Experimental Neuroendocrinology, Institute of Pharmacology,
Polish Academy of Sciences, 12 Smetna Street, 31-343, Krakow, Poland.
(2)Department of Cell Biology and Imaging, Confocal Microscopy Laboratory,
Institute of Zoology, Jagiellonian University, 9 Gronostajowa Street, 30-387,
Krakow, Poland.
(3)Department of Experimental Neuroendocrinology, Institute of Pharmacology,
Polish Academy of Sciences, 12 Smetna Street, 31-343, Krakow, Poland.
[email protected].

The neuroprotective potential of 3,3'-diindolylmethane (DIM), which is a
selective aryl hydrocarbon receptor modulator, has recently been shown in
cellular and animal models of Parkinson's disease and lipopolysaccharide-induced
inflammation. However, there are no data concerning the protective capacity and
mechanisms of DIM action in neuronal cells exposed to hypoxia. The aim of the
present study was to investigate the neuroprotective potential of DIM against the
hypoxia-induced damage in mouse hippocampal cells in primary cultures, with a
particular focus on DIM interactions with the aryl hydrocarbon receptor (AhR),
its nuclear translocator ARNT, and estrogen receptor β (ERβ). In the present
study, 18 h of hypoxia induced apoptotic processes, in terms of the mitochondrial
membrane potential, activation of caspase-3, and fragmentation of cell nuclei.
These effects were accompanied by substantial lactate dehydrogenase release and
neuronal cell death. The results of the present study demonstrated strong
neuroprotective and anti-apoptotic actions of DIM in hippocampal cells exposed to
hypoxia. In addition, DIM decreased the Ahr and Arnt mRNA expression and
stimulated Erβ mRNA expression level. DIM-induced mRNA alterations were mirrored
by changes in protein levels, except for ERβ, as detected by ELISA, Western
blotting, and immunofluorescence labeling. We also demonstrated that DIM
decreased the expression of AhR-regulated CYP1A1. Using specific siRNAs, we
provided evidence that impairment of AhR and ARNT, but not ERβ plays a key role
in the neuroprotective action of DIM against hypoxia-induced cell damage. This
study may have implication for identifying new agents that could protect neurons
against hypoxia by targeting AhR/ARNT signaling.

DOI: 10.1007/s12035-015-9471-0
PMID: 26476840

Curr Pharmacol Rep. 2015 May;1(3):179-196. Epub 2015 Jan 30.

Dietary Glucosinolates Sulforaphane, Phenethyl Isothiocyanate,
Indole-3-Carbinol/3,3'-Diindolylmethane: Anti-Oxidative Stress/Inflammation,
Nrf2, Epigenetics/Epigenomics and In Vivo Cancer Chemopreventive Efficacy.

Fuentes F(1), Paredes-Gonzalez X(1), Kong AT(1).

Author information:
(1)Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The
State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey
08854, USA.

Glucosinolates are a group of sulfur-containing glycosides found in many plant
species, including cruciferous vegetables such as broccoli, cabbage, brussels
sprouts, and cauliflower. Accumulating evidence increasingly supports the
beneficial effects of dietary glucosinolates on overall health, including as
potential anti-cancer agents, because of their role in the prevention of the
initiation of carcinogenesis via the induction of cellular defense
detoxifying/antioxidant enzymes and their epigenetic mechanisms, including
modification of the CpG methylation of cancer-related genes, histone modification
regulation and changes in the expression of miRNAs. In this context, the defense
mechanism mediated by Nrf2-antioxidative stress and anti-inflammatory signaling
pathways can contribute to cellular protection against oxidative stress and
reactive metabolites of carcinogens. In this review, we summarize the cancer
chemopreventive role of naturally occurring glucosinolate derivatives as
inhibitors of carcinogenesis, with particular emphasis on specific molecular
targets and epigenetic alterations in in vitro and in vivo human cancer animal
models.

DOI: 10.1007/s40495-015-0017-y
PMCID: PMC4596548
PMID: 26457242

Curr Med Chem. 2015;22(38):4412-33.

A Review of Bisindolylmethane as an Important Scaffold for Drug Discovery.

Imran S, Taha M(1), Ismail NH.

Author information:
(1)Atta-ur-Rahman Institute for Natural Product Discovery, Faculty of Applied
Science, Universiti Teknologi Mara P.O. Box 42300, Puncak Alam, Malaysia.
[email protected].

Bisindolylmethane and its derivatives are pharmacologically active and applicable
in the field of pharmaceutical chemistry. Bisindolylmethanes have a variety of
biological activities such as antihyperglycemic, antiinflammatory, antibacterial,
anticancer, and antileishmanial activities, including enzyme inhibition activity.
They play a crucial role in many diseases especially anticancer activity.
Modifying their structure had proven to be useful in the search of new
therapeutic agents. Extensive research carried out on bisindolylmethane and its
derivatives shows that they are pharmacologically significant. The present review
focuses on the pharmacological profile of bisindolylmethane derivatives. This
review includes the current literature with an update of research findings as
well as the perspectives that they hold for future research.

PMID: 26438249  [Indexed for MEDLINE]

Chemistry. 2015 Nov 9;21(46):16585-92. doi: 10.1002/chem.201502932. Epub 2015 Sep
29.

Exploration of the Chiral Recognition of Sugar-Based Diindolylmethane Receptors:
Anion and Receptor Structures.

Granda JM(1), Jurczak J(2).

Author information:
(1)Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52,
01-224 Warsaw (Poland).
(2)Institute of Organic Chemistry, Polish Academy of Sciences, Kasprzaka 44/52,
01-224 Warsaw (Poland). [email protected].

In this study, we have conducted a systematic investigation of the chiral
recognition of carboxylic anions by D-glucuronic acid/diindolylmethane receptors.
We investigate the influence of the anion structure on chiral recognition in the
diindolylmethane/glucuronic acid-based receptor 1 a. We found that presence of an
additional hydrogen-bond donor at the α position to the carboxylic function is
essential for effective chiral differentiation in these systems. Furthermore, we
present a synthetic procedure that allows for the synthesis of sugar-decorated
receptors that possess a modified substituent at the anomeric position. Four new
receptors 1 b-e have been synthesized, and their chiral-discrimination ability
toward model carboxylates is studied. The obtained results show that the chiral
recognition of these receptors can be fine-tuned by incorporation of a proper
substituent into the receptor structure.

DOI: 10.1002/chem.201502932
PMID: 26418487

Mol Cancer. 2015 Sep 17;14:172. doi: 10.1186/s12943-015-0443-9.

Aryl hydrocarbon receptor-microRNA-212/132 axis in human breast cancer suppresses
metastasis by targeting SOX4.

Hanieh H(1).

Author information:
(1)Laboratory of Physiology, Biological Sciences Department, College of Science,
King Faisal University, Faisal Bin Fahd road, Hofuf, 31982, Ahsaa, Saudi Arabia.
[email protected].

BACKGROUND: MicroRNAs (miRNAs) are a class of short non-coding RNAs that pave a
new avenue for understanding immune responses and cancer progression. Although
the miRNAs are involved in breast cancer development, their axis with the
transcription factors that show therapeutic potential in breast cancer is largely
unknown. Previous studies showed anti-metastatic roles of agonist-activated aryl
hydrocarbon receptor (Ahr) in various breast cancer cell lines. Recently, we
demonstrated that agonist-activated Ahr induced a highly conserved miRNA cluster,
named miR-212/132, in murine cellular immune compartment. Therefore, current
study was performed to examine if this miRNA cluster mediates the anti-metastatic
properties of Ahr agonists.
METHODS: The expression of miR-212/132 cluster and coding genes were examined by
real-time PCR, and the protein levels were detected by western blot. The
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3'-diindolylmethane (DIM) were
used to activate Ahr in MDA-MB-231 and T47D breast cancer cells. Chromatin
immunoprecipitation (ChIP) assay was used to identify the binding site(s) for Ahr
on miR-212/132 promoter. For prediction of potentially target gene of the miRNA
cluster, bioinformatics analysis was carried out, and to test targeting,
luciferase activity was quantified. Besides, biological effects of
Ahr-miR-212/132 axis were examined in vitro by cell migration, expansion and
invasion, and examined in vivo by orthotopic model of spontaneous metastasis.
RESULTS: The miR-212/132 cluster was transcriptionally activated in MDA-MB-231
and T47D cells by TCDD and DIM, and this activation was regulated by Ahr. A
reciprocal correlation was identified between Ahr agonists-induced miR-212/132
and the pro-metastatic SRY-related HMG-box4 (SOX4), and a new specific binding
sites for miR-212/132 were identified on the untranslated region (3'UTR) of SOX4.
Interestingly, miR-212/132 over-expression showed direct anti-migration,
anti-expansion and anti-invasion properties, and an inhibition of the miRNA
cluster mitigated the anti-invasive properties of TCDD and DIM. Further in vivo
studies demonstrated that the Ahr-miR-212/132-SOX4 module was induced by Ahr
activation.
CONCLUSION: Taken together, the findings provide the first evidences of the
synergistic anti-metastatic properties of miR-212/132 cluster through suppression
of SOX4. Also, current study suggest a new miRNA-based mechanism elucidating the
anti-metastatic properties of Ahr agonists, suggesting possibility of using
miR-212/132 to control metastasis in breast cancer patients.

DOI: 10.1186/s12943-015-0443-9
PMCID: PMC4573482
PMID: 26377202  [Indexed for MEDLINE]

BMC Cancer. 2015 Sep 3;15:611. doi: 10.1186/s12885-015-1627-9.

Sulindac, 3,3'-diindolylmethane and curcumin reduce carcinogenesis in the Pirc
rat, an Apc-driven model of colon carcinogenesis.

Femia AP(1), Soares PV(2), Luceri C(3), Lodovici M(4), Giannini A(5), Caderni
G(6).

Author information:
(1)NEUROFARBA Department, Section of Pharmacology and Toxicology, University of
Florence, 6 Viale Pieraccini, 50139, Florence, Italy. [email protected].
(2)Department of Pathology and Legal Medicine, Faculty of Medicine of Ribeirão
Preto, University of São Paulo, São Paulo, Brazil. [email protected].
(3)NEUROFARBA Department, Section of Pharmacology and Toxicology, University of
Florence, 6 Viale Pieraccini, 50139, Florence, Italy. [email protected].
(4)NEUROFARBA Department, Section of Pharmacology and Toxicology, University of
Florence, 6 Viale Pieraccini, 50139, Florence, Italy. [email protected].
(5)Department of Pathology, General Hospital of Prato, Prato, Italy.
[email protected].
(6)NEUROFARBA Department, Section of Pharmacology and Toxicology, University of
Florence, 6 Viale Pieraccini, 50139, Florence, Italy. [email protected].

BACKGROUND: Recently, we showed that Sulindac (SU; 320 ppm) reduces precancerous
lesions in the colon of Pirc rats, mutated in the Apc gene. Surprisingly,
previous data in Apc-mutated mice showed that SU, with reported efficacy in
Familial Adenomatous Polyposis (FAP), increases colon carcinogenesis. Therefore,
we assessed the effect of SU 320 ppm in a long-term carcinogenesis experiment in
Pirc rats. Moreover, since side effects of SU hamper its chronic use and a
combination of drugs could be more effective and less toxic than single agents,
we also studied whether two natural compounds, 3,3'-diindolylmethane (DIM;
250 ppm) and curcumin (CUR; 2000 ppm), with or without lower doses of SU could
affect carcinogenesis
METHODS: Pirc rats were fed an AIN76 diet containing SU, DIM and CUR and
sacrificed at 8 months of age to measure intestinal tumours. Apoptosis and
proliferation in the normal colon mucosa, as well as gene expression profile were
studied
RESULTS: Colon tumours were significantly reduced by SU 320 ppm (62 % reduction
over Controls), by DIM and CUR without or with SU 80 and 160 ppm (50, 53 and 58 %
reduction, respectively) but not by SU 80 ppm alone. Total tumours (colon and
small intestine) were reduced by SU (80 and 320 ppm) and by DIM and CUR.
Apoptosis in the normal mucosa was significantly increased by SU 320 ppm, and
slightly increased by DIM and CUR with or without SU. A slight reduction in
Survivin-Birc5 expression was observed with all the treatments compared to
Controls. Proliferative activity was not varied
CONCLUSIONS: The results on SU reinforce the validity of Pirc rats to identify
chemopreventive products. Moreover, the efficacy of the DIM and CUR combination
to lower colon tumours, suggests an alternative strategy to be exploited in
patients at risk.

DOI: 10.1186/s12885-015-1627-9
PMCID: PMC4559292
PMID: 26335331  [Indexed for MEDLINE]

Int J Clin Exp Pathol. 2015 Jun 1;8(6):6505-11. eCollection 2015.

Unfolded protein response mediated JNK/AP-1 signal transduction, a target for
ovarian cancer treatment.

Zheng GF(1), Cai Z(1), Meng XK(2), Zhang Y(2), Zhu W(2), Pang XY(2), Dou L(2).

Author information:
(1)The Bone Marrow Transplantation Center & Multiple Myeloma Treatment Center,
The First Affiliated Hospital of Medical College, Zhejiang University Hangzhou
310003, Zhejiang, China.
(2)Department of Gynecology, The First Hospital of China Medical University
Shenyang 110001, Liaoning, China.

Researches have revealed several stressors, which could activate unfolded protein
response (UPR) in cells. However, the survival or death pathway was determined by
the duration of UPR exposure. Based on the UPR mediated death pathway, our study
was aimed to investigate role of UPR on c-Jun N-terminal kinase (JNK)/activator
protein-1 (Ap-1) signal transduction in diindolylmethane (DIM) treated ovarian
cancer cell lines. Activation of UPR proteins, UPR mediated apoptotic signaling
proteins and expression level of EpCAM, JNK, Ap-1, Caspase-3 and Bcl-2 were
measured. Protein and gene expression, transcription factor activity, and protein
phosphorylation were measured using standard molecular biology techniques. Our
results demonstrated DIM treatment had significantly increased the expression of
Endoplasmic reticulum (ER) stress regulators such as Bip, IRE1, CHOP and
activation of UPR related apoptotic proteins in ovarian cancer cells. Decreased
expression of EpCAM and activity of AP-1 transcription factor were observed in
DIM treated cells. The pharmacologic inhibitors of the JNK signal transduction
pathway, suggest that the impact of EpCAM expression on AP-1 transcription factor
activity is mediated through the JNK pathway. Taken together, these results
suggest that UPR mediated JNK/Ap-1 signal transduction has a significant role in
the regulation of apoptosis in human ovarian cancer cells, and is a potential
molecular target to enhance sensitivity of ovarian cancer to chemotherapy.

PMCID: PMC4525862
PMID: 26261528  [Indexed for MEDLINE]

Hum Exp Toxicol. 2016 Jun;35(6):685-92. doi: 10.1177/0960327115597985. Epub 2015
Aug 6.

Antiproliferative and anti-inflammatory properties of diindolylmethane and lupeol
against N-butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in
experimental rats.

Prabhu B(1), Balakrishnan D(1), Sundaresan S(2).

Author information:
(1)Department of Medical Research, SRM Medical College Hospital and Research
Centre, SRM University, Kattankulathur, Kanchipuram District, Tamil Nadu, India.
(2)Department of Medical Research, SRM Medical College Hospital and Research
Centre, SRM University, Kattankulathur, Kanchipuram District, Tamil Nadu, India
[email protected].

INTRODUCTION: Chemoprevention may involve perturbation of a variety of steps in
tumor initiation, promotion, and progression.
OBJECTIVE: To investigate the antiproliferative and anti-inflammatory potential
effects of diindolylmethane (DIM) and lupeol on experimental bladder
carcinogenesis.
METHODS: Sixty healthy male Wistar rats were selected and randomly divided into
six groups, with 10 rats in each group. Group I: control; group II:
N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN; 150 mg/gavage/twice a week) for 8
weeks, and then they were given 100 ppm concentrations of dimethylarsenic acid
(DMA) in the drinking water for 28 weeks; group III: BBN + DMA + DIM (5 mg/kg
body weight (b.w.)/day) treatment was started after BBN treatment, and it was
orally administered for 28 weeks); group IV: BBN + DMA + lupeol (50 mg/kg
b.w./day) treatment was started after BBN treatment, and it was orally
administered for 28 weeks); and groups V and VI: DIM and lupeol treatment alone
for 36 weeks. Bladder tissues were collected after 36th week study protocol for
further analysis.
RESULTS: Our results revealed that DIM and lupeol treatment showed inhibition of
tumor growth in the bladder by histopathological confirmations as well as
significantly (p < 0.001) increased the expression of phosphotensin (PTEN) and
significantly (p < 0.001) decreased the expression of tumor necrosis factor α,
nuclear factor κβ (p65) were quantified using Western blot analysis. DIM and
lupeol treatment significantly (p < 0.001) decreased the levels of Cox-2 in
bladder tissue samples and NMP 22 in urine samples were quantified using
enzyme-linked immunosorbent assay method.
CONCLUSION: Preventive DIM and lupeol administration act as potent Cox-2
inhibitors, which activates the tumor suppressor protein PTEN against
experimental bladder carcinogenesis by antiproliferative and anti-inflammatory
properties.

DOI: 10.1177/0960327115597985
PMID: 26251508  [Indexed for MEDLINE]

Mater Sci Eng C Mater Biol Appl. 2015 Nov 1;56:233-40. doi:
10.1016/j.msec.2015.06.016. Epub 2015 Jun 12.

Desorption of 3,3'-diindolylmethane from imprinted particles: An impact of
cross-linker structure on binding capacity and selectivity.

Klejn D(1), Luliński P(1), Maciejewska D(2).

Author information:
(1)Department of Organic Chemistry, Faculty of Pharmacy, Medical University of
Warsaw, Banacha 1, 02-097 Warsaw, Poland.
(2)Department of Organic Chemistry, Faculty of Pharmacy, Medical University of
Warsaw, Banacha 1, 02-097 Warsaw, Poland. Electronic address:
[email protected].

Here, seven cross-linkers (six polar diacrylates or dimethacrylates of different
lengths between double bonds, and one aromatic-divinylbenzene) were used to
examine the impact of the cross-linker on binding capacity and selectivity of
3,3'-diindolylmethane (DIM) imprinted material. DIM participates in the
suppression of viability of human ovarian and human breast cancer cell lines, but
has low bioavailability. The investigations of novel imprinted polymer matrices
for improvement of DIM release could allow to utilize not only a potency of DIM
but also similar alkaloids, which are the important compounds with
pharmacological activity. The bulk, thermal radical copolymerization of the
cross-linkers in the presence of 3,3'-diindolylmethane (the template) and
allylamine (the functional monomer) in dimethyl sulfoxide or in carbon
tetrachloride (porogens) was carried out. The binding capacities of imprinted and
non-imprinted polymers were compared, and two polymers (these were prepared using
ethylene glycol dimethacrylate and polyethylene glycol dimethacrylate as the
cross-linkers) with the highest selectivity and binding capacity were selected to
desorption test. The desorption profile of polymer prepared using polyethylene
glycol dimethacrylate as the cross-linker revealed sustained release of
3,3'-diindolylmethane, and this system was selected for further optimization of
the cross-linker amounts. The morphology and structure of the selected particles
were analyzed using SEM micrographs, (13)C CP/MAS NMR spectroscopy, and BET
measurements. The desorption of 3,3'-diindolylmethane from
poly(allylamine-co-polyethylene glycol dimethacrylate) particles was in
accordance with pseudo-second-order kinetics and the simplified Higuchi model
indicated the diffusion controlled release of 3,3'-diindolylmethane.

DOI: 10.1016/j.msec.2015.06.016
PMID: 26249585  [Indexed for MEDLINE]

Int J Oncol. 2015 Sep;47(3):918-26. doi: 10.3892/ijo.2015.3089. Epub 2015 Jul 17.

Genome-wide transcriptome analysis reveals inactivation of Wnt/β-catenin by
3,3'-diindolylmethane inhibiting proliferation of colon cancer cells.

Leem SH(1), Li XJ(2), Park MH(3), Park BH(4), Kim SM(2).

Author information:
(1)Department of Biological Science, Dong-A University, Busan 602-760, Republic
of Korea.
(2)Department of Physiology, Institute for Medical Sciences, Chonbuk National
University Medical School, Jeonju 561-180, Republic of Korea.
(3)Catholic University of Pusan, Busan 609-757, Republic of Korea.
(4)Department of Biochemistry, Institute for Medical Sciences, Chonbuk National
University Medical School, Jeonju 561-180, Republic of Korea.

Multiple genetic and signaling pathway alterations underlie the development of
colon cancer. We utilized genome-wide transcriptome analysis to identify
important gene expression patterns following treatment with 3,3'-diindolylmethane
(DIM), a natural compound derived from cruciferous vegetables, on colon cancer
cells. Statistical analyses of gene expression data from DIM treated cells
revealed that 692 genes were significantly upregulated, while 731 genes were
down-regulated. Putative gene networks showed that several oncogenes (β-catenin,
Myc and FOS) were significantly suppressed by DIM treatment. Using clinical data
from colon cancer patients, activation of β-catenin was found to be significantly
associated with patient prognosis by Kaplan-Meir plot analysis. We validated the
mRNA and protein expression levels of c-Myc, β-catenin, and cyclin D1, all of
which were significantly suppressed after DIM treatment in DLD-1 and HCT116
cells. System level characterization of our findings suggests for the first time
that β-catenin and c-Myc, which are major genes involved in colon carcinogenesis,
were significantly downregulated by DIM treatment in colon cancer cells.
Therefore, targeting Wnt/β-catenin signaling by DIM may be an attractive strategy
for the prevention and treatment of colon cancer.

DOI: 10.3892/ijo.2015.3089
PMID: 26203047  [Indexed for MEDLINE]

Int J Clin Exp Pathol. 2015 May 1;8(5):5121-8. eCollection 2015.

DIM attenuates TGF-β1-induced myofibroblast differentiation in neonatal rat
cardiac fibroblasts.

Li J(1), Zhang W(1), Jiao R(2), Yang Z(1), Yuan Y(1), Wu Q(1), Hu Z(1), Xiang
S(1), Tang Q(1).

Author information:
(1)Department of cardiology, Renmin Hospital of Wuhan University Wuhan 430060,
China ; Cardiovascualar Research Institute of Wuhan University Wuhan 430060,
China.
(2)Department of cardiology, Renmin Hospital of Wuhan University Wuhan 430060,
China ; Cardiovascualar Research Institute of Wuhan University Wuhan 430060,
China ; Department of Xiangyang Hospital, Hubei University of Medicine Xiangyang
441000, China.

3,3'-Diindolylmethane (DIM) is a natural component of cruciferous plants.
Previous studies have shown that DIM has multiple physiological effects including
anti-angiogenic, anti-inflammatory and anti-cancer effect. However, little is
known about the role of DIM on myofibroblast differentiation and extracellular
matrix (ECM) production. This study investigated the effect of DIM on
myofibroblast differentiation and ECM production in neonatal rat cardiac
fibroblasts induced by transforming growth factor β1 (TGF-β1). We found that DIM
blunted TGF-β1 induced conversion of cardiac fibroblast into myofibroblast, and
reduced the mRNA and protein expressions of α-smooth muscle actin (α-SMA).
Furthermore, DIM also significantly decreased the mRNA expression of fibrosis
markers (Collagen I, Collagen III, connective tissue growth factor (CTGF) in
neonatal rat cardiac fibroblasts induced by TGF-β1. DIM attenuated the
phosphorylation AKT and glycogen synthase kinase-3β (GSK-3β) induced by TGF-β1.
Our results showed that DIM was a potential drug to attenuate myofibroblast
differentiation and excessive ECM production induced by TGF-β1 through
down-regulated AKT/GSK-3β signaling pathways.

PMCID: PMC4503079
PMID: 26191207  [Indexed for MEDLINE]

J Pharmacokinet Pharmacodyn. 2015 Aug;42(4):401-8. doi:
10.1007/s10928-015-9421-5. Epub 2015 Jul 3.

Pharmacokinetics and pharmacodynamics of 3,3'-diindolylmethane (DIM) in
regulating gene expression of phase II drug metabolizing enzymes.

Wu TY(1), Huang Y, Zhang C, Su ZY, Boyanapalli S, Khor TO, Wang H, Lin H, Gounder
M, Kagan L, Androulakis IP, Kong AN.

Author information:
(1)Department of Pharmacology, Tzu Chi University, Hualien City, Taiwan.

3,3'-Diindolylmethane (DIM) has been investigated as a potential anti-cancer
chemopreventive agent in many preclinical and clinical studies. In this study, we
sought to characterize the pharmacokinetics of DIM and to build a pharmacokinetic
(PK) and pharmacodynamic (PD) model of the DIM-induced gene expression of phase
II drug metabolizing enzymes (DME), which potentially links DIM's molecular
effects to its in vivo chemopreventive efficacy. DIM (10 mg/kg) was administered
intravenously (i.v.) to male Sprague-Dawley rats and blood samples were collected
at selected time points for 48 h. The plasma concentration of DIM was determined
using a validated HPLC method. The mRNA expression of NQO1, GSTP1 and UGT1A1 in
blood lymphocytes was measured using quantitative PCR. An indirect response model
was employed to relate the concentration of DIM to the expression of the genes
NQO1, GSTP1 and UGT1A1, which were chosen as PD markers for DIM. After i.v.
administration, the plasma concentration of DIM declined quickly, and the
expression of target genes increased significantly, peaking at 1-2 h and then
returning to basal levels after 24 h. The parameters in the PK-PD model were
estimated. The PK-PD model aptly described the time delay and magnitude of gene
expression induced by DIM. Our results indicate that DIM is effective at inducing
various phase II DME, which are capable of detoxify carcinogens. This PK-PD
modeling approach provides a framework for evaluating the acute effects of DIM or
other similar drugs in clinical trials.

DOI: 10.1007/s10928-015-9421-5
PMID: 26138223  [Indexed for MEDLINE]

Oncol Lett. 2015 May;9(5):2393-2397. Epub 2015 Mar 3.

3,3'-diindolylmethane potentiates tumor necrosis factor-related
apoptosis-inducing ligand-induced apoptosis of gastric cancer cells.

Ye Y(1), Miao S(2), Wang Y(3), Zhou J(4), Lu R(5).

Author information:
(1)Department of Preventive Medicine, School of Medical Science and Laboratory
Medicine, Jiangsu University, Zhenjiang, Jiangsu 212001, P.R. China ; Department
of Molecular Cell Biology and Toxicology, Cancer Center, School of Public Health,
Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
(2)Department of Molecular Cell Biology and Toxicology, Cancer Center, School of
Public Health, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China ;
Department of Health Care, The Fourth Affiliated Hospital, Jiangsu University,
Zhenjiang, Jiangsu 212001, P.R. China.
(3)Department of Preventive Medicine, School of Medical Science and Laboratory
Medicine, Jiangsu University, Zhenjiang, Jiangsu 212001, P.R. China.
(4)Department of Molecular Cell Biology and Toxicology, Cancer Center, School of
Public Health, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
(5)Department of Preventive Medicine, School of Medical Science and Laboratory
Medicine, Jiangsu University, Zhenjiang, Jiangsu 212001, P.R. China ; Department
of Public Health Laboratory Science, School of Medical Science and Laboratory
Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) specifically
kills cancer cells without destroying the majority of healthy cells. However,
numerous types of cancer cell, including gastric cancer cells, tend to be
resistant to TRAIL. The bioactive product 3,3'-diindolylmethane (DIM), which is
derived from cruciferous vegetables, is also currently recognized as a candidate
anticancer agent. In the present study, a Cell Counting Kit 8 cell growth assay
and an Annexin V-fluorescein isothiocyanate apoptosis assay were performed to
investigate the potentiating effect of DIM on TRAIL-induced apoptosis in gastric
cancer cells, and the possible mechanisms of this potentiation. The results
obtained demonstrated that, compared with TRAIL or DIM treatment alone,
co-treatment with TRAIL (25 or 50 ng/ml) and DIM (10 µmol/l) induced cytotoxic
and apoptotic effects in BGC-823 and SGC-7901 gastric cancer cells. Furthermore,
western blot analysis revealed that the protein expression levels of death
receptor 5 (DR5), CCAAT/enhancer binding protein homologous protein (CHOP) and
glucose-regulated protein 78 (GRP78) were upregulated in the co-treated gastric
cancer cells. To the best of our knowledge, the present study is the first to
provide evidence that DIM sensitizes TRAIL-induced inhibition of proliferation
and apoptosis in gastric cancer cells, accompanied by the upregulated expression
of DR5, CHOP and GRP78 proteins, which may be involved in endoplasmic reticulum
stress mechanisms.

DOI: 10.3892/ol.2015.3008
PMCID: PMC4467362
PMID: 26137077

J Control Release. 2015 Sep 10;213:18-26. doi: 10.1016/j.jconrel.2015.06.014.
Epub 2015 Jun 14.

Evaluation of self-emulsified DIM-14 in dogs for oral bioavailability and in
Nu/nu mice bearing stem cell lung tumor models for anticancer activity.

Patel AR(1), Doddapaneni R(1), Andey T(1), Wilson H(2), Safe S(3), Singh M(4).

Author information:
(1)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University,
Tallahassee, FL 32307, USA.
(2)Institutes of Biosciences and Technology, Houston, TX 77843, USA.
(3)Institutes of Biosciences and Technology, Houston, TX 77843, USA; Texas A & M
Health Sciences Center, Houston, TX 77030, USA.
(4)College of Pharmacy and Pharmaceutical Sciences, Florida A&M University,
Tallahassee, FL 32307, USA.

3, 3-Diindolylmethane-14 (DIM-14), a novel lipophilic derivative of DIM, has
demonstrated anticancer activity in different types of cancers. However, poor
solubility and low oral bioavailability of DIM-14 limit its translational
benefits in vivo. This study was carried out to improve the oral bioavailability
of DIM-14 via self-emulsifying drug (SED) delivery system in dogs and to evaluate
pharmacodynamic characteristics of SED against H1650 stem cell tumor models.
DIM-14 was incorporated into an oil, surfactant, and co-surfactant mixture using
labrafil and tween-80 to obtain SED. SED were characterized by droplet size,
polydispersitiy index (PDI), zeta potential, entrapment efficiency (EE), in vitro
permeability and drug release (investigated with Caco-2 monolayers and
dissolution apparatus respectively). Pharmacokinetic parameters in dogs were
evaluated and analyzed using Winonlin. Anti-tumor activity was carried out in
H1650 lung tumor model. Particle size of SED was between 230 and 246 nm and
surface charge was negative and ranged from 26.50 to 28.69 mV. Entrapment
efficiency of SED was 85%. Pharmacokinetic evaluation in dogs showed increased
Cmax (39.18 ± 7.34 vs 21.68 ± 6.3 μg·dL-1), higher AUC0-t (34,481.34 ± 1125.46 vs
14,159.53 ± 702.20 μg·min·dL-1) and improved absorption with 3 times more
bioavailability of SED compared to DIM-14 solution. SED showed ~30-59% tumor
volume/weight reduction in H1650 tumor model compared to DIM-P solution. Our
studies demonstrate the potential application of self-emulsifying drug delivery
system (SEDDS), that enhances oral absorption of DIM-14 and increased anti-tumor
activity against lung tumor models.

Published by Elsevier B.V.

DOI: 10.1016/j.jconrel.2015.06.014
PMID: 26079185  [Indexed for MEDLINE]

Oncotarget. 2015 Sep 15;6(27):23776-92.

3'3-Diindolylmethane inhibits migration, invasion and metastasis of
hepatocellular carcinoma by suppressing FAK signaling.

Li WX(1), Chen LP(2), Sun MY(1), Li JT(1), Liu HZ(1), Zhu W(1).

Author information:
(1)Dearpartmant of Toxicology, Guangzhou Center for Disease Control and
Prevention, Guangzhou, China.
(2)Faculty of Toxicology, School of Public Health, Sun Yet-sen University,
Guangzhou, China.

Late stage hepatocellular carcinoma (HCC) usually has a low survival rate because
it has high potential of metastases and there is no effective cure.
3'3-Diindolylmethane (DIM) is the major product of the acid-catalyzed
oligomerization of indole-3-carbinol present in cruciferous vegetables. DIM has
been proved to exhibit anticancer properties. In this study, we explored the
effects and molecular mechanisms of anti-metastasis of DIM on HCC cells both in
vitro and in vivo. We chose two HCC cell lines SMMC-7721 and MHCC-97H that have
high potential of invasion. The results showed that DIM inhibited the
proliferation, migration and invasion of these two cell lines in vitro. In
addition, in vivo study demonstrated that DIM significantly decreased the volumes
of SMMC-7721 orthotopic liver tumor and suppressed lung metastasis in nude mice.
Focal Adhesion Kinase (FAK) is found over activated in HCC cells. We found that
DIM decreased the level of phospho-FAK (Tyr397) both in vitro and in vivo. DIM
inhibition of phospho-FAK (Tyr397) led to down-regulation of MMP2/9 and decreased
potential of metastasis. DIM also repressed the migration and invasion induced by
vitronectin through inactivation of FAK pathway and down-regulation of MMP2/9 in
vitro. We also found that pTEN plays a role in down-regulation of FAK by DIM.
These results demonstrated that DIM blocks HCC cell metastasis by suppressing
tumor cell migration and invasion. The anti-metastasis effect of DIM could be
explained to be its down-regulated expression and activation of MMP2/9 partly
induced by up-regulation of pTEN and inhibition of phospho-FAK (Tyr397).

DOI: 10.18632/oncotarget.4196
PMCID: PMC4695151
PMID: 26068982  [Indexed for MEDLINE]

Eur J Pharm Sci. 2015 Sep 18;77:141-53. doi: 10.1016/j.ejps.2015.06.003. Epub
2015 Jun 9.

Impact of structural differences in carcinopreventive agents indole-3-carbinol
and 3,3'-diindolylmethane on biological activity. An X-ray, ¹H-¹⁴N NQDR, ¹³C
CP/MAS NMR, and periodic hybrid DFT study.

Latosińska JN(1), Latosińska M(2), Szafrański M(2), Seliger J(3), Žagar V(4),
Burchardt DV(5).

Author information:
(1)Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań,
Poland. Electronic address: [email protected].
(2)Faculty of Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań,
Poland.
(3)Faculty of Mathematics and Physics, University of Ljubljana, Jadranska 19,
1000 Ljubljana, Slovenia; "Jozef Stefan" Institute, Jamova 39, 1000 Ljubljana,
Slovenia.
(4)"Jozef Stefan" Institute, Jamova 39, 1000 Ljubljana, Slovenia.
(5)Department of Paediatric Dentistry, Poznan University of Medical Sciences,
Bukowska 70, 60-812 Poznań, Poland.

Three experimental techniques (1)H-(14)N NQDR, (13)C CP/MAS NMR and X-ray and
Density Functional Theory (GGA/BLYP with PBC) and Hirshfeld surfaces were applied
for the structure-activity oriented studies of two phyto-antioxidants and
anticarcinogens: indole-3-carbinol, I3C, and 3,3'-diindolylmethane, DIM, (its
bioactive metabolite). One set of (14)N NQR frequencies for DIM (2.310, 2.200 and
0.110 MHz at 295K) and I3C (2.315, 1.985 and 0.330 MHz at 160K) was recorded. The
multiplicity of NQR lines recorded at RT revealed high symmetry (chemical and
physical equivalence) of both methyl indazole rings of DIM. Carbonyl (13)C CSA
tensor components were calculated from the (13)C CP/MAS solid state NMR spectrum
of I3C recorded under fast and slow spinning. At room temperature the crystal
structure of I3C is orthorhombic: space group Pca21, Z=4, a=5.78922(16),
b=15.6434(7) and c=8.4405(2)Å. The I3C molecules are aggregated into ribbons
stacked along [001]. The oxygen atomsare disorderedbetween the two sites of
different occupancy factors. It implies that the crystal is built of about 70%
trans and 30% gauche conformers, and apart from the weak OH⋯O hydrogen bonds
(O⋯O=3.106Å) the formation of alternative O'H⋯O bonds (O'⋯O=2.785Å) is possible
within the 1D ribbons. The adjacent ribbons are further stabilised by O'H⋯O bonds
(O'⋯O=2.951Å). The analysis of spectra and intermolecular interactions pattern by
experimental techniques was supported by solid (periodic) DFT calculations. The
knowledge of the topology and competition of the interactions in crystalline
state shed some light on the preferred conformations of CH2OH in I3C and steric
hindrance of methyl indole rings in DIM. A comparison of the local environment in
gas phase and solid permitted drawing some conclusions on the nature of the
interactions required for effective processes of recognition and binding of a
given anticarcinogen to the protein or nucleic acid.

DOI: 10.1016/j.ejps.2015.06.003
PMID: 26066413  [Indexed for MEDLINE]

Oncol Rep. 2015 Jul;34(1):495-503. doi: 10.3892/or.2015.3970. Epub 2015 May 11.

3-(2-Bromoethyl)-indole inhibits the growth of cancer cells and NF-κB activation.

Fadlalla K(1), Elgendy R(2), Gilbreath E(1), Pondugula SR(3), Yehualaeshet T(1),
Mansour M(3), Serbessa T(4), Manne U(5), Samuel T(1).

Author information:
(1)Department of Pathobiology, College of Veterinary Medicine, Nursing and Allied
Health, Tuskegee University, Tuskegee, AL, USA.
(2)Department of Pharmacology, Faculty of Veterinary Medicine, Suez Canal
University, Ismailia, Egypt.
(3)Department of Anatomy, Physiology and Pharmacology, College of Veterinary
Medicine, Auburn University, Auburn, AL, USA.
(4)Department of Natural Sciences, Elizabeth City State University, Elizabeth
City, NC, USA.
(5)Department of Pathology and Comprehensive Cancer Center, University of Alabama
at Birmingham, Birmingham, AL, USA.

Indole-3-carbinol (I3C) and diindolylmethane (DIM), found in cruciferous
vegetables, have chemopreventive and anticancer properties. In the present study,
14 substituted indoles were tested for activity against SW480 colon cancer cells.
Among these, 3-(2-bromoethyl)-indole, named BEI-9, showed the greatest
inhibition. The effects of BEI-9 on cancer cells were analyzed by MTS and
CellTiter-Glo assays for effects on cell viability, by microscopy for phenotypic
changes, by scratch wound assays for effects on migration, by flow cytometry for
changes in the cell cycle, by immunoblotting for cyclin D and A to assess effects
on cell cycle regulation, and by NF-κB reporter assays for effects on basal and
drug-induced NF-κB activation. BEI-9 inhibited the growth of SW480 and HCT116
colon cancer cells at concentrations of 12.5 and 5 µM, respectively. BEI-9 also
inhibited cell motility as determined with scratch wound assays, and reduced the
levels of cyclin D1 and A. Furthermore, in reporter cells, BEI-9 (0.8 µM)
inhibited basal and induced NF-κB activation and increased cell death when
combined with the cytokine TNFα or the drug camptothecin (CPT), both of which
activate NF-κB. Preliminary experiments to identify a safe dose range for
immunodeficient mice showed that BEI-9, administered intraperitoneally, was
tolerable at doses below 10 mg/kg. Thus, BEI-9 and other indole derivatives may
be useful in chemoprevention or as chemosensitizers. Since NF-κB activation is
implicated in carcinogenesis and in reducing sensitivity to anticancer drugs,
BEI-9 should be investigated in combination with drugs such as CPT, which
activate NF-κB.

DOI: 10.3892/or.2015.3970
PMCID: PMC4484612
PMID: 26063116  [Indexed for MEDLINE]

Mol Divers. 2015 Nov;19(4):685-93. doi: 10.1007/s11030-015-9605-3. Epub 2015 May
28.

Iodine-catalyzed [Formula: see text] C-H bond activation by selenium dioxide:
synthesis of diindolylmethanes and di(3-indolyl)selanides.

Naidu PS(1), Majumder S(1), Bhuyan PJ(2).

Author information:
(1)Medicinal Chemistry Division, CSIR-North East Institute of Science &
Technology, Jorhat, 785006, Assam, India.
(2)Medicinal Chemistry Division, CSIR-North East Institute of Science &
Technology, Jorhat, 785006, Assam, India. [email protected].

An efficient reaction protocol was developed for the synthesis of several
diindolylmethane derivatives via the [Formula: see text] C-H bond activation of
aryl methyl ketones by [Formula: see text] and indoles in the presence of
catalytic amounts of [Formula: see text] at 80 [Formula: see text] using dioxane
as solvent. Unexpectedly, an interesting class of di(3-indolyl)selenide compounds
was isolated when the reaction was carried out at room temperature.

DOI: 10.1007/s11030-015-9605-3
PMID: 26016723  [Indexed for MEDLINE]

Mol Med Rep. 2015 Sep;12(3):3815-20. doi: 10.3892/mmr.2015.3836. Epub 2015 May
25.

Pro-apoptotic and anti-proliferative effects of 3,3'-diindolylmethane in
nasopharyngeal carcinoma cells via downregulation of telomerase activity.

Li F(1), Xu Y(2), Chen C(1), Chen SM(2), Xiao BK(1), Tao ZZ(1).

Author information:
(1)Research Institute of Otolaryngology‑Head and Neck Surgery, Renmin Hospital of
Wuhan University, Wuhan, Hubei 430060, P.R. China.
(2)Department of Otolaryngology‑Head and Neck Surgery, Renmin Hospital of Wuhan
University, Wuhan, Hubei 430060, P.R. China.

The pro-apoptotic and anti-proliferative effects of 3,3'-diindolylmethane (DIM)
in various tumor cell types have been widely investigated. The underlying
mechanisms were suggested to include cell cycle arrest, cell signaling inhibition
and downregulation of the androgen receptor. The present study demonstrated that
DIM induced apoptosis and inhibited proliferation in nasopharyngeal carcinoma
cells by downregulating the activity of telomerase. The nasopharyngeal carcinoma
cell line 5‑8F was selected for this purpose. A cell counting kit‑8 assay and
flow cytometry were performed to detect apoptosis and proliferation of 5‑8F
cells, respectively, which revealed the pro‑apoptotic and anti‑proliferative
effects of DIM. Telomerase activity was detected using a telomeric repeat
amplification protocol assay, which revealed that the telomerase activity was
inhibited by DIM in a dose‑dependent manner. Reverse transcription polymerase
chain reaction was used to detect the mRNA expression levels of human telomerase
reverse transcriptase (hTERT) and human telomerase RNA (hTR), and western blot
analysis was used to detect the protein expression of hTERT. The results showed
that the mRNA and protein expression of hTERT were downregulated in 5‑8F cells
following treatment with DIM; however, the mRNA expression of hTR remained
unchanged, suggesting that hTERT was the target of DIM. To further identify the
target, the length of telomeres was continually measured using a telomere length
detection kit, revealing that the telomeres were shortened by DIM in an
concentration‑dependent manner. The present study confirmed that DIM had
pro‑apoptotic and anti‑proliferative effects in nasopharyngeal carcinoma cells by
regulating telomerase.

DOI: 10.3892/mmr.2015.3836
PMID: 26004560  [Indexed for MEDLINE]

Org Lett. 2015 Jun 5;17(11):2610-3. doi: 10.1021/acs.orglett.5b00882. Epub 2015
May 18.

Gene-inspired mycosynthesis of skeletally new indole alkaloids.

Lin LP(1), Yuan P(1), Jiang N(2), Mei YN(3), Zhang WJ(1), Wu HM(1), Zhang AH(1),
Cao JM(1), Xiong ZX(2), Lu Y, Tan RX(1).

Author information:
(1)†State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional
Biomolecules, Nanjing University, Nanjing 210093, China.
(2)§School of Pharmacy, Nanjing Medical University, Nanjing 210029, China.
(3)∥Department of Clinical Laboratory, the First Affiliated Hospital of Nanjing
Medical University, Nanjing 210029, China.

Dalesindole, an antibacterial and anti-inflammatory indole alkaloid with an
undescribed carbon skeleton, was stereoselectively constructed by Daldinia
eschscholzii through class II aldolase catalyzed Michael addition of fungal
chromone with 3,3'-diindolylmethane (DIM) formed in situ from indole-3-carbinol
(I3C) under catalyses of monooxygenase and 8-amino-7-oxononanoate synthase
(AONS). Dalesindole isomerizes via a retro-Michael reaction to give stereoisomers
with bioactivities. The work provides an access to new bioactive hybrids of
fungal oligoketide with microbially decorated exogenous chemistry.

DOI: 10.1021/acs.orglett.5b00882
PMID: 25985278  [Indexed for MEDLINE]

Oncol Rep. 2015 Jun;33(6):3171-7. doi: 10.3892/or.2015.3938. Epub 2015 Apr 28.

FOXM1-mediated downregulation of uPA and MMP9 by 3,3'-diindolylmethane inhibits
migration and invasion of human colorectal cancer cells.

Jin H(1), Li XJ(1), Park MH(2), Kim SM(1).

Author information:
(1)Department of Physiology, Institute for Medical Sciences, Chonbuk National
University Medical School, Jeonju, Republic of Korea.
(2)Catholic University of Pusan, Busan, Republic of Korea.

Although 3,3'-diindolylmethane (DIM) has been suggested to reduce the risk of
colorectal cancer, the underlying biological mechanism is not clearly understood.
In the present study, we investigated the effect of DIM on the migratory and
invasive activities of the human colorectal cancer cell lines DLD-1 and HCT116.
DIM significantly inhibited the migration and invasion of colorectal cancer cells
as assessed by wound healing and Matrigel invasion assays. The migratory ability
of the DLD-1 and HCT116 cells was significantly reduced by DIM at 24 and 48 h.
DIM also significantly inhibited the invasion rate of the DLD-1 and HCT116 cells
in a dose-dependent manner. The mRNA expression levels of urokinase type
plasminogen activator (uPA) and matrix metalloprotease 9 (MMP9) were
significantly attenuated, whereas expression of E-cadherin mRNA was significantly
enhanced, following DIM treatment. DIM also decreased the protein levels of uPA
and MMP9, yet significantly increased E-cadherin protein expression. In addition,
DIM significantly reduced the mRNA and protein levels of FOXM1 in the DLD-1 and
HCT116 cells. Our results suggest that DIM can influence the cell migratory and
invasive properties of human colorectal cancer cells and may decrease the
invasive capacity of colorectal cancer through downregulation of uPA and MMP9
mediated by suppression of the transcription factor FOXM1.

DOI: 10.3892/or.2015.3938
PMID: 25962429  [Indexed for MEDLINE]

Int J Mol Med. 2015 Jul;36(1):301-8. doi: 10.3892/ijmm.2015.2202. Epub 2015 May
5.

3,3'-Diindolylmethane inhibits VEGF expression through the HIF-1α and NF-κB
pathways in human retinal pigment epithelial cells under chemical hypoxic
conditions.

Park H(1), Lee DS(2), Yim MJ(2), Choi YH(3), Park S(4), Seo SK(4), Choi JS(5),
Jang WH(6), Yea SS(6), Park WS(7), Lee CM(8), Jung WK(9), Choi IW(4).

Author information:
(1)Department of Urology, Kangwon National University Hospital, Kangwon National
University School of Medicine, Chuncheon, Gangwon-do, Republic of Korea.
(2)Marine Biodiversity Institute of Korea, Seocheon, Chungcheongnam-do, Republic
of Korea.
(3)Department of Biochemistry, College of Oriental Medicine, Dongeui University,
Busan, Republic of Korea.
(4)Department of Microbiology, College of Medicine, Inje University, Busan,
Republic of Korea.
(5)Department of Internal Medicine, Busan Paik Hospital, College of Medicine,
Inje University, Busan, Republic of Korea.
(6)Department of Biochemistry, College of Medicine, Inje University, Busan,
Republic of Korea.
(7)Department of Physiology, Kangwon National University School of Medicine,
Chuncheon, Gangwon-do, Republic of Korea.
(8)Department of Molecular Microbiology and Immunology, Warren Alpert School of
Medicine, Providence, RI, USA.
(9)Department of Biomedical Engineering and Center for Marine-Integrated
Biomedical Technology (BK21 Plus), Pukyong National University, Busan, Republic
of Korea.

Oxidative stress in the retinal pigment epithelium (RPE) can lead to the
pathological causes of age-related macular degeneration (AMD). Hypoxia induces
oxidative damage in retinal pigment epithelial cells (RPE cells). In this study,
we investigated the capacity of 3,3'-diindolylmethane (DIM) to reduce the
expression of vascular endothelial growth factor (VEGF) under hypoxic conditions,
as well as the molecular mechanisms involved. Human RPE cells (ARPE-19 cells)
were treated with cobalt chloride (CoCl2, 200 µM) and/or DIM (10 and 20 µM). The
production of VEGF was measured by enzyme-linked immunosorbent assay. The
translocation of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB
(NF-κB) was determined by western blot analysis. The binding activity of HIF-1α
and NF-κB was analyzed by electrophoretic mobility shift assay. The
phosphorylation levels of mitogen-activated protein kinases (MAPKs) were measured
by western blot analysis. The levels of mitochondrial reactive oxygen species
(ROS) were detected by fluorescence microplate assay. The results revealed that
DIM significantly attenuated the CoCl2-induced expression of VEGF in the ARPE-19
cells. The CoCl2-induced translocation and activation of HIF-1α and NF-κB were
also attenuated by treatment with DIM. In addition, DIM inhibited the
CoCl2-induced activation of p38 MAPK in the ARPE-19 cells. Pre-treatment with
YCG063, a mitochondrial ROS inhibitor, led to the downregulation of the
CoCl2-induced production of VEGF by suppressing HIF-1α and NF-κB activity. Taken
together, the findings of our study demonstrate that DIM inhibits the
CoCl2-induced production of VEGF by suppressing mitochondrial ROS production,
thus attenuating the activation of HIF-1α and p38 MAPK/NF-κB.

DOI: 10.3892/ijmm.2015.2202
PMID: 25955241  [Indexed for MEDLINE]

Curr Med Chem. 2015;22(17):2051-64.

Impact of Epigenetic Dietary Components on Cancer through Histone Modifications.

Gao Y, Tollefsbol TO(1).

Author information:
(1)CH175, 1300 University Boulevard, Birmingham, AL 35294-1170. [email protected].

Epigenetics, the study of heritable changes in gene expression without modifying
the nucleotide sequence, is among the most important topics in medicinal
chemistry and cancer prevention and therapy. Among those changes, DNA methylation
and histone modification have been shown to be associated with various types of
cancers in a number of ways, many of which are regulated by dietary components
that are mostly found in plants. Although mechanisms of nutrient components
affecting histone acetylation/deacetylation in cancer are widely studied, how
those natural compounds affect cancer through other histone modifications, such
as methylation, phosphorylation and ubiquitylation, is rarely reviewed. Thus,
this review article discusses impacts on histone acetylation as well as other
histone modifications by nutrient components, such as genistein, resveratrol,
curcumin, epigallocatechin-3-gallate (EGCG), 3,3'-diindolylmethane (DIM), diallyl
disulfide, garcinol, procyanidin B3, quercetin, sulforaphane and other
isothiocyanates that have been recently reported in vivo as well as in various
types of cancer cell lines.

PMCID: PMC5012963
PMID: 25891109  [Indexed for MEDLINE]

J Agric Food Chem. 2015 May 6;63(17):4297-304. doi: 10.1021/jf506098s. Epub 2015
Apr 22.

Synergistic anticancer activity of capsaicin and 3,3'-diindolylmethane in human
colorectal cancer.

Clark R(1), Lee J(1), Lee SH(1).

Author information:
(1)Department of Nutrition and Food Science, College of Agriculture and Natural
Resources, University of Maryland, College Park, Maryland 20742, United States.

Cancer is a leading cause of morbidity and mortality worldwide. A promising area
of cancer research is focused on chemoprevention by nutritional compounds.
Epidemiological studies have shown a strong negative correlation between fruit,
vegetable, and spice intake and rates of cancer. Although individual active
compounds have demonstrated significant anticancer activity, an emerging area of
research is focusing on the combination of multiple dietary compounds that act
synergistically on cancer to exert greater effects. The current study evaluated
the potential synergistic effects of capsaicin, an active compound from red chili
peppers, in combination with 3,3'-diindolylmethane (DIM), from cruciferous
vegetables. A synergistic induction of apoptosis and inhibition of cell
proliferation was observed in human colorectal cancer cells treated with the
combination of capsaicin and DIM. It was also observed that these two compounds
activated transcriptional activity of NF-κB and p53 synergistically. Combination
treatment stabilized nuclear p53 and up- or down-regulated expression of several
target genes that are downstream of NF-κB and p53. The present study suggests
capsaicin and DIM work synergistically to inhibit cell proliferation and induce
apoptosis in colorectal cancer through modulating transcriptional activity of
NF-κB, p53, and target genes associated with apoptosis.

DOI: 10.1021/jf506098s
PMID: 25876645  [Indexed for MEDLINE]

Mol Pharmacol. 2015 Jun;87(6):1021-34. doi: 10.1124/mol.114.095398. Epub 2015 Apr
9.

The Nurr1 Activator 1,1-Bis(3'-Indolyl)-1-(p-Chlorophenyl)Methane Blocks
Inflammatory Gene Expression in BV-2 Microglial Cells by Inhibiting Nuclear
Factor κB.

De Miranda BR(1), Popichak KA(1), Hammond SL(1), Jorgensen BA(1), Phillips AT(1),
Safe S(1), Tjalkens RB(2).

Author information:
(1)Center for Environmental Medicine, Department of Environmental and
Radiological Health Sciences, Colorado State University, Fort Collins, Colorado
(B.R.D.M., K.A.P., S.L.H., B.A.J., A.T.P., R.B.T.); Center for Environmental and
Genetic Medicine, Institute of Biosciences and Technology, Texas A&M Health
Science Center, Houston, Texas; and Department of Veterinary Physiology and
Pharmacology, Texas A&M University, College Station, Texas (S.S.).
(2)Center for Environmental Medicine, Department of Environmental and
Radiological Health Sciences, Colorado State University, Fort Collins, Colorado
(B.R.D.M., K.A.P., S.L.H., B.A.J., A.T.P., R.B.T.); Center for Environmental and
Genetic Medicine, Institute of Biosciences and Technology, Texas A&M Health
Science Center, Houston, Texas; and Department of Veterinary Physiology and
Pharmacology, Texas A&M University, College Station, Texas (S.S.)
[email protected].

NR4A family orphan nuclear receptors are an important class of transcription
factors for development and homeostasis of dopaminergic neurons that also inhibit
expression of inflammatory genes in glial cells. The identification of NR4A2
(Nurr1) as a suppressor of nuclear factor κB (NF-κB)-related neuroinflammatory
genes in microglia and astrocytes suggests that this receptor could be a target
for pharmacologic intervention in neurologic disease, but compounds that promote
this activity are lacking. Selected diindolylmethane compounds (C-DIMs) have been
shown to activate or inactivate nuclear receptors, including Nurr1, in cancer
cells and also suppress astrocyte inflammatory signaling in vitro. Based upon
these data, we postulated that C-DIM12 [1,1-bis(3'-indolyl)-1-(p-chlorophenyl)
methane] would suppress inflammatory signaling in microglia by a Nurr1-dependent
mechanism. C-DIM12 inhibited lipopolysaccharide (LPS)-induced expression of
NF-κB-regulated genes in BV-2 microglia including nitric oxide synthase (NOS2),
interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2), and the effects
were attenuated by Nurr1-RNA interference. Additionally, C-DIM12 decreased NF-κB
activation in NF-κB-GFP (green fluorescent protein) reporter cells and enhanced
nuclear translocation of Nurr1 primary microglia. Chromatin immunoprecipitation
assays indicated that C-DIM12 decreased lipopolysaccharide-induced p65 binding to
the NOS2 promoter and concurrently enhanced binding of Nurr1 to the p65-binding
site. Consistent with these findings, C-DIM12 also stabilized binding of the
Corepressor for Repressor Element 1 Silencing Transcription Factor (CoREST) and
the Nuclear Receptor Corepressor 2 (NCOR2). Collectively, these data identify
C-DIM12 as a modulator of Nurr1 activity that results in inhibition of
NF-κB-dependent gene expression in glial cells by stabilizing nuclear corepressor
proteins, which reduces binding of p65 to inflammatory gene promoters.

DOI: 10.1124/mol.114.095398
PMCID: PMC4429718
PMID: 25858541  [Indexed for MEDLINE]

Mol Med Rep. 2015 Jul;12(1):1247-52. doi: 10.3892/mmr.2015.3523. Epub 2015 Mar
20.

3,3'-Diindolylmethane attenuates cardiac H9c2 cell hypertrophy through
5'-adenosine monophosphate-activated protein kinase-α.

Zong J(1), Wu QQ(1), Zhou H(1), Zhang JY(1), Yuan Y(1), Bian ZY(1), Deng W(1),
Dai J(1), Li FF(1), Xu M(1), Fang Y(1), Tang QZ(1).

Author information:
(1)Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, Hubei
430060, P.R. China.

3,3'-Diindolylmethane (DIM) is the major product of the acid-catalyzed
condensation of indole-3-carbinol (I3C), a component of extracts of Brassica food
plants. Numerous studies have suggested that DIM has several beneficial
biological activities, including elimination of free radicals, antioxidant and
anti-angiogenic effects and activation of apoptosis of various tumor cells. In
the present study, an in vitro model was established, using 1 µM angiotensin II
(Ang II) in cultured rat cardiac H9c2 cells, to observe the effects of DIM on
cardiac hypertrophy. Following 24 h stimulation with DIM (1, 5, and 10 µM) with
or without Ang II, cells were characterized by immunofluorescence to analyze
cardiac α-actinin expression. Cardiomyocyte hypertrophy and molecular markers of
cardiac hypertrophy were assessed by quantitative polymerase chain reaction.
Atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain β
mRNA expression were induced by Ang II in H9c2 cells treated with the optimal
concentration of DIM for 6, 12, and 24 h. The levels of phosphorylated and total
proteins of the 5' AMP-activated protein kinase α (AMPKα)/mitogen-activated
protein kinase (MAPK)/mechanistic target of rapamycin (mTOR) signaling pathways
in H9c2 cells treated with DIM for 0, 15, 30, and 60 min induced by Ang II were
determined by western blot analysis. The results showed that DIM attenuated
cellular hypertrophy in vitro, enhanced the phosphorylation of AMPKα and
inhibited the MAPK‑mTOR signaling pathway in response to hypertrophic stimuli.

DOI: 10.3892/mmr.2015.3523
PMID: 25816057  [Indexed for MEDLINE]

Mutat Res. 2015 Apr;774:25-32. doi: 10.1016/j.mrfmmm.2015.02.003. Epub 2015 Feb
25.

Effect of phytochemical intervention on dibenzo[a,l]pyrene-induced DNA adduct
formation.

Russell GK(1), Gupta RC(2), Vadhanam MV(3).

Author information:
(1)Department of Pharmacology and Toxicology, University of Louisville School of
Medicine, Louisville, KY 40202, United States.
(2)Department of Pharmacology and Toxicology, University of Louisville School of
Medicine, Louisville, KY 40202, United States; James Graham Brown Cancer Center,
University of Louisville School of Medicine, Louisville, KY 40202, United States.
(3)James Graham Brown Cancer Center, University of Louisville School of Medicine,
Louisville, KY 40202, United States. Electronic address: [email protected].

Dibenzo[a,l]pyrene (DBP) has been found to be the most potent carcinogen of the
polycyclic aromatic hydrocarbons (PAHs). Primary sources for DBP in the
environment are combustion of wood and coal burning, gasoline and diesel exhaust,
and tires. Given the likelihood of environmental exposure to DBP and strong
experimental evidence of its potency, it is likely to contribute to lung cancer
development. Intervention with compounds of natural origin ("phytochemicals") is
considered an effective means to prevent cancer development and favorably
modulate the underlying mechanisms, including DNA adduct formation. In this
study, several agents have been identified that inhibit environmental
carcinogen-induced DNA adduct formation using a cell-free microsomal system. Of
the ten agents tested, resveratrol (648 ± 26 adducts/10(9) nucleotides), oltipraz
(1007 ± 348 adducts/10(9) nucleotides), delphinidin (1252 ± 142 adducts/10(9)
nucleotides), tanshinone I (1981 ± 213 adducts/10(9) nucleotides), tanshinone IIA
(2606 ± 478 adducts/10(9) nucleotides) and diindoylmethane (3643 ± 469
adducts/10(9) nucleotides) were the most effective compared to vehicle treatment
(14,062 ± 1097 adducts/10(9) nucleotides). DBP is metabolized by phase I
metabolizing enzymes CYP1A1, CYP1A2, and CYP1B1. DBP-induced DNA adducts can be
inhibited by several mechanisms. We found that all the test agents inhibited DNA
adducts by inhibiting one or more of these enzymes. Oltipraz inhibited DNA
adducts entirely by inhibiting the CYP450s, while resveratrol and delphinidin
inhibited DNA adducts by also interacting directly with the carcinogenic
metabolite, anti-dibenzo(a,l)pyrene-11,12-dihydrodiol-13,14-epoxide.

DOI: 10.1016/j.mrfmmm.2015.02.003
PMCID: PMC4402140
PMID: 25794985  [Indexed for MEDLINE]

Chem Biol Interact. 2015 May 5;232:58-67. doi: 10.1016/j.cbi.2015.03.008. Epub
2015 Mar 17.

A therapeutically relevant, 3,3'-diindolylmethane derivative NGD16 attenuates
angiogenesis by targeting glucose regulated protein, 78kDa (GRP78).

Nayak D(1), Amin H(2), Rah B(2), Ur Rasool R(2), Sharma D(3), Gupta AP(3),
Kushwaha M(3), Mukherjee D(3), Goswami A(4).

Author information:
(1)Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute
of Integrative Medicine, Jammu, India; Cancer Pharmacology Division, CSIR-Indian
Institute of Integrative Medicine, Jammu, India.
(2)Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine,
Jammu, India.
(3)Natural Product Chemistry Division, CSIR-Indian Institute of Integrative
Medicine, Jammu, India.
(4)Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute
of Integrative Medicine, Jammu, India; Cancer Pharmacology Division, CSIR-Indian
Institute of Integrative Medicine, Jammu, India. Electronic address:
[email protected].

Angiogenesis remain a critical procedure for tumor progression and malignancy.
Anticancer agents targeting angiogenic cascades have been proved to be an
effective strategy in the field of cancer therapeutics. The current study aims to
explore the mechanistic prevention of angiogenesis and cancer cell proliferation
by 1,1'-β-d-glucopyranosyl-3,3'-bis(5-bromoindolyl)-octyl methane (NGD16), a
novel N-glycosylated derivative of 3,3'-diindolylmethane (DIM). NGD16 suppressed
the viability of prostate cancer (PC-3), pancreatic adenocarcinoma (MiaPaca-2),
colorectal cancer (COLO-205) and human umbilical vein endothelial cells (HUVECs)
effectively with IC50 values 0.8 μM, 2.8 μM, 5.3 μM and 2.5 μM respectively.
Abrogation of angiogenesis by NGD16 was promising in in vivo mouse Matrigel plug
assay as well as in ex vivo sprouting of rat thoracic aorta. At the molecular
level, NGD16 inhibited the expression of glucose regulated protein, 78 kDa
(GRP78), vascular endothelial growth factor receptor-2 (VEGFR2) and matrix
metalloproteinase-9 (MMP-9) expression, the main mediators of angiogenesis and
neovessel formation. Overexpression of GRP78 upregulated the expression of MMP-9
and VEGFR2 in PC-3 and HUVECs. Antibody blocking of GRP78 further potentiated
NGD16 in attenuating angiogenesis through inhibition of MMP-9. NGD16 depicted its
promising biodistribution profile in a pharmacokinetic study with 46.9%
intraperitoneal bioavailability. Our findings suggest NGD16 is a potent inhibitor
of neo-angiogenesis with a desirable pharmacokinetic profile, which can be taken
forward in its development as an anticancer drug.

DOI: 10.1016/j.cbi.2015.03.008
PMID: 25794856  [Indexed for MEDLINE]

Mini Rev Med Chem. 2015;15(3):203-10.

Topoisomerase 1B as a target against leishmaniasis.

D'Annessa I, Castelli S, Desideri A(1).

Author information:
(1)Department of Biology, University of Rome Tor Vergata, Via Della Ricerca
Scientifica, Rome 00133, Italy. [email protected].

Leishmaniasis affects more than 12 million people in 98 countries, the infection
being caused by more than 20 species of protozoan parasites belonging to the
genus Leishmania and spread by sandflies bite. Poor sanitary conditions,
malnutrition, deforestation and urbanization increase the risk for leishmaniasis.
Leishmaniasis is the only tropical disease treated with non-anti-leishmanial
drugs, among which liposomal amphotericin B, a combination of pentavalent
antimonials and paromomycin and miltefosine, that are highly toxic, represent the
most used ones. Drug resistance is now widespread and the search for new
molecular targets is open. Topoisomerase 1B, that controls the topological state
of DNA and is essential for the parasites viability, has been detected as a
promising target for anti-leishmaniasis therapy. The enzyme presents
structural/functional differences with the human counterpart, making it unique
among Eukarya. Here we review the structural features of this enzyme and the
drugs that can be developed and used for this specific targeting.

PMID: 25769969  [Indexed for MEDLINE]

PLoS One. 2015 Feb 23;10(2):e0118506. doi: 10.1371/journal.pone.0118506.
eCollection 2015.

Natural indoles, indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM),
attenuate staphylococcal enterotoxin B-mediated liver injury by downregulating
miR-31 expression and promoting caspase-2-mediated apoptosis.

Busbee PB(1), Nagarkatti M(2), Nagarkatti PS(1).

Author information:
(1)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, South Carolina, United States of America.
(2)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, South Carolina, United States of America;
WJB Dorn Veterans Affairs Medical Center, Columbia, South Carolina, United States
of America.

Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing
inflammation characterized by robust immune cell activation and proinflammatory
cytokine release. Exposure to SEB can result in food poisoning as well as fatal
conditions such as toxic shock syndrome. In the current study, we investigated
the effect of natural indoles including indole-3-carbinol (I3C) and
3,3'-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into
D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as
indicated by an increase in enzyme aspartate transaminase (AST) levels, induction
of inflammatory cytokines, and massive infiltration of immune cells into the
liver. Administration of I3C and DIM (40 mg/kg), by intraperitonal injection,
attenuated SEB-induced acute liver injury, as evidenced by decrease in AST
levels, inflammatory cytokines and cellular infiltration in the liver. I3C and
DIM triggered apoptosis in SEB-activated T cells primarily through activation of
the intrinsic mitochondrial pathway. In addition, inhibitor studies involving
caspases revealed that I3C and DIM-mediated apoptosis in these activated cells
was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition,
I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also
down-regulated miR-31, which directly targets caspase-2 and influences apoptosis
in SEB-activated cells. Our data demonstrate for the first time that indoles can
effectively suppress acute hepatic inflammation caused by SEB and that this may
be mediated by decreased expression of miR-31 and consequent caspase-2-dependent
apoptosis in T cells.

DOI: 10.1371/journal.pone.0118506
PMCID: PMC4338211
PMID: 25706292  [Indexed for MEDLINE]

J Pharmacol Sci. 2015 Jan;127(1):75-82. doi: 10.1016/j.jphs.2014.11.006. Epub
2014 Nov 20.

3,3'-Diindolylmethane increases bone mass by suppressing osteoclastic bone
resorption in mice.

Yu TY(1), Pang WJ(2), Yang GS(2).

Author information:
(1)Division of Integrative Pathophysiology, Proteo-Science Center, Graduate
School of Medicine, Ehime University, Ehime 791-0295, Japan; Laboratory of
Epigenetic Skeletal Diseases, Institute of Molecular and Cellular Biosciences,
The University of Tokyo, Tokyo 113-0032, Japan. Electronic address:
[email protected].
(2)College of Animal Science and Technology, Northwest A&F University, Yangling,
Shaanxi Province, 712100, PR China.

3,3'-Diindolylmethane (DIM), a major acid-condensation product or metabolite of
indole-3-carbinol which is found in cruciferous vegetables, has been shown to
have anticancer, anti-inflammatory, and multiple immune stimulating effects.
However, its function in bone metabolism is poorly understood. This study
evaluated the effect of DIM on bone mass in mice under physiological and
pathological conditions. Eight-week-old female mice received injections of a
vehicle or 0.1mg/g of DIM, twice a week for four weeks. We found that DIM
treatment significantly increased bone mass as assessed by dual-energy X-ray
absorptiometry (DEXA) and micro-computed tomography (μCT). Further, Bone
histomorphometric analyses showed that this treatment significantly reduced bone
resorption parameters, but did not increase bone formation parameters.
Furthermore, we use ovariectomized (OVX)-induced osteoporotic mouse model, and
explore function of DIM in skeletal pathological processes. Bone phenotype
analyses revealed that the administration of DIM in this study effectively
prevented OVX-induced bone loss resulting from increased bone resorption. Our
results demonstrated that DIM increased bone mass by suppressing osteoclastic
bone resorption in bone metabolism under both physiological and pathological
conditions. Accordingly, DIM may be of value in the treatment and the possible
prevention of bone diseases characterized by bone loss, such as postmenopausal
osteoporosis.

DOI: 10.1016/j.jphs.2014.11.006
PMID: 25704022  [Indexed for MEDLINE]

Oncol Rep. 2015 Apr;33(4):2031-6. doi: 10.3892/or.2015.3758. Epub 2015 Jan 28.

3,3'-Diindolylmethane potentiates paclitaxel-induced antitumor effects on gastric
cancer cells through the Akt/FOXM1 signaling cascade.

Jin H(1), Park MH(2), Kim SM(1).

Author information:
(1)Department of Physiology, Institute for Medical Sciences, Chonbuk National
University Medical School, Jeonju, Republic of Korea.
(2)Catholic University of Pusan, Busan, Republic of Korea.

Gastric cancer is the fourth most common cancer and is one of the leading causes
of cancer-related mortality worldwide. Forkhead box M1 (FOXM1) is overexpressed
in gastric cancer, suggesting that it is important in gastric cancer oncogenesis.
However, no studies have investigated the role of 3,3'-diindolylmethane (DIM), a
component of cruciferous vegetables, in the regulation of FOXM1 and its signaling
pathway in gastric cancer. Here, we report for the first time that DIM
effectively downregulated Akt/FOXM1 in gastric cancer cells. Combination
treatment with DIM and paclitaxel significantly and dose-dependently inhibited
the proliferation of SNU638 cells when compared to treatment with DIM or
paclitaxel alone. Colony formation of SNU638 cells was significantly attenuated
by treatment with DIM and paclitaxel, and DIM potentiated the inhibition of
colony formation in SNU638 cells by paclitaxel when compared to treatment with a
single agent. Treatment with DIM plus paclitaxel substantially increased
apoptosis as indicated by increased levels of cleaved polyADP-ribose polymerase
(PARP) and cleaved caspase-9 protein. DIM dose-dependently sensitized gastric
cancer cells through downregulation of FOXM1 and potentiated the effects of
paclitaxel. FOXM1 effector genes such as CDK4, p53 and cyclin D1 were
downregulated in gastric cancer cells by combination treatment with DIM and
paclitaxel. In addition, DIM significantly and dose-dependently inhibited
phosphorylation of Akt and potentiated paclitaxel-induced inhibition of Akt
function in gastric cancer cells. Therefore, our results indicate that DIM
effectively potentiates the efficacy of chemotherapeutic agents such as
paclitaxel by downregulation of the Akt/FOXM1 signaling cascade in gastric cancer
cells. Our findings suggest that DIM enhances the therapeutic efficacy of
paclitaxel in gastric cancer and is a potential clinical anticancer agent for the
prevention and/or treatment of gastric cancer.

DOI: 10.3892/or.2015.3758
PMID: 25633416  [Indexed for MEDLINE]

Fam Cancer. 2015 Jun;14(2):281-6. doi: 10.1007/s10689-015-9783-2.

The effect of oral 3,3'-diindolylmethane supplementation on the 2:16α-OHE ratio
in BRCA1 mutation carriers.

Nikitina D(1), Llacuachaqui M, Sepkovic D, Bradlow HL, Narod SA, Kotsopoulos J.

Author information:
(1)Women's College Research Institute, 790 Bay St, Toronto, ON, M5G 1N8, Canada,
[email protected].

Hormonal exposures are known to influence breast cancer risk among women with a
BRCA1 mutation. Thus, dietary factors that increase the 2-hydroxyestrone
(OHE):16α-OHE ratio, a biomarker inversely related to breast cancer development,
may also influence cancer risk. We conducted a dietary intervention study to
evaluate the ability of 300 mg/day of 3,3'-diindolylmethane (DIM) to increase the
urinary 2:16α-OHE ratio in 20 women with a BRCA1 mutation. BRCA1 mutation
carriers (n = 15) were assigned to receive 300 mg/day of Rx Balance BioREsponse
DIM for 4-6 weeks (intervention group) and five BRCA1 mutation carriers did not
take DIM (control group). The urinary 2:16α-OHE ratio was assessed at baseline
and after 4-6 weeks by immunoassay. There was no significant effect of DIM on the
2:16α-OHE ratio (2.4 at baseline vs. 3.0 after the intervention, P = 0.35). A
short dietary intervention with DIM did not significantly increase the 2:16α-OHE
ratio in female BRCA1 mutation carriers. Larger studies investigating the effect
of dietary or lifestyle interventions on circulating hormone levels in these
high-risk women are warranted.

DOI: 10.1007/s10689-015-9783-2
PMID: 25613194  [Indexed for MEDLINE]

Toxicol Lett. 2015 Feb 3;232(3):580-9. doi: 10.1016/j.toxlet.2014.12.015. Epub
2014 Dec 24.

Diindolylmethane, a naturally occurring compound, induces CYP3A4 and MDR1 gene
expression by activating human PXR.

Pondugula SR(1), Flannery PC(2), Abbott KL(2), Coleman ES(3), Mani S(4), Samuel
T(5), Xie W(6).

Author information:
(1)Department of Anatomy, Physiology and Pharmacology, College of Veterinary
Medicine, Auburn University, AL, United States; Auburn University Research
Initiative in Cancer, Auburn University, Auburn, AL, United States. Electronic
address: [email protected].
(2)Department of Anatomy, Physiology and Pharmacology, College of Veterinary
Medicine, Auburn University, AL, United States; Auburn University Research
Initiative in Cancer, Auburn University, Auburn, AL, United States.
(3)Department of Anatomy, Physiology and Pharmacology, College of Veterinary
Medicine, Auburn University, AL, United States.
(4)Albert Einstein Cancer Center, Albert Einstein College of Medicine, New York,
NY, United States.
(5)Department of Pathobiology, College of Veterinary Medicine, Nursing and Allied
Health, Tuskegee University, AL, United States.
(6)Center for Pharmacogenetics and Department of Pharmaceutical Sciences,
University of Pittsburgh, Pittsburgh, PA, United States.

Activation of human pregnane X receptor (hPXR)-regulated expression of cytochrome
P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1) plays an important
role in mediating adverse drug interactions. Given the common use of natural
products as part of adjunct human health behavior, there is a growing concern
about natural products for their potential to induce undesired drug interactions
through the activation of hPXR-regulated CYP3A4 and MDR1. Here, we studied
whether 3,3'-diindolylmethane (DIM), a natural health supplement, could induce
hPXR-mediated regulation of CYP3A4 and MDR1 in human hepatocytes and intestinal
cells. DIM, at its physiologically relevant concentrations, not only induced hPXR
transactivation of CYP3A4 promoter activity but also induced gene expression of
CYP3A4 and MDR1. DIM decreased intracellular accumulation of MDR1 substrate
rhodamine 123, suggesting that DIM induces the functional expression of MDR1.
Pharmacologic inhibition or genetic knockdown of hPXR resulted in attenuation of
DIM induced CYP3A4 and MDR1 gene expression, suggesting that DIM induces CYP3A4
and MDR1 in an hPXR-dependent manner. Together, these results support our
conclusion that DIM induces hPXR-regulated CYP3A4 and MDR1 gene expression. The
inductive effects of DIM on CYP3A4 and MDR1 expression caution the use of DIM in
conjunction with other medications metabolized and transported via CYP3A4 and
MDR1, respectively.

DOI: 10.1016/j.toxlet.2014.12.015
PMCID: PMC4568078
PMID: 25542144  [Indexed for MEDLINE]

Br J Pharmacol. 2015 Apr;172(8):2133-47. doi: 10.1111/bph.13036. Epub 2015 Mar
17.

3,3'-Diindolylmethane attenuates LPS-mediated acute liver failure by regulating
miRNAs to target IRAK4 and suppress Toll-like receptor signalling.

Tomar S(1), Nagarkatti M, Nagarkatti PS.

Author information:
(1)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, SC, USA.

BACKGROUND AND PURPOSE: Acute liver failure (ALF) is a severe and potentially
lethal clinical syndrome. 3,3'-Diindolylmethane (DIM) is a natural plant-derived
compound with anti-cancer activities. Recently, DIM has also been shown to have
anti-inflammatory properties. Here, we tested the hypothesis that DIM would
suppress endotoxin-induced ALF.
EXPERIMENTAL APPROACH: We investigated the therapeutic potential of DIM in a
mouse model of D-galactosamine/Lipopolysaccharide (GalN/LPS)-induced ALF. The
efficacy of DIM treatment was assessed by survival, liver histopathology, serum
levels of alanine transaminase, pro-inflammatory cytokines and number of
activated liver macrophages. Effects of DIM on the expression of two miRNAs, 106a
and 20b, and their predicted target gene were measured by qRT-PCR and Western
blotting. Effects of DIM on the release of TNF-α from RAW264.7 macrophages
transfected with mimics of these miRNAs and activated by LPS was assessed by
elisa.
KEY RESULTS: DIM treatment protected mice from ALF symptoms and reduced the
number of activated liver macrophages. DIM increased expression of miR-106a and
miR-20b in liver mononuclear cells and decreased expression of their predicted
target gene IL-1 receptor-associated kinase 4 (IRAK4), involved in signalling
from Toll-like receptor 4 (TLR4). In vitro transfection of RAW264.7 cells using
miRNA mimics of miR-106a and 20b decreased expression of IRAK4 and of TNF-α
secretion, following LPS stimulation.
CONCLUSIONS AND IMPLICATIONS: DIM attenuated GalN/LPS-induced ALF by regulating
the expression of unique miRNAs that target key molecules in the TLR4
inflammatory pathway. DIM may represent a potential novel hepatoprotective agent.

DOI: 10.1111/bph.13036
PMCID: PMC4386987
PMID: 25521277  [Indexed for MEDLINE]

Int J Cardiol. 2014 Nov 15;177(1):235-7. doi: 10.1016/j.ijcard.2014.09.037. Epub
2014 Sep 28.

3,3'-Diindolylmethane improves myocardial energy metabolism imbalance induced by
pressure overload via AMPKα in mice.

Deng W(1), Zong J(2), Wei L(3), Guo H(4), Cheng Z(5), Zhang R(1), Lin Y(1), Tang
Q(6).

Author information:
(1)Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060,
China; Cardiovascular Research Institute of Wuhan University, Wuhan 430060,
China.
(2)Department of Cardiology, The affiliated hospital of Xuzhou Medical College,
Xuzhou 221000, China.
(3)Cardiovascular Research Institute of Wuhan University, Wuhan 430060, China;
Department of Pediatrics, Renmin Hospital of Wuhan University, Wuhan 430060,
China.
(4)The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese
Ministry of Education, Qilu Hospital of Shandong University, Jinan 250012, China;
Department of Critical Care Medicine, Qilu Hospital of Shandong University, Jinan
250012, China.
(5)National Pharmaceutical Engineering Research Center, Shanghai 201203, China.
(6)Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060,
China; Cardiovascular Research Institute of Wuhan University, Wuhan 430060,
China. Electronic address: [email protected].

DOI: 10.1016/j.ijcard.2014.09.037
PMID: 25499385  [Indexed for MEDLINE]

J Biochem. 2015 May;157(5):311-20. doi: 10.1093/jb/mvu075. Epub 2014 Nov 25.

Mutation analysis and molecular modeling for the investigation of ligand-binding
modes of GPR84.

Nikaido Y(1), Koyama Y(1), Yoshikawa Y(1), Furuya T(1), Takeda S(2).

Author information:
(1)Faculty of Science and Technology, Division of Molecular Science, Gunma
University, Gunma 376-8515, Japan and Drug Discovery Department, Research &
Development Division, PharmaDesign Inc., Tokyo 104-0032, Japan.
(2)Faculty of Science and Technology, Division of Molecular Science, Gunma
University, Gunma 376-8515, Japan and Drug Discovery Department, Research &
Development Division, PharmaDesign Inc., Tokyo 104-0032, Japan
[email protected].

GPR84 is a G protein-coupled receptor for medium-chain fatty acids. Capric acid
and 3,3'-diindolylmethane are specific agonists for GPR84. We built a homology
model of a GPR84-capric acid complex to investigate the ligand-binding mode using
the crystal structure of human active-state β2-adrenergic receptor. We performed
site-directed mutagenesis to subject ligand-binding sites to our model using
GPR84-Giα fusion proteins and a [(35)S]GTPγS-binding assay. We compared the
activity of the wild type and mutated forms of GPR84 by [(35)S]GTPγS binding to
capric acid and diindolylmethane. The mutations L100D `Ballesteros-Weinstein
numbering: 3.32), F101Y (3.33) and N104Q (3.36) in the transmembrane helix III
and N357D (7.39) in the transmembrane helix VII resulted in reduced capric acid
activity but maintained the diindolylmethane responses. Y186F (5.46) and Y186H
(5.46) mutations had no characteristic effect on capric acid but with
diindolylmethane they significantly affected the G protein activation efficiency.
The L100D (3.32) mutant responded to decylamine, a fatty amine, instead of a
natural agonist, the fatty acid capric acid, suggesting that we have identified a
mutated G protein-coupled receptor-artificial ligand pairing. Our molecular model
provides an explanation for these results and interactions between GPR84 and
capric acid. Further, from the results of a double stimulation assay, we
concluded that diindolylmethane was a positive allosteric modulator for GPR84.

DOI: 10.1093/jb/mvu075
PMID: 25425658  [Indexed for MEDLINE]

Ophthalmic Surg Lasers Imaging Retina. 2014 Nov-Dec;45(6):589-91. doi:
10.3928/23258160-20141118-16.

Bilateral central serous chorioretinopathy associated with estrogen modulator
diindolylmethane.

Bussel II, Lally DR, Waheed NK.

Central serous chorioretinopathy (CSCR) is an idiopathic disease associated with
states of hypercortisolism that causes fluid to collect under the retina
resulting in visual impairment. The authors describe an otherwise healthy female
patient who presented with headaches and blurry vision after a 2-month history of
excessive daily consumption of diindolylmethane (DIM), an over-the-counter
estrogen modulator medication used to treat facial acne. Imaging demonstrated
asymmetric, bilateral CSCR with active leakage in the left eye. She was
instructed to discontinue DIM and reported that visual improvement began 2 weeks
after, with resolution to baseline after 8 weeks.

DOI: 10.3928/23258160-20141118-16
PMID: 25423641  [Indexed for MEDLINE]

Cancer Prev Res (Phila). 2015 Jan;8(1):77-85. doi:
10.1158/1940-6207.CAPR-14-0245. Epub 2014 Nov 17.

Dietary diindolylmethane suppresses inflammation-driven lung squamous cell
carcinoma in mice.

Song JM(1), Qian X(1), Teferi F(1), Pan J(2), Wang Y(2), Kassie F(3).

Author information:
(1)Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota.
(2)Department of Pharmacology and Toxicology and Cancer Center, Medical College
of Wisconsin, Milwaukee, Wisconsin.
(3)Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota.
College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota.
[email protected].

Inflammatory conditions of the lung such as chronic obstructive pulmonary disease
(COPD) are known to increase lung cancer risk, particularly lung squamous cell
carcinoma (LSCC). In the present study, we developed a mouse model of
inflammation-driven LSCC that was induced by N-nitroso-trischloroethylurea (NTCU)
and enhanced by lipopolysaccharide (LPS), a potent proinflammatory agent
contained in tobacco and tobacco smoke, and determined the chemopreventive
effects of diindolylmethane (DIM) in the same model. Compared with
mice treated with NTCU alone, mice treated with the combination of NTCU and LPS
had a 9-fold increase in the number of bronchioles with LSCC. Also, compared with
mice treated with LPS alone, mice treated with NTCU plus LPS showed significantly
increased expression of the inflammatory cytokines IL1α, IL6, and TNFα (all three
increased about 7-fold). Parallel to the increased cytokine gene expression, the
NTCU plus LPS-treated group exhibited significantly enhanced activation of NF-κB,
STAT3, ERK, p-38, and Akt, expression of p53, COX-2, and Mcl-1, and NF-κB- and
STAT3-DNA binding in the lung. Dietary administration of DIM (10 μmol/g diet or
2,460 ppm) to mice treated with NTCU plus LPS reduced the incidence of LSCC by
2-fold, suppressed activation/expression of proinflammatory and procarcinogenic
proteins and NF-κB- and STAT3-DNA binding, but not the expression of cytokines
and p53. This study highlights the potential significance of our mouse model to
identify promising drugs or dietary agents for the chemoprevention of human LSCC
and that DIM is a very good candidate for clinical lung cancer chemoprevention
trials.

DOI: 10.1158/1940-6207.CAPR-14-0245
PMCID: PMC4289649
PMID: 25403850  [Indexed for MEDLINE]

J Biomed Res. 2014 Sep;28(5):339-48. doi: 10.7555/JBR.28.20140008. Epub 2014 Apr
20.

Multiple therapeutic and preventive effects of 3,3'-diindolylmethane on cancers
including prostate cancer and high grade prostatic intraepithelial neoplasia.

Zhang WW(1), Feng Z(2), Narod SA(3).

Author information:
(1)Division of Urology, Sunnybrook Health Sciences Centre, Sunnybrook Research
Institute, University of Toronto, Toronto, Ontario M4N 3M5, Canada.
(2)Department of Pathology, Nanjing Medical University, Nanjing, Jiangsu 210029,
China.
(3)Department of Public Health Sciences, Women's College Hospital, Women's
College Research Institute, University of Toronto, Toronto, Ontario M4N 3M5,
Canada.

Cruciferous vegetables belong to the plant family that has flowers with four
equal-sized petals in the pattern of a crucifer cross. These vegetables are an
abundant source of dietary phytochemicals, including glucosinolates and their
hydrolysis products such as indole-3-carbinol (I3C) and 3,3'-diindolylmethane
(DIM). By 2013, the total number of natural glucosinolates that have been
documented is estimated to be 132. Recently, cruciferous vegetable intake has
garnered great interest for its multiple health benefits such as anticancer,
antiviral infections, human sex hormone regulation, and its therapeutic and
preventive effects on prostate cancer and high grade prostatic intraepithelial
neoplasia (HGPIN). DIM is a hydrolysis product of glucosinolates and has been
used in various trials. This review is to provide an insight into the latest
developments of DIM in treating or preventing both prostate cancer and HGPIN.

DOI: 10.7555/JBR.28.20140008
PMCID: PMC4197384
PMID: 25332705

Int J Pharm. 2014 Dec 30;477(1-2):96-101. doi: 10.1016/j.ijpharm.2014.10.018.
Epub 2014 Oct 11.

Intranasal delivery of liposomal indole-3-carbinol improves its pulmonary
bioavailability.

Song JM(1), Kirtane AR(2), Upadhyaya P(1), Qian X(1), Balbo S(1), Teferi F(1),
Panyam J(3), Kassie F(4).

Author information:
(1)Masonic Cancer Center, University of Minnesota, MN, USA.
(2)Department of Pharmaceutics, University of Minnesota, MN, USA.
(3)Masonic Cancer Center, University of Minnesota, MN, USA; Department of
Pharmaceutics, University of Minnesota, MN, USA.
(4)Masonic Cancer Center, University of Minnesota, MN, USA; College of Veterinary
Medicine, University of Minnesota, MN, USA. Electronic address: [email protected].

Indole-3-carbinol (I3C), a constituent of commonly consumed Brassica vegetables,
has been shown to have anticancer effects in a variety of preclinical models of
lung cancer. However, it has shown only limited efficacy in clinical trials,
likely due to its poor oral bioavailability. Intranasal administration of I3C has
the potential to enhance the pulmonary accumulation of the drug, thereby
improving its availability at the target site of action. In this study, we
developed a liposomal formulation of I3C and evaluated its lung delivery and
chemopreventive potential in tobacco smoke carcinogen
[4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK)]-treated mice. Intranasal
administration of I3C liposomes led to a ∼100-fold higher lung exposure of I3C
than the oral route of administration. Further, intranasal delivery of liposomal
I3C led to a significant reduction (37%; p<0.05) in the levels of the DNA adduct
formation induced by NNK treatment. Liposomal I3C also significantly increased
(by 10-fold) the expression of CYP1A1, a cytochrome P450 enzyme known to increase
the detoxification of chemical carcinogens by enhancing their metabolism.
Overall, our findings demonstrate that intranasal administration of liposomal I3C
has the potential to significantly improve the efficacy of I3C for lung cancer
chemoprevention.

DOI: 10.1016/j.ijpharm.2014.10.018
PMCID: PMC4268270
PMID: 25311179  [Indexed for MEDLINE]

EPMA J. 2014 Oct 8;5(1):18. doi: 10.1186/1878-5085-5-18. eCollection 2014.

Safety and tolerability of DIM-based therapy designed as personalized approach to
reverse prostatic intraepithelial neoplasia (PIN).

Paltsev M(1), Kiselev V(2), Muyzhnek E(3), Drukh V(2), Kuznetsov I(4),
Pchelintseva O(2).

Author information:
(1)National Research Centre (NRC 'Kurchatov Institute'), 1, Akademika Kurchatova
pl., Moscow 123182, Russia.
(2)Peoples' Friendship University of Russia, Miklukho-Maklaya str. 6, 117198
Moscow, Russia.
(3)ZAO 'MiraxBioPharma', 12 Kutuzovsky av., 121248 Moscow, Russia.
(4)Moscow State Medical Stomatological University (MGMSU), Delegatskaya St. 2/1,
127473 Moscow, Russia.

BACKGROUND: It has been shown previously that novel formulation of
3,3'-diindolylmethane (DIM) substance with high bioavailability (Infemin)
inhibits tumor development due to the tumor growth rate reduction in the
xenograft model of prostate cancer. Prostatic intraepithelial neoplasia (PIN) is
considered to be promising as a personalized and preventive treatment strategy of
prostate cancer (PC). We assessed the safety of Infemin in men with PIN and
discussed the interim results.
MATERIALS AND METHODS: A total of 14 patients with PIN were enrolled. They were
randomized to 900 mg DIM or placebo daily for 3 months. Safety was evaluated by
adverse events (AEs), laboratory tests and physical examinations.
RESULTS AND CONCLUSION: The trial revealed that Infemin treatment is associated
with minimal toxicity and no serious adverse events when administered orally for
3 months. We noted three adverse events including nausea and diarrhea in two
patients (14%). Combined 95% confidence interval (CI) was 1.8%-42.8%. Therapy was
continued in all cases of adverse events. Good tolerability of DIM-based
formulation allows us to recommend it for further clinical trials among men
diagnosed with PIN for its efficacy and long-term safety parameters.

DOI: 10.1186/1878-5085-5-18
PMCID: PMC4193909
PMID: 25309637

Prostate. 2015 Feb;75(2):161-74. doi: 10.1002/pros.22901. Epub 2014 Oct 13.

Androgen receptor splice variants contribute to prostate cancer aggressiveness
through induction of EMT and expression of stem cell marker genes.

Kong D(1), Sethi S, Li Y, Chen W, Sakr WA, Heath E, Sarkar FH.

Author information:
(1)Department of Pathology, Karmanos Cancer Institute, Wayne State University
School of Medicine, Detroit, Michigan.

BACKGROUND: The mechanism(s) by which androgen receptor (AR) splice variants
contribute to castration-resistant prostate cancer (CRPC) is still lacking.
METHODS: Expressions of epithelial-to-mesenchymal transition (EMT) and stem cell
markers were molecularly tested using prostate cancer (PCa) cells transfected
with AR and AR3 (also known as AR-V7) plasmids or siRNA, and also cultured cells
under androgen deprivation therapy (ADT) condition. Cell migration,
clonogenicity, sphere-forming capacity was assessed using PCa cells under all
experimental conditions and 3,3'-diindolylmethane (DIM) treatment. Human
PCa samples from DIM untreated or treated patients were also used for
assessing the expression of AR3 and stem cell markers.
RESULTS: Overexpression of AR led to the induction of EMT phenotype, while
overexpression of AR3 not only induced EMT but also led to the expression of stem
cell signature genes. More importantly, ADT enhanced the expression of AR and AR3
concomitant with up-regulated expression of EMT and stem cell marker genes.
Dihydrotestosterone (DHT) treatment decreased the expression of AR and AR3, and
reversed the expression of these EMT and stem cell marker genes. DIM
administered to PCa patients prior to radical prostatectomy inhibited the
expression of cancer stem cell markers consistent with inhibition of self-renewal
of PCa cells after DIM treatment.
CONCLUSION: AR variants could contribute to PCa progression through induction of
EMT and acquisition of stem cell characteristics, which could be attenuated by
DIM, suggesting that DIM could become a promising agent for the prevention
of CRPC and/or for the treatment of PCa.

DOI: 10.1002/pros.22901
PMCID: PMC4270852
PMID: 25307492  [Indexed for MEDLINE]

Hepatology. 2015 Feb;61(2):548-60. doi: 10.1002/hep.27547. Epub 2015 Jan 5.

Lineage-dependent effects of aryl hydrocarbon receptor agonists contribute to
liver tumorigenesis.

Harrill JA(1), Parks BB, Wauthier E, Rowlands JC, Reid LM, Thomas RS.

Author information:
(1)Institute for Chemical Safety Sciences, Hamner Institutes for Health Sciences,
Research Triangle Park, NC.

Rodent cancer bioassays indicate that the aryl hydrocarbon receptor (AHR)
agonist, 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD), causes increases in both
hepatocytic and cholangiocytic tumors. Effects of AHR activation have been
evaluated on rodent hepatic stem cells (rHpSCs) versus their descendants,
hepatoblasts (rHBs), two lineage stages of multipotent, hepatic precursors with
overlapping but also distinct phenotypic traits. This was made possible by
defining the first successful culture conditions for ex vivo maintenance of
rHpScs consisting of a substratum of hyaluronans and Kubota's medium (KM), a
serum-free medium designed for endodermal stem/progenitor cells. Supplementation
of KM with leukemia inhibitory factor elicited lineage restriction to rHBs.
Cultures were treated with various AHR agonists including TCDD,
6-formylindolo-[3,2-b]carbazole (FICZ), and 3-3'-diindolylmethane (DIM) and then
analyzed with a combination of immunocytochemistry, gene expression, and
high-content image analysis. The AHR agonists increased proliferation of rHpSCs
at concentrations producing a persistent AHR activation as indicated by induction
of Cyp1a1. By contrast, treatment with TCDD resulted in a rapid loss of viability
of rHBs, even though the culture conditions, in the absence of the agonists, were
permissive for survival and expansion of rHBs. The effects were not observed with
FICZ and at lower concentrations of DIM.CONCLUSION: Our findings are consistent
with a lineage-dependent mode of action for AHR agonists in rodent liver
tumorigenesis through selective expansion of rHpSCs in combination with a
toxicity-induced loss of viability of rHBs. These lineage-dependent effects
correlate with increased frequency of liver tumors.

DOI: 10.1002/hep.27547
PMCID: PMC4303521
PMID: 25284723  [Indexed for MEDLINE]

Int Immunopharmacol. 2014 Dec;23(2):489-98. doi: 10.1016/j.intimp.2014.09.024.
Epub 2014 Oct 1.

3,3'-Diindolylmethane alleviates steatosis and the progression of NASH partly
through shifting the imbalance of Treg/Th17 cells to Treg dominance.

Liu Y(1), She W(1), Wang F(1), Li J(2), Wang J(1), Jiang W(3).

Author information:
(1)Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai
200032, China.
(2)Department of Gastroenterology, Tongji Hospital Tongji University, Shanghai
China.
(3)Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai
200032, China. Electronic address: [email protected].

This study was designed to discuss the effects of 3, 3'-diindolylmethane (DIM) on
methionine-choline-deficient (MCD)-diet induced mouse nonalcoholic
steatohepatitis (NASH) and the potential mechanisms. NASH mice were administrated
with or without DIM at different concentrations for 8 weeks. Both the in-vivo and
in-vitro effects of DIM on Treg/Th17 imbalance during NASH progression were
analyzed. The in-vivo blocking of CD25 or IL-17 was performed to respectively
deplete respective function of Treg or Th17 subset. Besides, with the assistance
of AhR antagonist CH223191 and anti-TLR4 neutralizing antibody, we designed the
in-vitro DIM-incubation experiments to discuss the roles of aryl hydrocarbon
receptor (AhR) (CYP1A1, CYP1B1) and toll-like receptor 4 (TLR4) on DIM's effects
when shifting Treg/Th17 imbalance. Notably, in NASH mouse models, DIM alleviated
hepatic steatosis and inflammation, and shifted the Treg/Th17 imbalance from MCD
diet-induced Th17 dominance to Treg dominance. In-vitro, DIM not only
significantly up-regulated the mRNAs of Foxp3 (Treg-specific) in purified spleen
CD4(+) T cells, but also enhanced the immunosuppressive function of these Treg
cells. Besides, DIM significantly up-regulated the proteins of CYP1A1 and CYP1B1
whereas down-regulated those of TLR4 on CD4(+) T cells from MCD-diet mice.
Moreover, blocking AhR attenuated while blocking TLR4 enhanced the effects of DIM
when regulating Treg/Th17 imbalance. Conclusively, DIM could be used as a
potential therapeutic candidate to treat NASH based on its dramatic induction of
Treg dominance to alleviate intra-hepatic inflammation, suggesting us a clue that
the dietary cruciferous vegetables (containing abundant DIM) might exist as a
protective factor for patients with NASH-related liver diseases.

DOI: 10.1016/j.intimp.2014.09.024
PMID: 25281898  [Indexed for MEDLINE]

Biomed Res Int. 2014;2014:804510. doi: 10.1155/2014/804510. Epub 2014 Sep 1.

Regulation of microRNAs by natural agents: new strategies in cancer therapies.

Phuah NH(1), Nagoor NH(2).

Author information:
(1)Institute of Biological Science (Genetics and Molecular Biology), Faculty of
Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
(2)Institute of Biological Science (Genetics and Molecular Biology), Faculty of
Science, University of Malaya, 50603 Kuala Lumpur, Malaysia ; Centre for Research
in Biotechnology for Agriculture (CEBAR), University of Malaya, 50603 Kuala
Lumpur, Malaysia.

MicroRNAs (miRNAs) are short noncoding RNA which regulate gene expression by
messenger RNA (mRNA) degradation or translation repression. The plethora of
published reports in recent years demonstrated that they play fundamental roles
in many biological processes, such as carcinogenesis, angiogenesis, programmed
cell death, cell proliferation, invasion, migration, and differentiation by
acting as tumour suppressor or oncogene, and aberrations in their expressions
have been linked to onset and progression of various cancers. Furthermore, each
miRNA is capable of regulating the expression of many genes, allowing them to
simultaneously regulate multiple cellular signalling pathways. Hence, miRNAs have
the potential to be used as biomarkers for cancer diagnosis and prognosis as well
as therapeutic targets. Recent studies have shown that natural agents such as
curcumin, resveratrol, genistein, epigallocatechin-3-gallate, indole-3-carbinol,
and 3,3'-diindolylmethane exert their antiproliferative and/or proapoptotic
effects through the regulation of one or more miRNAs. Therefore, this review will
look at the regulation of miRNAs by natural agents as a means to potentially
enhance the efficacy of conventional chemotherapy through combinatorial
therapies. It is hoped that this would provide new strategies in cancer therapies
to improve overall response and survival outcome in cancer patients.

DOI: 10.1155/2014/804510
PMCID: PMC4165563
PMID: 25254214  [Indexed for MEDLINE]

Pharmacol Res. 2014 Nov;89:46-56. doi: 10.1016/j.phrs.2014.08.005. Epub 2014 Aug
30.

Indole-3-carbinol induces cMYC and IAP-family downmodulation and promotes
apoptosis of Epstein-Barr virus (EBV)-positive but not of EBV-negative Burkitt's
lymphoma cell lines.

Perez-Chacon G(1), de Los Rios C(2), Zapata JM(3).

Author information:
(1)Instituto de Investigaciones Biomedicas "Alberto Sols", CSIC/UAM, Spain.
Electronic address: [email protected].
(2)Instituto Teofilo Hernando, Spain; Departamento de Farmacologia y Terapeutica,
Facultad de Medicina, UAM, 28029 Madrid, Spain. Electronic address:
[email protected].
(3)Instituto de Investigaciones Biomedicas "Alberto Sols", CSIC/UAM, Spain.
Electronic address: [email protected].

Indole-3-carbinol (I3C) is a natural product found in broadly consumed plants of
the Brassica genus, such as broccoli, cabbage, and cauliflower, which exhibits
anti-tumor effects through poorly defined mechanisms. I3C can be orally
administered and clinical trials have demonstrated that I3C and derivatives are
safe in humans. In this study we show that I3C efficiently induces apoptosis in
cell lines derived from EBV-positive Burkitt's lymphomas (virus latency I/II),
while it does not have any cytotoxic activity against EBV-negative Burkitt's
lymphomas and immortalized EBV-infected lymphoblastoid cell lines (virus latency
III). The effect of I3C in EBV-positive Burkitt's lymphoma is very specific,
since only I3C and its C6-methylated derivative, but not other 3-substituted
indoles, have an effect on cell viability. I3C treatment caused apoptosis
characterized by loss of mitochondria membrane potential and caspase activation.
I3C alters the expression of proteins involved in the control of apoptosis and
transcription regulation in EBV-positive Burkitt's lymphoma cell lines. Among
those, cMYC, cIAP1/2 and XIAP downmodulation at mRNA and protein level precede
apoptosis induction, thus suggesting a role in I3C cytotoxicity. We also showed
that I3C and, more particularly, its condensation dimer 3,3'-diindolylmethane
(DIM) prolonged survival and reduced tumor burden of mice xenotransplanted with
EBV-positive Burkitt's lymphoma Daudi cells. In summary these results, together
with previous reports from clinical trials indicating the lack of toxicity in
humans of I3C and derivatives, support the use of these compounds as a new
therapeutic approach for treating patients with endemic (EBV-positive) Burkitt's
lymphoma.

DOI: 10.1016/j.phrs.2014.08.005
PMID: 25180456  [Indexed for MEDLINE]

Chem Commun (Camb). 2014 Oct 21;50(82):12293-6. doi: 10.1039/c4cc05901h.

Copper-catalyzed tandem annulation/arylation for the synthesis of
diindolylmethanes from propargylic alcohols.

Li H(1), Li X, Wang HY, Winston-McPherson GN, Geng HM, Guzei IA, Tang W.

Author information:
(1)School of Pharmacy, University of Wisconsin, Madison, WI 53705, USA.
[email protected].

Various highly substituted 2,3'-diindolylmethane heterocycles were prepared from
propargylic alcohols and indole nucleophiles via a transition metal-catalyzed
tandem indole annulation/arylation reaction for the first time. Among the metal
catalysts we examined, the most economical copper(I) catalyst provided the
highest efficiency. The indole nucleophiles could also be replaced by other
electron-rich arenes or alcohols.

DOI: 10.1039/c4cc05901h
PMCID: PMC4169321
PMID: 25178910  [Indexed for MEDLINE]

Asian Pac J Cancer Prev. 2014;15(16):6463-75.

The mTOR signalling pathway in cancer and the potential mTOR inhibitory
activities of natural phytochemicals.

Tan HK(1), Moad AI, Tan ML.

Author information:
(1)Malaysian Institute of Pharmaceuticals and Nutraceuticals, Ministry of
Science, Technology and Innovation (MOSTI), Halaman Bukit Gambir, Malaysia E-mail
: [email protected], [email protected].

The mammalian target of rapamycin (mTOR) kinase plays an important role in
regulating cell growth and cell cycle progression in response to cellular
signals. It is a key regulator of cell proliferation and many upstream activators
and downstream effectors of mTOR are known to be deregulated in various types of
cancers. Since the mTOR signalling pathway is commonly activated in human
cancers, many researchers are actively developing inhibitors that target key
components in the pathway and some of these drugs are already on the market.
Numerous preclinical investigations have also suggested that some herbs and
natural phytochemicals, such as curcumin, resveratrol, timosaponin III, gallic
acid, diosgenin, pomegranate, epigallocatechin gallate (EGCC), genistein and
3,3'-diindolylmethane inhibit the mTOR pathway either directly or indirectly.
Some of these natural compounds are also in the clinical trial stage. In this
review, the potential anti-cancer and chemopreventive activities and the current
status of clinical trials of these phytochemicals are discussed.

PMID: 25169472  [Indexed for MEDLINE]

Mol Endocrinol. 2014 Oct;28(10):1729-39. doi: 10.1210/me.2014-1102. Epub 2014 Aug
6.

Diindolylmethane analogs bind NR4A1 and are NR4A1 antagonists in colon cancer
cells.

Lee SO(1), Li X, Hedrick E, Jin UH, Tjalkens RB, Backos DS, Li L, Zhang Y, Wu Q,
Safe S.

Author information:
(1)Department of Food Science and Technology (S.-O.L.), Keimyung University,
Daegu 704-701, Republic of Korea; College of Medicine (X.L.), Texas A&M Health
Science Center, and Department of Veterinary Physiology and Pharmacology (E.H.,
S.S.), Texas A&M University, College Station, TX 77843; Institute of Bioscience
and Technology (U.-H.J., S.S.), Texas A&M Health Science Center, Houston, Texas
77030; Center for Environmental Medicine (R.B.T.), Department of Environmental
and Radiological Health Sciences, College of Veterinary Medicine and Biomedical
Sciences, Colorado State University, Fort Collins, Colorado 80523; Department of
Pharmaceutical Sciences (D.S.B.), University of Colorado Anschutz Medical Campus,
Aurora, Colorado 80045; and School of Life Sciences (L.L., Y.Z., Q.W.),
University of Xiamen, Xiamen, 361005 Fujian, China.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methane (C-DIM) compounds exhibit
antineoplastic activity in multiple cancer cell lines and the p-hydroxyphenyl
analog (DIM-C-pPhOH) inactivates nuclear receptor 4A1 (NR4A1) in lung and
pancreatic cancer cell lines. Using a series of 14 different p-substituted phenyl
C-DIMs, we show that several compounds including DIM-C-pPhOH directly interacted
with the ligand binding domain of NR4A1. Computational-based molecular modeling
studies showed high-affinity interactions of DIM-C-pPhOH and related compounds
within the ligand binding pocket of NR4A1, and these same compounds decreased
NR4A1-dependent transactivation in colon cancer cells transfected with a
construct containing 3 tandem Nur77 binding response elements linked to a
luciferase reporter gene. Moreover, we also show that knockdown of NR4A1 by RNA
interference (small interfering NR4A1) or treatment with DIM-C-pPhOH and related
compounds decreased colon cancer cell growth, induced apoptosis, decreased
expression of survivin and other Sp-regulated genes, and inhibited mammalian
target of rapamycin signaling. Thus, C-DIMs such as DIM-C-pPhOH directly bind
NR4A1 and are NR4A1 antagonists in colon cancer cells, and their antineoplastic
activity is due, in part, to their interactions with nuclear NR4A1.

DOI: 10.1210/me.2014-1102
PMCID: PMC4179635
PMID: 25099012  [Indexed for MEDLINE]

PLoS One. 2014 Aug 1;9(8):e103407. doi: 10.1371/journal.pone.0103407. eCollection
2014.

Exogenous methyl jasmonate treatment increases glucosinolate biosynthesis and
quinone reductase activity in kale leaf tissue.

Ku KM(1), Jeffery EH(2), Juvik JA(1).

Author information:
(1)Department of Crop Sciences, University of Illinois at Urbana-Champaign,
Urbana, Illinois, United States of America.
(2)Department of Food Science and Human Nutrition, University of Illinois at
Urbana-Champaign, Urbana, Illinois, United States of America.

Methyl jasmonate (MeJA) spray treatments were applied to the kale varieties
'Dwarf Blue Curled Vates' and 'Red Winter' in replicated field plantings in 2010
and 2011 to investigate alteration of glucosinolate (GS) composition in harvested
leaf tissue. Aqueous solutions of 250 µM MeJA were sprayed to saturation on
aerial plant tissues four days prior to harvest at commercial maturity. The MeJA
treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%),
and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these
genotypes over two seasons. Induction of quinone reductase (QR) activity, a
biomarker for anti-carcinogenesis, was significantly increased by the extracts
from the leaf tissue of these two cultivars. Extracts of apical leaf tissues had
greater MeJA mediated increases in phenolics, glucosinolate concentrations, GS
hydrolysis products, and QR activity than extracts from basal leaf tissue
samples. The concentration of the hydrolysis product of glucoraphanin,
sulforphane was significantly increased in apical leaf tissue of the cultivar
'Red Winter' in both 2010 and 2011. There was interaction between exogenous MeJA
treatment and environmental conditions to induce endogenous JA. Correlation
analysis revealed that indole-3-carbanol (I3C) generated from the hydrolysis of
glucobrassicin significantly correlated with QR activity (r = 0.800, P<0.001).
Concentrations required to double the specific QR activity (CD values) of I3C was
calculated at 230 µM, which is considerably weaker at induction than other
isothiocyanates like sulforphane. To confirm relationships between GS hydrolysis
products and QR activity, a range of concentrations of MeJA sprays were applied
to kale leaf tissues of both cultivars in 2011. Correlation analysis of these
results indicated that sulforaphane, NI3C, neoascorbigen, I3C, and
diindolylmethane were all significantly correlated with QR activity. Thus,
increased QR activity may be due to combined increases in phenolics (quercetin
and kaempferol) and GS hydrolysis product concentrations rather than by
individual products alone.

DOI: 10.1371/journal.pone.0103407
PMCID: PMC4118879
PMID: 25084454  [Indexed for MEDLINE]

BMC Cancer. 2014 Jul 21;14:524. doi: 10.1186/1471-2407-14-524.

Low levels of 3,3'-diindolylmethane activate estrogen receptor α and induce
proliferation of breast cancer cells in the absence of estradiol.

Marques M, Laflamme L, Benassou I, Cissokho C, Guillemette B, Gaudreau L(1).

Author information:
(1)Département de Biologie, Université de Sherbrooke, J1K 2R1 Sherbrooke, QC,
Canada. [email protected].

BACKGROUND: 3,3'-diindolylmethane (DIM) is an acid-catalyzed dimer of
idole-3-carbinol (I3C), a phytochemical found in cruciferous vegetables that
include broccoli, Brussels sprouts and cabbage. DIM is an aryl hydrocarbon
receptor (AhR) ligand and a potential anticancer agent, namely for the treatment
of breast cancer. It is also advertised as a compound that regulates sex hormone
homeostasis.
METHODS: Here we make use of RNA expression assays coupled to Chromatin
Immunoprecipitation (ChIP) in breast cancer cell lines to study the effect of DIM
on estrogen signaling. We further make use of growth assays, as well as
fluorescence-activated cell sorting (FACS) assays, to monitor cell growth.
RESULTS: In this study, we report that 'physiologically obtainable'
concentrations of DIM (10 μM) activate the estrogen receptor α (ERα) signaling
pathway in the human breast cancer cell lines MCF7 and T47D, in a 17β-estradiol
(E2)-independent manner. Accordingly, we observe induction of ERα target genes
such as GREB1 and TFF1, and an increase in cellular proliferation after treatment
with 10 μM DIM in the absence of E2. By using an ERα specific inhibitor (ICI 182
780), we confirm that the transcriptional and proliferative effects of DIM
treatment are mediated by ERα. We further show that the protein kinase A
signaling pathway participates in DIM-mediated activation of ERα. In contrast,
higher concentrations of DIM (e.g. 50 μM) have an opposite and expected effect on
cells, which is to inhibit proliferation.
CONCLUSIONS: We document an unexpected effect of DIM on cell proliferation, which
is to stimulate growth by inducing the ERα signaling pathway. Importantly, this
proliferative effect of DIM happens with potentially physiological concentrations
that can be provided by the diet or by taking caplet supplements.

DOI: 10.1186/1471-2407-14-524
PMCID: PMC4223525
PMID: 25048790  [Indexed for MEDLINE]

Br J Cancer. 2014 Sep 23;111(7):1269-74. doi: 10.1038/bjc.2014.391. Epub 2014 Jul
15.

BRCA1 mRNA levels following a 4-6-week intervention with oral
3,3'-diindolylmethane.

Kotsopoulos J(1), Zhang S(2), Akbari M(1), Salmena L(3), Llacuachaqui M(2),
Zeligs M(4), Sun P(2), Narod SA(1).

Author information:
(1)1] Familial Breast Cancer Research Unit, Women's College Research Institute,
Toronto, Ontario, M5G 1N8 Canada [2] Dalla Lana School of Public Health,
University of Toronto, Toronto, Ontario, M5T 3M7 Canada.
(2)Familial Breast Cancer Research Unit, Women's College Research Institute,
Toronto, Ontario, M5G 1N8 Canada.
(3)Department of Medical Biophysics, University of Toronto, Princess Margaret
Cancer Centre, Toronto, Ontario, M5T 2M9 Canada.
(4)BioResponse, LLC, Boulder, CO 80303, USA.

BACKGROUND: Haploinsufficiency may contribute to the development of breast cancer
among women with a BRCA1 mutation. Thus, interventions that enhance BRCA1
expression may represent avenues for prevention. Studies have shown that
3,3'-diindolylmethane (DIM) can upregulate BRCA1 expression in breast cancer
cells. This has yet to be demonstrated in vivo.
METHODS: We conducted a study to evaluate the ability of oral DIM to upregulate
BRCA1 mRNA expression in white blood cells. A total of 18 women were enroled in
the study, including 13 BRCA1 mutation carriers who received 300 mg per day of
DIM for 4-6 weeks (intervention group) and 5 BRCA1 mutation
carriers who did not take DIM (control group). BRCA1 mRNA expression was assessed
at baseline and at 4-6 weeks by real-time, quantitative PCR and the relative
change in BRCA1 mRNA expression (that is, 2(-ΔΔCT)) was calculated.
RESULTS: The relative change in BRCA1 mRNA expression among women in the
intervention group achieved borderline significance (P paired t-test=0.05). In
the intervention group, BRCA1 mRNA expression increased in 10 of the
participants, decreased in 2 and remained unchanged in 1 of the participants
following DIM intervention (P sign test=0.02). On average, women in the
intervention group experienced a 34% increase in BRCA1 mRNA expression (range -24
to 194%). There was no significant difference in the relative change in BRCA1
mRNA expression among women in the control group (P paired t-test=0.45).
CONCLUSIONS: Under the tested conditions, oral DIM was associated with an
increase in BRCA1 mRNA expression in women with a BRCA1 mutation. The possibility
of mitigating the effect of an inherited deleterious BRCA1 mutation by increasing
the physiologic expression of the gene and normalising protein levels represents
a clinically important paradigm shift in the prevention strategies available to
these high-risk women. Future studies with a larger sample size and higher doses
of DIM are warranted.

DOI: 10.1038/bjc.2014.391
PMCID: PMC4183839
PMID: 25025957  [Indexed for MEDLINE]

Bioorg Med Chem Lett. 2014 Aug 15;24(16):4023-5. doi: 10.1016/j.bmcl.2014.06.009.
Epub 2014 Jun 18.

Synthesis and biological evaluation of 2,3'-diindolylmethanes as agonists of aryl
hydrocarbon receptor.

Winston-McPherson GN(1), Shu D(2), Tang W(3).

Author information:
(1)School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI
53705, United States.
(2)School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI
53705, United States; Department of Chemistry, University of Wisconsin, 1101
University Avenue, Madison, WI 53706, United States.
(3)School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI
53705, United States; Department of Chemistry, University of Wisconsin, 1101
University Avenue, Madison, WI 53706, United States. Electronic address:
[email protected].

Recent studies suggest that arylhydrocarbon receptor (AhR) may be a target for a
number of diseases. Natural product malassezin is a AhR agonist with an
interesting 2,3'-diindolylmethane skeleton. We have prepared a series of
analogues of natural product malassezin using our recently developed method and
tested the activity of these analogues against AhR in a cell-based assay. We
found that a methyl substituent at 1'-N can significantly increase the activity
and the 2-formyl group is not critical for some diindolylmethanes.

DOI: 10.1016/j.bmcl.2014.06.009
PMCID: PMC4130779
PMID: 24997686  [Indexed for MEDLINE]

Adv Pharmacol Sci. 2014;2014:832161. doi: 10.1155/2014/832161. Epub 2014 May 8.

Attenuation of Carcinogenesis and the Mechanism Underlying by the Influence of
Indole-3-carbinol and Its Metabolite 3,3'-Diindolylmethane: A Therapeutic Marvel.

Maruthanila VL(1), Poornima J(1), Mirunalini S(1).

Author information:
(1)Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai
University, Annamalainagar, Tamilnadu 608 002, India.

Rising evidence provides credible support towards the potential role of bioactive
products derived from cruciferous vegetables such as broccoli, cauliflower, kale,
cabbage, brussels sprouts, turnips, kohlrabi, bok choy, and radishes. Many
epidemiological studies point out that Brassica vegetable protects humans against
cancer since they are rich sources of glucosinolates in addition to possessing a
high content of flavonoids, vitamins, and mineral nutrients. Indole-3-carbinol
(I3C) belongs to the class of compounds called indole glucosinolate, obtained
from cruciferous vegetables, and is well-known for tits anticancer properties. In
particular, I3C and its dimeric product, 3,3'-diindolylmethane (DIM), have been
generally investigated for their value against a number of human cancers in vitro
as well as in vivo. This paper reviews an in-depth study of the anticancer
activity and the miscellaneous mechanisms underlying the anticarcinogenicity
thereby broadening its therapeutic marvel.

DOI: 10.1155/2014/832161
PMCID: PMC4060499
PMID: 24982671

Exp Oncol. 2014 Jun;36(2):90-3.

Preclinical antitumor activity of the diindolylmethane formulation in xenograft
mouse model of prostate cancer.

Kiselev VI(1), Drukh VM(1), Muyzhnek EL(2), Kuznetsov IN(3), Pchelintseva OI(1),
Paltsev MA(4).

Author information:
(1)Peoples' Friendship University of Russia, Moscow 117198, Russia.
(2)ZAO "MiraxBioPharma", Moscow 121248, Russia.
(3)Moscow State Medical Stomatological University (MGMSU), Moscow 127473, Russia.
(4)National Research Centre (NRC "Kurchatov Institute"), Moscow 123182, Russia.

AIM: Preclinical study of the specific anticancer pharmacological activity of the
formulation containing active substance 3,3ʹ-diindolylmethane (DIM), cod liver
oil, polysorbate 80 and α-tocopherol acetate (vitamin E), in vivo in a xenograft
animal model of LNCaP.
MATERIALS AND METHODS: The DIM, cod liver oil, polysorbate 80 and α-tocopherol
acetate (vitamin E) formulation was intragastrically administered to BALB/c-nude
(nu/nu) mice during 33 days post inoculation at the dose of 133 mg/kg/day.
Antitumor activity of the test drug was estimated by the rate of tumor growth
inhibition (T/C% - treated versus control), dividing the tumor volumes from
treatment groups with the control groups.
RESULTS: Statistically significant tumor xenograft regressions have been shown in
group which received the DIM, cod liver oil, polysorbate 80 and α-tocopherol
acetate (vitamin E) on the 37(th) day of observation post inoculation. The
highest antitumor activity was achieved on the 39(th) day (T/C = 16,8%).
Therapeutic effect lasts for 6 days after the end of therapy period.
CONCLUSION: Our findings demonstrate inhibitory effect of the formulation on
tumor development in the xenograft animal model due to the tumor growth rate
reduction.

PMID: 24980762  [Indexed for MEDLINE]

Exp Ther Med. 2014 Jun;7(6):1635-1638. Epub 2014 Mar 28.

3,3'-Diindolylmethane inhibits the invasion and metastasis of nasopharyngeal
carcinoma cells in vitro and in vivo by regulation of epithelial mesenchymal
transition.

Wu T(1), Chen C(1), Li F(1), Chen Z(1), Xu Y(1), Xiao B(1), Tao Z(1).

Author information:
(1)Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan
University, Wuhan, Hubei 430060, P.R. China.

Nasopharyngeal carcinoma (NPC) is characterized by silent progression and
atypical early symptoms. Early metastasis to the neck lymph nodes is common.
However, conventional chemoradiotherapy is limited and unable to effectively
control cervical lymph node metastasis of NPC. In addition, toxicities caused by
chemoradiotherapy often induce damage to normal tissues and organs. Thus, the aim
of this study was to investigate the ability of 3,3'-diindolylmethane (DIM) to
inhibit the invasion and metastasis of NPC cells in vitro and in vivo. The
migration and invasive abilities of the 5-8F human NPC cell line were detected
using a Transwell assay. Lymph node metastasis in nude mice was observed
following the implantation of xenograft tumors for 8 weeks. In addition, western
blot analysis was used to detect the expression levels of epithelial mesenchymal
transition (EMT)-associated key proteins in NPC cells treated with DIM in vitro
and in vivo. The results demonstrated that DIM effectively inhibited the
migration and invasion of NPC cells in vitro and the effect was
concentration-dependent. In addition, DIM significantly delayed and reduced the
occurrence of lymph node metastasis in the animal model. The expression levels of
a number of key proteins associated with EMT were affected by DIM treatment. In
the animal model, there were no signs of toxicity in the vital organs, including
the heart, liver and kidney, of animals fed a diet containing DIM. Therefore, the
results of the present study indicate that DIM affects the expression levels of a
number of EMT-associated key proteins and induces the inhibition of invasion and
metastasis of NPC cells in vitro and in vivo.

DOI: 10.3892/etm.2014.1649
PMCID: PMC4043566
PMID: 24926357

J Pharmacol Exp Ther. 2014 Aug;350(2):341-52. doi: 10.1124/jpet.114.214742. Epub
2014 Jun 4.

3,3'-diindolylmethane ameliorates experimental autoimmune encephalomyelitis by
promoting cell cycle arrest and apoptosis in activated T cells through microRNA
signaling pathways.

Rouse M(1), Rao R(1), Nagarkatti M(1), Nagarkatti PS(2).

Author information:
(1)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, South Carolina.
(2)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, South Carolina [email protected].

3,3'-Diindolylmethane (DIM) is a naturally derived indole found in cruciferous
vegetables that has great potential as a novel and effective therapeutic agent.
In the current study, we investigated the effects of DIM post-treatment on the
regulation of activated T cells during the development of experimental autoimmune
encephalomyelitis (EAE), a murine model of multiple sclerosis. We demonstrated
that the administration of DIM 10 days after EAE induction was effective at
ameliorating disease parameters, including inflammation and central nervous
system cellular infiltration. MicroRNA (miRNA) microarray analysis revealed an
altered miRNA profile in brain infiltrating CD4(+) T cells following DIM
post-treatment of EAE mice. Additionally, bioinformatics analysis suggested the
involvement of DIM-induced miRNAs in pathways and processes that halt cell cycle
progression and promote apoptosis. Additional studies confirmed that DIM impacted
these cellular processes in activated T cells. Further evidence indicated that
DIM treatment significantly upregulated several miRNAs (miR-200c, miR-146a,
miR-16, miR-93, and miR-22) in brain CD4(+) T cells during EAE while suppressing
their associated target genes. Similarly, we found that overexpression of miR-16
in primary CD4(+) T cells led to significant downregulation of both mRNA and
protein levels of cyclin E1 and B-cell lymphoma-2, which play important roles in
regulating cell cycle progression and apoptosis. Collectively, these studies
demonstrate that DIM post-treatment leads to the amelioration of EAE development
by suppressing T-cell responses through the induction of select miRNAs that
control cell cycle progression and mediate apoptosis.

DOI: 10.1124/jpet.114.214742
PMCID: PMC4109492
PMID: 24898268  [Indexed for MEDLINE]

Integr Cancer Ther. 2014 Sep;13(5):386-95. doi: 10.1177/1534735414534728. Epub
2014 May 26.

Beyond androgen deprivation: ancillary integrative strategies for targeting the
androgen receptor addiction of prostate cancer.

McCarty MF(1), Hejazi J(2), Rastmanesh R(3).

Author information:
(1)Catalytic Longevity, Carlsbad, CA, USA [email protected].
(2)Shahid Beheshti University of Medical Sciences, Tehran, Iran.
(3)National Nutrition and Food Sciences Technology Research Institute, Tehran,
Iran.

The large majority of clinical prostate cancers remain dependent on androgen
receptor (AR) activity for proliferation even as they lose their responsiveness
to androgen deprivation or antagonism. AR activity can be maintained in these
circumstances by increased AR synthesis--often reflecting increased NF-κB
activation; upregulation of signaling pathways that promote AR activity in the
absence of androgens; and by emergence of AR mutations or splice variants lacking
the ligand-binding domain, which render the AR constitutively active. Drugs
targeting the N-terminal transactivating domain of the AR, some of which are now
in preclinical development, can be expected to inhibit the activity not only of
unmutated ARs but also of the mutant forms and splice variants selected for by
androgen deprivation. Concurrent measures that suppress AR synthesis or boost AR
turnover could be expected to complement the efficacy of such drugs. A number of
nutraceuticals that show efficacy in prostate cancer xenograft models--including
polyphenols from pomegranate, grape seed, and green tea, the crucifera metabolite
diindolylmethane, and the hormone melatonin--have the potential to suppress AR
synthesis via downregulation of NF-κB activity; clinical doses of salicylate may
have analogous efficacy. The proteasomal turnover of the AR is abetted by diets
with a high ratio of long-chain omega-3 to omega-6 fatty acids, which are
beneficial in prostate cancer xenograft models; berberine and sulforaphane, by
inhibiting AR's interaction with its chaperone Hsp90, likewise promote AR
proteasomal degradation and retard growth of human prostate cancer in nude mice.
Hinge region acetylation of the AR is required for optimal transactivational
activity, and low micromolar concentrations of the catechin
epigallocatechin-3-gallate (EGCG) can inhibit such acetylation--possibly
explaining the ability of EGCG administration to suppress androgenic activity and
cell proliferation in prostate cancer xenografts. Hence, it is proposed that
regimens featuring an N-terminal domain-targeting drug, various
nutraceuticals/drugs that downregulate NF-κB activity, and/or supplemental
intakes of fish oil, berberine, sulforaphane, and EGCG have potential for
blocking proliferation of prostate cancer by targeting its characteristic
addiction to androgen receptor activity.

DOI: 10.1177/1534735414534728
PMID: 24867960  [Indexed for MEDLINE]

Can J Physiol Pharmacol. 2014 May;92(5):363-8. doi: 10.1139/cjpp-2014-0032. Epub
2014 Mar 10.

Vitronectin [correction of Vitronetcin] promotes cell growth and inhibits
apoptotic stimuli in a human hepatoma cell line via the activation of caspases.

Zhu W(1), Liu Y, Hu K, Li W, Chen J, Li J, Yang G, Wu J, Liang X, Fu C, Hu Q.

Author information:
(1)a Department of Toxicology, Guangzhou Center for Disease Control and
Prevention, Guangzhou, China.

This study sought to understand the effects of vitronectin (VTN) on the growth of
SMMC-7721 hepatoma cells. In addition, this study examined how VTN inhibits the
induction of apoptosis in SMMC-7721 cells by 3,3'-diindolylmethane (DIM), a
metabolite of natural phytochemicals, and preliminarily investigated the
signaling molecules involved in this process. A cell proliferation reagent was
used to observe the effects of VTN on cell proliferation rates. Laser scanning
confocal microscopy was performed to observe the effects of VTN on the morphology
of tubulin, a component of the cytoskeleton. Flow cytometry and Western blotting
assays were used to observe the inhibitory effects of VTN on DIM-induced
apoptosis in SMMC-7721 cells and changes in the expression levels of the
signaling molecules involved in this process. VTN promoted tumor cell growth in a
concentration-dependent manner and inhibited apoptosis caused by the effects of
apoptosis-inducing agents. Under in vitro experimental conditions, VTN
contributed to the growth of SMMC-7721 hepatoma cells and protected them from the
effects of an apoptosis-inducing agent. These findings suggest that during
hepatocellular carcinogenesis, VTN may promote tumor cell growth and inhibit
chemically induced apoptosis.

DOI: 10.1139/cjpp-2014-0032
PMID: 24784470  [Indexed for MEDLINE]

J Physiol Biochem. 2014 Jun;70(2):525-34. doi: 10.1007/s13105-014-0332-5. Epub
2014 Apr 9.

Attenuation of hyperglycemia-mediated oxidative stress by indole-3-carbinol and
its metabolite 3, 3'- diindolylmethane in C57BL/6J mice.

Jayakumar P(1), Pugalendi KV, Sankaran M.

Author information:
(1)Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai
University, Annamalainagar, 608002, Tamilnadu, India.

In this study, we have investigated the effect of the nutritive phytochemicals,
indole-3-carbinol (I3C) and its metabolite, 3, 3'- diindolylmethane (DIM) on
oxidative stress developed in type 2 diabetes mellitus (T2DM). This work was
carried out in the genetically modified mouse (C57BL/6J mice) that closely
simulated the metabolic abnormalities of the human disease after the
administration of high fat diet (HFD). Glucose, insulin, hemoglobin (Hb),
glycated hemoglobin (HbA1c), thiobarbituric acid reactive substances (TBARS),
lipid hydroperoxides (LOOH), conjugated dienes (CD), superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPx), vitamin C, vitamin E, and reduced
glutathione (GSH) levels were monitored in all the groups. Treatments positively
modulate the glucose, insulin, and Hb and HbA1c levels in HFD mice. TBARS, LOOH,
and CD were decreased in treatment groups when compared to the HFD group.
Treatments increase SOD, CAT, GPx levels (erythrocyte, liver, kidney, and heart)
and vitamin C, vitamin E, and GSH levels (plasma, liver, kidney, and heart) in
diabetic mice. From the study, it was clear that the antioxidant-scavenging
action were accelerated in mice treated with DIM than the I3C treatment group
which was comparable with the standard drug metformin.

DOI: 10.1007/s13105-014-0332-5
PMID: 24715233  [Indexed for MEDLINE]

J Chromatogr B Analyt Technol Biomed Life Sci. 2014 May 1;958:1-9. doi:
10.1016/j.jchromb.2014.02.026. Epub 2014 Mar 12.

Development and validation of an HPLC method for the simultaneous quantification
of indole-3-carbinol acetate, indole-3-carbinol, and 3,3'-diindolylmethane in
mouse plasma, liver, and kidney tissues.

Moussata J(1), Wang Z(2), Wang J(3).

Author information:
(1)Department of Pharmaceutical Sciences, College of Pharmacy, Western University
of Health Sciences, 309 E. 2(nd) Street, Pomona, CA 91766, USA.
(2)Center for Advancement of Drug Research and Evaluation, College of Pharmacy,
Western University of Health Sciences, 309 E. 2(nd) Street, Pomona, CA 91766,
USA. Electronic address: [email protected].
(3)Department of Pharmaceutical Sciences, College of Pharmacy, Western University
of Health Sciences, 309 E. 2(nd) Street, Pomona, CA 91766, USA; Center for
Advancement of Drug Research and Evaluation, College of Pharmacy, Western
University of Health Sciences, 309 E. 2(nd) Street, Pomona, CA 91766, USA.
Electronic address: [email protected].

A novel Indole-3-carbinol derivative (I3C) prodrug, indole-3-carbinol acetate
(I3CA), was synthesized and a rapid high-performance liquid chromatography (HPLC)
method for the quantification of I3CA, I3C, and the major metabolite of I3C,
diindolylmethane (DIM), in mouse plasma, liver and kidney tissues was developed
and validated. 4-Methoxy-1-methylindole was used as the internal standard.
Chromatographic separation was achieved on a Symmetry(®) C18 column (75mm×4.6mm,
3.5μm) and the detection was made at 280nm. A gradient elution was programmed
with the mobile phases of water (A) and acetonitrile (B) and a flow rate of
1ml/min. The total run time was 15min. The calibration curves were linear over
the range of 0.06-1.6μg/ml for both I3C and DIM with a correlation coefficient
(r(2)) higher than 0.997 and the lower limit of quantitation (LLOQ) of 0.06μg/ml.
The calibration curve of I3CA was linear over the range of 0.15-4.0μg/ml, with a
r(2)>0.995 and LLOQ of 0.15μg/ml. I3CA, I3C, and DIM intra-day accuracy values of
plasma, liver and kidney samples ranged from 90.0 to 101.3%, while the inter-day
ones were between 93.3 and 101.9%. Precision evaluated by the relative standard
deviation was ranged from 2.0 to 14.8% for intra-day and 1.9 to 14.4% for
inter-day variability. I3CA, I3C, and DIM were stable in mouse plasma, liver and
kidney samples containing an esterase inhibitor dichlorvos. This method was
successfully applied to a pharmacokinetic study in mice following oral and
intravenous administration of I3C and I3CA.

DOI: 10.1016/j.jchromb.2014.02.026
PMID: 24686233  [Indexed for MEDLINE]

Biomed Res Int. 2014;2014:520763. doi: 10.1155/2014/520763. Epub 2014 Jan 6.

Toward understanding the role of aryl hydrocarbon receptor in the immune system:
current progress and future trends.

Hanieh H(1).

Author information:
(1)Biological Sciences Department, King Faisal University, P.O. Box 380, Hofouf
31982, Saudi Arabia.

The immune system is regulated by distinct signaling pathways that control the
development and function of the immune cells. Accumulating evidence suggest that
ligation of aryl hydrocarbon receptor (Ahr), an environmentally responsive
transcription factor, results in multiple cross talks that are capable of
modulating these pathways and their downstream responsive genes. Most of the
immune cells respond to such modulation, and many inflammatory response-related
genes contain multiple xenobiotic-responsive elements (XREs) boxes upstream.
Active research efforts have investigated the physiological role of Ahr in
inflammation and autoimmunity using different animal models. Recently formed
paradigm has shown that activation of Ahr by 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) or 3,3'-diindolylmethane (DIM) prompts the differentiation of
CD4(+)Foxp3(+) regulatory T cells (Tregs) and inhibits T helper (Th)-17
suggesting that Ahr is an innovative therapeutic strategy for autoimmune
inflammation. These promising findings generate a basis for future clinical
practices in humans. This review addresses the current knowledge on the role of
Ahr in different immune cell compartments, with a particular focus on
inflammation and autoimmunity.

DOI: 10.1155/2014/520763
PMCID: PMC3914515
PMID: 24527450  [Indexed for MEDLINE]

PLoS One. 2014 Jan 22;9(1):e86787. doi: 10.1371/journal.pone.0086787. eCollection
2014.

Effects of sulforaphane and 3,3'-diindolylmethane on genome-wide promoter
methylation in normal prostate epithelial cells and prostate cancer cells.

Wong CP(1), Hsu A(1), Buchanan A(2), Palomera-Sanchez Z(1), Beaver LM(1),
Houseman EA(2), Williams DE(3), Dashwood RH(3), Ho E(4).

Author information:
(1)School of Biological & Population Health Sciences, Oregon State University,
Corvallis, Oregon, United States of America ; Linus Pauling Institute, Oregon
State University, Corvallis, Oregon, United States of America.
(2)School of Biological & Population Health Sciences, Oregon State University,
Corvallis, Oregon, United States of America.
(3)Linus Pauling Institute, Oregon State University, Corvallis, Oregon, United
States of America ; Department of Environmental and Molecular Toxicology, Oregon
State University, Corvallis, Oregon, United States of America.
(4)School of Biological & Population Health Sciences, Oregon State University,
Corvallis, Oregon, United States of America ; Linus Pauling Institute, Oregon
State University, Corvallis, Oregon, United States of America ; Moore Family
Center for Whole Grain Foods, Nutrition and Preventive Health, Oregon State
University, Corvallis, Oregon, United States of America.

Epigenetic changes, including aberrant DNA methylation, result in altered gene
expression and play an important role in carcinogenesis. Phytochemicals such as
sulforaphane (SFN) and 3,3'-diindolylmethane (DIM) are promising chemopreventive
agents for the treatment of prostate cancer. Both have been shown to induce
re-expression of genes, including tumor suppressor genes silenced in cancer
cells, via modulation of epigenetic marks including DNA methylation. However, it
remained unclear the effects SFN and DIM on DNA methylation at a genomic scale.
The goal of this study was to determine the genome-wide effects of SFN and DIM on
promoter methylation in normal prostate epithelial cells and prostate cancer
cells. Both SFN and DIM treatment decreased DNA methyltransferase expression in
normal prostate epithelial cells (PrEC), and androgen-dependent (LnCAP) and
androgen-independent (PC3) prostate cancer cells. The effects of SFN and DIM on
promoter methylation profiles in normal PrEC, LnCAP and PC3 prostate cancer cells
were determined using methyl-DNA immunoprecipitation followed by genome-wide DNA
methylation array. We showed widespread changes in promoter methylation patterns,
including both increased and decreased methylation, in all three prostate cell
lines in response to SFN or DIM treatments. In particular, SFN and DIM altered
promoter methylation in distinct sets of genes in PrEC, LnCAP, and PC3 cells, but
shared similar gene targets within a single cell line. We further showed that SFN
and DIM reversed many of the cancer-associated methylation alterations, including
aberrantly methylated genes that are dysregulated or are highly involved in
cancer progression. Overall, our data suggested that both SFN and DIM are
epigenetic modulators that have broad and complex effects on DNA methylation
profiles in both normal and cancerous prostate epithelial cells. Results from our
study may provide new insights into the epigenetic mechanisms by which SFN and
DIM exert their cancer chemopreventive effects.

DOI: 10.1371/journal.pone.0086787
PMCID: PMC3899342
PMID: 24466240  [Indexed for MEDLINE]

Cancer Metastasis Rev. 2014 Sep;33(2-3):629-40. doi: 10.1007/s10555-013-9478-9.

Recent progress on nutraceutical research in prostate cancer.

Li Y(1), Ahmad A, Kong D, Bao B, Sarkar FH.

Author information:
(1)Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, 740 Hudson Webber Cancer Research Center, 4100
John R, Detroit, MI, 48201, USA.

Recently, nutraceuticals have received increasing attention as the agents for
cancer prevention and supplement with conventional therapy. Prostate cancer (PCa)
is the most frequently diagnosed cancer and second leading cause of
cancer-related death in men in the US. Growing evidences from epidemiological
studies, in vitro experimental studies, animal studies, and clinical trials have
shown that nutraceuticals could be very useful for the prevention and treatment
of PCa. Several nutraceuticals including isoflavone, indole-3-carbinol,
3,3'-diindolylmethane, lycopene, (-)-epigallocatechin-3-gallate, and curcumin are
known to downregulate the signal transductions in AR, Akt, NF-κB, and other
signal transduction pathways which are vital for the development of PCa and the
progression of PCa from androgen-sensitive to castrate-resistant PCa. Therefore,
nutraceutical treatment in combination with conventional therapeutics could
achieve better treatment outcome in prostate cancer therapy. Interestingly, some
nutraceuticals could regulate the function of cancer stem cell (CSC)-related
miRNAs and associated molecules, leading to the inhibition of prostatic CSCs
which are responsible for drug resistance, tumor progression, and recurrence of
PCa. Hence, nutraceuticals may serve as powerful agents for the prevention of PCa
progression and they could also be useful in combination with chemotherapeutics
or radiotherapy. Such strategy could become a promising newer approach for the
treatment of metastatic PCa with better treatment outcome by improving overall
survival.

DOI: 10.1007/s10555-013-9478-9
PMCID: PMC4074449
PMID: 24375392  [Indexed for MEDLINE]

Cancer Epidemiol Biomarkers Prev. 2014 Feb;23(2):282-7. doi:
10.1158/1055-9965.EPI-13-0645. Epub 2013 Dec 19.

Urinary 3,3'-diindolylmethane: a biomarker of glucobrassicin exposure and
indole-3-carbinol uptake in humans.

Fujioka N(1), Ainslie-Waldman CE, Upadhyaya P, Carmella SG, Fritz VA, Rohwer C,
Fan Y, Rauch D, Le C, Hatsukami DK, Hecht SS.

Author information:
(1)Authors' Affiliations: Divisions of Hematology, Oncology, and Transplantation
and Epidemiology and Community Health; Masonic Cancer Center, and Departments of
Urology and Psychiatry, University of Minnesota, Minneapolis; Departments of Food
Science and Nutrition and Horticultural Science, University of Minnesota, St.
Paul; and Southern Research and Outreach Center, University of Minnesota, Waseca,
Minnesota.

BACKGROUND: Brassica vegetable consumption may confer a protective effect against
cancer, possibly attributable to their glucosinolates. Glucobrassicin is a
predominant glucosinolate and is the precursor of indole-3-carbinol (I3C), a
compound with anticancer effects. However, objective assessments of I3C uptake
from Brassica vegetables have not been successful.
METHODS: We conducted a randomized, crossover trial to test whether
3,3'-diindolylmethane (DIM, a metabolite of I3C) excreted in the urine after
consumption of raw Brassica vegetables with divergent glucobrassicin
concentrations is a marker of I3C uptake from such foods. Twenty-five subjects
were fed 50 g of either raw "Jade Cross" Brussels sprouts (high glucobrassicin
concentration) or "Blue Dynasty" cabbage (low glucobrassicin concentration) once
daily for 3 days. All urine was collected for 24 hours after vegetable
consumption each day. After a washout period, subjects crossed over to the
alternate vegetable. Urinary DIM was measured using a novel liquid
chromatography-electrospray ionization-tandem mass spectrometry-selected reaction
monitoring (LC-ESI-MS/MS-SRM) method with [(2)H2]DIM as internal standard.
RESULTS: Urinary DIM was consistently and significantly higher after Brussels
sprouts feeding than after cabbage feeding, as evidenced by an average difference
of 8.73 pmol/mg creatinine (95% confidence interval, 5.36-12.10; P = 0.00002).
CONCLUSION: We have successfully quantified urinary DIM after uptake of I3C from
food, and demonstrated that differences in glucobrassicin exposure are reflected
in urinary DIM levels.
IMPACT: Our LC-ESI-MS/MS-SRM method and the results of our study indicate urinary
DIM is a measure of I3C uptake from Brassica vegetables, a finding that can be
utilized in prospective epidemiologic and chemoprevention studies.

DOI: 10.1158/1055-9965.EPI-13-0645
PMCID: PMC3935417
PMID: 24357105  [Indexed for MEDLINE]

EPMA J. 2013 Dec 10;4(1):25. doi: 10.1186/1878-5085-4-25.

Comparative preclinical pharmacokinetics study of 3,3'-diindolylmethane
formulations: is personalized treatment and targeted chemoprevention in the
horizon?

Paltsev M, Kiselev V, Muyzhnek E, Drukh V(1), Kuznetsov I, Pchelintseva O.

Author information:
(1)Peoples' Friendship University of Russia, Miklukho-Maklaya St,, 6, Moscow
117198, Russia. [email protected].

BACKGROUND: 3,3'-Diindolylmethane (DIM) is known as an agent of natural origin
that provides protection against different cancers due to the broad spectrum of
its biological activities in vivo. However, this substance has a very poor
biodistribution and absorption in animal tissues. This preclinical trial was
conducted to evaluate the pharmacokinetics and bioavailability of various DIM
formulations in animal model.
MATERIALS AND METHODS: The pharmacokinetic parameters of one crystalline DIM
formulation and one liquid DIM formulation (oil solution) compared to
non-formulated crystalline DIM (control) were tested in 200 rats. The
formulations were orally administered to animals by gavage at doses of 200 mg/kg
per DIM (crystalline DIM formulation and non-formulated crystalline DIM) and
0.1 mg/kg per DIM (DIM in oil solution). DIM plasma elimination was measured
using HPLC method; after that, the area under the curve (AUC), relative
bioavailability, and absolute bioavailability were estimated for two formulations
in relation to non-formulated crystalline DIM.
RESULTS AND CONCLUSION: The highest bioavailability was achieved by administering
liquid DIM (oil solution), containing cod liver oil and polysorbate. The level of
DIM in rat blood plasma was about fivefold higher, though the 2,000-fold lower
dose was administered compared to crystalline DIM forms. The novel
pharmacological DIM substance with high bioavailability may be considered as a
promising targeted antitumor chemopreventive agent. It could be used to prevent
breast and ovarian cancer development in patients with heterozygous inherited and
sporadic BRCA1 gene mutations. Further preclinical and clinical trials are needed
to prove this concept.

DOI: 10.1186/1878-5085-4-25
PMCID: PMC4029298
PMID: 24325835

Am J Cancer Res. 2013 Nov 1;3(5):465-77. eCollection 2013.

Down-regulation of miR-221 inhibits proliferation of pancreatic cancer cells
through up-regulation of PTEN, p27(kip1), p57(kip2), and PUMA.

Sarkar S(1), Dubaybo H, Ali S, Goncalves P, Kollepara SL, Sethi S, Philip PA, Li
Y.

Author information:
(1)Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine Detroit, MI, USA.

Pancreatic cancer is the fourth leading cause of cancer related death in the US
and exhibits aggressive features with short survival rate and high mortality.
Therefore, it is important to understand the molecular mechanism(s) involved in
the aggressive growth of pancreatic cancers, and further design novel targeted
therapies for its treatment with better treatment outcome. In the present study,
we found that the expression of miR-221 was significantly up-regulated in
pancreatic cancer cell lines and tumor tissues compared to normal pancreatic duct
epithelial cells and normal pancreas tissues. Moreover, we found that the
pancreatic cancer patients with high miR-221 expression had a relatively shorter
survival compared to those with lower expression, suggesting that miR-221 could
be an oncogenic miRNA and a prognostic factor for poor survival of patients.
Interestingly, transfection of miR-221 inhibitor suppressed the proliferative
capacity of pancreatic cancer cells with concomitant up-regulation of PTEN,
p27(kip1), p57(kip2), and PUMA, which are the tumor suppressors and the predicted
targets of miR-221. Most importantly, we found that the treatment of pancreatic
cancer cells with isoflavone mixture (G2535), formulated 3,3'-diindolylmethane
(DIM), or synthetic curcumin analogue (CDF) could down-regulate the expression
of miR-221 and consequently up-regulate the expression of PTEN, p27(kip1),
p57(kip2), and PUMA, leading to the inhibition of cell proliferation and
migration of MiaPaCa-2 and Panc-1 cells. These results provide experimental
evidence in support of the oncogenic role of miR-221 and also demonstrate the
role of isoflavone, DIM, and CDF as potential non-toxic agents that are
capable of down-regulation of miR-221. Therefore, these agents combined with
conventional chemotherapeutics could be useful in designing novel targeted
therapeutic strategy for the treatment of pancreatic cancer for which there is no
curative therapy.

PMCID: PMC3816966
PMID: 24224124

Toxicol Appl Pharmacol. 2014 Jan 1;274(1):7-16. doi: 10.1016/j.taap.2013.10.022.
Epub 2013 Nov 5.

Natural indoles, indole-3-carbinol and 3,3'-diindolymethane, inhibit T cell
activation by staphylococcal enterotoxin B through epigenetic regulation
involving HDAC expression.

Busbee PB(1), Nagarkatti M(1), Nagarkatti PS(2).

Author information:
(1)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, SC 29208, USA.
(2)Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, SC 29208, USA. Electronic address:
[email protected].

Staphylococcal enterotoxin B (SEB) is a potent exotoxin produced by the
Staphylococcus aureus. This toxin is classified as a superantigen because of its
ability to directly bind with MHC-II class molecules followed by activation of a
large proportion of T cells bearing specific Vβ-T cell receptors. Commonly
associated with classic food poisoning, SEB has also been shown to induce toxic
shock syndrome, and is also considered to be a potential biological warfare agent
because it is easily aerosolized. In the present study, we assessed the ability
of indole-3-carbinol (I3C) and one of its byproducts, 3,3'-diindolylmethane
(DIM), found in cruciferous vegetables, to counteract the effects of SEB-induced
activation of T cells in mice. Both I3C and DIM were found to decrease the
activation, proliferation, and cytokine production by SEB-activated Vβ8(+) T
cells in vitro and in vivo. Interestingly, inhibitors of histone deacetylase
class I (HDAC-I), but not class II (HDAC-II), showed significant decrease in
SEB-induced T cell activation and cytokine production, thereby suggesting that
epigenetic modulation plays a critical role in the regulation of SEB-induced
inflammation. In addition, I3C and DIM caused a decrease in HDAC-I but not
HDAC-II in SEB-activated T cells, thereby suggesting that I3C and DIM may inhibit
SEB-mediated T cell activation by acting as HDAC-I inhibitors. These studies not
only suggest for the first time that plant-derived indoles are potent suppressors
of SEB-induced T cell activation and cytokine storm but also that they may
mediate these effects by acting as HDAC inhibitors.

DOI: 10.1016/j.taap.2013.10.022
PMCID: PMC3874587
PMID: 24200994  [Indexed for MEDLINE]

Toxicol Sci. 2014 Jan;137(1):158-67. doi: 10.1093/toxsci/kft240. Epub 2013 Oct
26.

3,3'-Diindolylmethane inhibits lipopolysaccharide-induced microglial
hyperactivation and attenuates brain inflammation.

Kim HW(1), Kim J, Kim J, Lee S, Choi BR, Han JS, Lee KW, Lee HJ.

Author information:
(1)* WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul
National University, Seoul 151-742, Republic of Korea;

Recent studies have revealed that microglial hyperactivation and
neuroinflammation are implicated in development and progression of
neurodegenerative diseases. In this study, we examined the beneficial effects of
3,3'-diindolylmethane (DIM) and indole-3-carbinol (I3C), dietary components found
in cruciferous vegetables, on brain inflammation. DIM, a major metabolite of I3C,
suppressed lipopolysaccharide (LPS)-induced expression of inducible nitric oxide
synthase and cyclooxygenase-2 in BV-2 microglia, but I3C did not. DIM, but not
I3C, attenuated DNA-binding activity of nuclear factor-κB (NF-κB) and
phosphorylation of inhibitor of κB, suggesting that DIM might inhibit microglial
hyperactivation by attenuating inflammatory transcription factor NF-κB. In
addition, DIM, but not I3C, protected primary cortical neurons from inflammatory
toxicity induced by the conditioned media from LPS-stimulated BV-2 microglia,
indicating that DIM might attenuate microglial hyperactivation-mediated neuronal
death. In an in vivo model of neuroinflammation, DIM suppressed LPS-induced brain
inflammation in mouse hippocampus, as determined by the number of Iba-1-positive
cells and the mRNA expression of F4/80. Taken together, these results suggest
that DIM may have beneficial potential against brain inflammation and
neurodegenerative diseases through the negative regulation of the NF-κB signal
pathway in microglia.

DOI: 10.1093/toxsci/kft240
PMID: 24162184  [Indexed for MEDLINE]

Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18650-5. doi:
10.1073/pnas.1308206110. Epub 2013 Oct 14.

DIM (3,3'-diindolylmethane) confers protection against ionizing radiation by a
unique mechanism.

Fan S(1), Meng Q, Xu J, Jiao Y, Zhao L, Zhang X, Sarkar FH, Brown ML, Dritschilo
A, Rosen EM.

Author information:
(1)Departments of Oncology, Radiation Medicine, and Biochemistry and Molecular
and Cell Biology, Georgetown University Medical Center, Washington, DC 20057.

Comment in
    Proc Natl Acad Sci U S A. 2013 Nov 12;110(46):18355-6.

DIM (3,3'-diindolylmethane), a small molecule compound, is a proposed cancer
preventive agent that can be safely administered to humans in repeated doses. We
report that administration of DIM in a multidose schedule protected rodents
against lethal doses of total body irradiation up to 13 Gy, whether DIM dosing
was initiated before or up to 24 h after radiation. Physiologic submicromolar
concentrations of DIM protected cultured cells against radiation by a unique
mechanism: DIM caused rapid activation of ataxia-telangiectasia mutated (ATM), a
nuclear kinase that regulates responses to DNA damage (DDR) and oxidative stress.
Subsequently, multiple ATM substrates were phosphorylated, suggesting that DIM
induces an ATM-dependent DDR-like response, and DIM enhanced radiation-induced
ATM signaling and NF-κB activation. DIM also caused activation of ATM in rodent
tissues. Activation of ATM by DIM may be due, in part, to inhibition of protein
phosphatase 2A, an upstream regulator of ATM. In contrast, DIM did not protect
human breast cancer xenograft tumors against radiation under the conditions
tested. In tumors, ATM was constitutively phosphorylated and was not further
stimulated by radiation and/or DIM. Our findings suggest that DIM is a potent
radioprotector and mitigator that functions by stimulating an ATM-driven DDR-like
response and NF-κB survival signaling.

DOI: 10.1073/pnas.1308206110
PMCID: PMC3831962
PMID: 24127581  [Indexed for MEDLINE]

Int J Oncol. 2013 Dec;43(6):1992-8. doi: 10.3892/ijo.2013.2121. Epub 2013 Oct 3.

Regulation of YAP through an Akt-dependent process by 3, 3'-diindolylmethane in
human colon cancer cells.

Li XJ(1), Leem SH, Park MH, Kim SM.

Author information:
(1)Department of Physiology, Institute for Medical Sciences, Chonbuk National
University Medical School, Jeonju, Republic of Korea.

Cancer development is a complex process governed by the interaction of several
signaling pathways. The Hippo and PI3K/Akt pathways have been shown to play a
critical role in controlling tissue growth involved in the regulation of cell
proliferation. 3, 3'-diindolylmethane (DIM) is a natural compound that
selectively kills cancer cells without causing toxicity to normal cells. This
study aims to investigate whether DIM has an effect on the Hippo signaling
pathway mediated via the PI3K/Akt signaling pathway in colon cancer cells. Our
study provides new insights into the mechanisms of crosstalk between Hippo
signaling and the Akt pathway controlling cell proliferation by PI3K inhibitor
and DIM treatment in colon cancer cells. DIM strongly potentiates the lethality
of LY294002 in HCT116 cells and inhibits proliferation of colon cancer cells via
inactivation of Akt and YAP. Thus, DIM has dramatic therapeutic effects when it
is combined with the PI3K inhibitor in the treatment of colon cancer cells. These
findings highlight the potential usefulness of DIM and can help develop
therapeutic strategies for the prevention and treatment of colon cancer.

DOI: 10.3892/ijo.2013.2121
PMID: 24100865  [Indexed for MEDLINE]

Mol Cell Biochem. 2014 Jan;385(1-2):7-15. doi: 10.1007/s11010-013-1808-2. Epub
2013 Sep 27.

Regulation of carbohydrate metabolism by indole-3-carbinol and its metabolite
3,3'-diindolylmethane in high-fat diet-induced C57BL/6J mice.

Poornima J(1), Mirunalini S.

Author information:
(1)Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai
University, Annamalainagar, 608002, Tamil Nadu, India.

Indole glucosinolates, present in cruciferous vegetables have been investigated
for their putative pharmacological properties. The current study was designed to
analyse whether the treatment of the indole glucosinolates-indole-3-carbinol
(I3C) and its metabolite 3,3'-diindolylmethane (DIM) could alter the carbohydrate
metabolism in high-fat diet (HFD)-induced C57BL/6J mice. The plasma glucose,
insulin, haemoglobin (Hb), glycosylated haemoglobin (HbA1c), glycogen and the
activities of glycolytic enzyme (hexokinase), hepatic shunt enzyme
(glucose-6-phosphate dehydrogenase), gluconeogenic enzymes (glucose-6-phosphatase
and fructose-1,6-bisphosphatase) were analysed in liver and kidney of the treated
and HFD mice. Histopathological examination of liver and pancreases were also
carried out. The HFD mice show increased glucose, insulin and HbA1c and decreased
Hb and glycogen levels. The elevated activity of glucose-6-phosphatase and
fructose-1,6-bisphosphatase and subsequent decline in the activity of glucokinase
and glucose-6-phosphate dehydrogenase were seen in HFD mice. Among treatment
groups, the mice administered with I3C and DIM, DIM shows decreased glucose,
insulin and HbA1c and increased Hb and glycogen content in liver when compared to
I3C, which was comparable with the standard drug metformin. The similar result
was also obtained in case of carbohydrate metabolism enzymes; treatment with DIM
positively regulates carbohydrate metabolic enzymes by inducing the activity of
glucokinase and glucose-6-phosphate dehydrogenase and suppressing the activity of
glucose-6-phosphatase and fructose-1,6-bisphosphatase when compared to I3C, which
were also supported by our histopathological observations.

DOI: 10.1007/s11010-013-1808-2
PMID: 24072613  [Indexed for MEDLINE]

Oncol Rep. 2013 Nov;30(5):2419-26. doi: 10.3892/or.2013.2717. Epub 2013 Sep 4.

3,3'-Diindolylmethane suppresses the growth of gastric cancer cells via
activation of the Hippo signaling pathway.

Li XJ(1), Park ES, Park MH, Kim SM.

Author information:
(1)Department of Physiology, Institute for Medical Sciences, Chonbuk National
University Medical School, Jeonju, Republic of Korea.

Recent studies have revealed that 3,3-diindolylmethane (DIM) has antitumor
effects in both in vivo and in vitro tumor models. However, the biological
function of DIM in human gastric cancer cells is unknown. Genetic and biological
studies have confirmed the importance of the novel Hippo tumor-suppressor pathway
in regulating cell proliferation, apoptosis, organ size and tumorigenesis in
mammals. Thus, the purpose of this study was to investigate the cytotoxic effects
of DIM in human gastric cancer cells and to elucidate whether DIM induces cell
death by activating the Hippo signaling pathway. Two human gastric cancer cell
lines (SNU-1 and SNU-484) were used to investigate the DIM response. DIM
significantly inhibited the proliferation of human gastric cancer cells in a
dose-dependent manner. The percentage of G1 phase cells increased 24 h following
DIM treatment. DIM reduced CDK2, CDK4, CDK6 and cyclin D1 protein levels, while
increasing p53 protein levels. DIM induced the levels of cleaved poly(ADP-ribose)
polymerase, cleaved-caspase-9, and diminished pro-caspase-3 protein production.
In addition, DIM increased pLATS1, Mob1, pMob1, pYAP and Ras association domain
family 1 (RASSF1) protein levels and reduced Yap protein production levels. DIM
stimulated the binding of RASSF1 with the Mst1/2-LATS1-Mob1 complex, promoting an
active Hippo signaling pathway and favoring YAP phosphorylation (pYAP) that
inactivates cell proliferation. Furthermore, DIM inhibited the growth of human
gastric tumors in a xenograft mouse model. These results indicate that DIM
suppresses the growth of gastric cancer cells by activating the Hippo signaling
pathway.

DOI: 10.3892/or.2013.2717
PMID: 24008339  [Indexed for MEDLINE]

Food Chem Toxicol. 2013 Dec;62:188-93. doi: 10.1016/j.fct.2013.08.055. Epub 2013
Aug 30.

Contamination of deconjugation enzymes derived from Helix pomatia with the plant
bioactive compounds 3,3'-diindolylmethane, 5-methoxypsoralen, and
8-methoxypsoralen.

Ainslie-Waldman CE(1), Simpkins SW, Upadhyaya P, Carmella SG, Hecht SS, Trudo SP.

Author information:
(1)Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles
Ave., St. Paul, MN 55108, USA; Division of Epidemiology and Community Health,
University of Minnesota, 1300 2nd St. S, Minneapolis, MN 55455, USA.

Bioactive compounds from plant foods are intensely investigated for effects on
disease prevention. β-Glucuronidase/arylsulfatase from Helix pomatia (snail) is
commonly used when quantifying exposure to metabolized dietary components.
However, we describe here the contamination of multiple formulations of this
enzyme preparation with 3,3'-diindolylmethane (DIM), 8-methoxypsoralen (8-MOP),
and 5-methoxypsoralen (5-MOP), bioactives from cruciferous and apiaceous
vegetables under investigation as putative cancer chemopreventive agents. We
identified an Escherichia coli preparation of β-glucuronidase as free from
contamination with any of the compounds tested. These results demonstrate the
importance of selecting appropriate enzyme preparations when quantifying
naturally occurring, trace level compounds in biological fluids.

DOI: 10.1016/j.fct.2013.08.055
PMCID: PMC3842406
PMID: 23994708  [Indexed for MEDLINE]

Org Lett. 2013 Sep 20;15(18):4730-3. doi: 10.1021/ol402074u. Epub 2013 Aug 29.

Sweet anion receptors: recognition of chiral carboxylate anions by
D-glucuronic-acid-decorated diindolylmethane.

Granda JM(1), Jurczak J.

Author information:
(1)Institute of Organic Chemistry, Polish Academy of Sciences , Kasprzaka 44/52,
01-224 Warsaw, Masovian, Poland.

Anion receptors containing glucuronic acid were synthesized, and their anion
binding ability studied. Chirality of anionic guests derived from mandelic acid
and amino acids can be distinguished not only in terms of stability constants but
also by significant differences in chemical shift changes for sugar moiety
protons.

DOI: 10.1021/ol402074u
PMID: 23988260  [Indexed for MEDLINE]

ChemMedChem. 2013 Nov;8(11):1873-84. doi: 10.1002/cmdc.201300273. Epub 2013 Aug
28.

Structural elaboration of a natural product: identification of
3,3'-diindolylmethane aminophosphonate and urea derivatives as potent anticancer
agents.

Kandekar S(1), Preet R, Kashyap M, Renu Prasad MU, Mohapatra P, Das D, Satapathy
SR, Siddharth S, Jain V, Choudhuri M, Kundu CN, Guchhait SK, Bharatam PV.

Author information:
(1)National Institute of Pharmaceutical Education & Research (NIPER), Sector 67,
S.A.S. Nagar (Mohali), Punjab 160062 (India).

An approach involving rational structural elaboration of the biologically active
natural product diindolylmethane (DIM) with the incorporation of aminophosphonate
and urea moieties toward the discovery of potent anticancer agents was
considered. A four-step approach for the synthesis of DIM aminophosphonate and
urea derivatives was established. These novel compounds showed potent anticancer
activities in two representative kidney and colon cancer cell lines, low toxicity
to normal cells, higher potency than the parent natural product DIM and
etoposide, and potent inhibition of cancer cell migration. Biophysical and
immunological studies, including DAPI nuclear staining, western blot analysis
with apoptotic protein markers, flow cytometry, immunocytochemistry, and comet
assays of the two most potent compounds revealed good efficacies in apoptosis and
DNA damage. It was found that down-regulation of nuclear factor κB (NF-κB p65)
could be an important mode of action in apoptosis, and the two most potent
derivatives were found to be more potent than parent compound DIM in the
down-regulation of NF-κB. Our results show the importance of structural
elaboration of DIM by rational incorporation of aminophosphonate and urea
moieties to produce potent anticancer agents; they also suggest that this
approach using other structurally simple bioactive natural products as scaffolds
holds promise for future drug discovery and development.

DOI: 10.1002/cmdc.201300273
PMID: 23983049  [Indexed for MEDLINE]

J Nutr Biochem. 2013 Nov;24(11):1882-8. doi: 10.1016/j.jnutbio.2013.05.004. Epub
2013 Aug 19.

3,3'-diindolylmethane rapidly and selectively inhibits hepatocyte growth
factor/c-Met signaling in breast cancer cells.

Nicastro HL(1), Firestone GL, Bjeldanes LF.

Author information:
(1)Department of Nutritional Science & Toxicology, University of California
Berkeley, Berkeley, CA 94720-3104. Electronic address: [email protected].

3,3'-Diindolylmethane (DIM), an indole derivative from vegetables of the Brassica
genus, has antiproliferative activity in breast cancer cells. Part of this
activity is thought to be due to DIM inhibition of Akt signaling, but an upstream
mechanism of DIM-induced Akt inhibition has not been described. The goals of this
study were to investigate the kinetics of inhibition of Akt by physiologically
relevant concentrations of DIM and to identify an upstream factor that mediates
this effect. Here we report that DIM (5-25 μM) inhibited Akt activation from 30
min to 24h in tumorigenic MDA-MB-231 cells but did not inhibit Akt activation in
non-tumorigenic preneoplastic MCF10AT cells. DIM inhibited hepatocyte growth
factor (HGF)-induced Akt activation by up to 46%, cell migration by 66% and cell
proliferation by up to 54%, but did not inhibit induction of Akt by epidermal
growth factor or insulin-like growth factor-1. DIM decreased phosphorylation of
the HGF receptor, c-Met, at tyrosines 1234 and 1235, indicating decreased
activation of the receptor. This decrease was reversed by pretreatment with
inhibitors of p38 or calcineurin. Our results demonstrate the important role of
HGF and c-Met in DIM's anti-proliferative effect on breast cancer cells and
suggest that DIM could have preventive or clinical value as an inhibitor of c-Met
signaling.

DOI: 10.1016/j.jnutbio.2013.05.004
PMCID: PMC3808088
PMID: 23968581  [Indexed for MEDLINE]

Org Lett. 2013 Aug 16;15(16):4162-5. doi: 10.1021/ol4018408. Epub 2013 Aug 5.

Platinum-catalyzed tandem indole annulation/arylation for the synthesis of
diindolylmethanes and indolo[3,2-b]carbazoles.

Shu D(1), Winston-McPherson GN, Song W, Tang W.

Author information:
(1)School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705, USA.

Various diindolylmethanes were prepared from propargylic ethers and substituted
indoles via a platinum-catalyzed tandem indole annulation/arylation cascade. The
resulting diindolylmethanes could be converted to natural product malassezin by
formylation or indolo[3,2-b]carbazoles by cyclization.

DOI: 10.1021/ol4018408
PMCID: PMC3776426
PMID: 23909946  [Indexed for MEDLINE]

Br J Pharmacol. 2013 Oct;170(3):649-60. doi: 10.1111/bph.12323.

3,3'-Diindolylmethane ameliorates experimental hepatic fibrosis via inhibiting
miR-21 expression.

Zhang Z(1), Gao Z, Hu W, Yin S, Wang C, Zang Y, Chen J, Zhang J, Dong L.

Author information:
(1)State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing, China.

BACKGROUND AND PURPOSE: Hepatic fibrosis is a type of liver disease characterized
by excessive collagen deposition produced by activated hepatic stellate cells
(HSCs), and no appropriate drug treatment is available clinically. The microRNA,
miR-21 exhibits an important role in the pathogenesis and progression of hepatic
fibrosis. 3,3'-Diindolylmethane (DIM) is a natural autolytic product in plants
and can down-regulate miR-21 expression. Here we have assessed the therapeutic
effects of DIM against hepatic fibrosis and investigated the underlying
mechanisms.
EXPERIMENTAL APPROACH: The effects of DIM on HSC activation were measured by
analysing the expression of α-smooth muscle actin and collagen I in both HSC-T6
cell line and primary HSCs. Expression of miR-21 was also measured after DIM
treatment and the therapeutic effect of DIM was further studied in vivo, using
the model of hepatic fibrosis induced by thioacetamide in mice. The antagonist
oligonucleotide, antagomir-21, was also used to suppress the effects of miR-21.
KEY RESULTS: DIM suppressed the central TGF-β signalling pathway underlying HSC
activation by down-regulating the expression of miR-21. The decreased miR-21
expression was achieved by inhibiting the activity of the transcription factor,
AP-1. Moreover, DIM blunted the activation phenotype of primary HSCs.
Administration of DIM in vivo attenuated liver fibrosis induced by thioacetamide,
as assessed by collagen deposition and profiles of profibrogenic markers.
CONCLUSIONS AND IMPLICATIONS: DIM shows potential as a therapeutic agent for the
treatment of hepatic fibrosis.

DOI: 10.1111/bph.12323
PMCID: PMC3792002
PMID: 23902531  [Indexed for MEDLINE]

Proc Natl Acad Sci U S A. 2013 Jul 23;110(30):12391-6. doi:
10.1073/pnas.1302856110. Epub 2013 Jul 8.

Modulation of natural killer cell antitumor activity by the aryl hydrocarbon
receptor.

Shin JH(1), Zhang L, Murillo-Sauca O, Kim J, Kohrt HE, Bui JD, Sunwoo JB.

Author information:
(1)Department of Otolaryngology-Head, and Neck Surgery, Stanford Cancer
Institute, Stanford Institute for Stem Cell Biology and Regenerative Medicine,
Stanford University, Stanford, CA 94305, USA.

The aryl hydrocarbon receptor (AhR) has become increasingly recognized for its
role in the differentiation and activity of immune cell subsets; however, its
role in regulating the activity of natural killer (NK) cells has not been
described. Here, we show that AhR expression is induced in murine NK cells upon
cytokine stimulation. We show that in the absence of AhR, NK cells have reduced
cytolytic activity and reduced capacity to control RMA-S tumor formation in vivo,
despite having normal development and maturation markers. Although AhR was first
identified to bind the xenobiotic compound dioxin, AhR is now known to bind a
variety of natural exogenous (e.g., dietary) and endogenous ligands. We show that
activation of AhR with an endogenous tryptophan derivative,
6-formylindolo[3,2-b]carbazole, potentiates NK cell IFN-γ production and
cytolytic activity. Further, administration of 6-formylindolo[3,2-b]carbazole in
vivo enhances NK cell control of tumors in an NK cell- and AhR-dependent manner.
Finally, similar effects on NK cell potency occur with AhR dietary ligands,
potentially explaining the numerous associations that have been observed in the
past between diet and NK cell function. Our studies introduce AhR as another
regulator of NK cell activity in vivo.

DOI: 10.1073/pnas.1302856110
PMCID: PMC3725066
PMID: 23836658  [Indexed for MEDLINE]

Curr Drug Targets. 2013 Sep;14(10):1167-74.

Regulating miRNA by natural agents as a new strategy for cancer treatment.

Sethi S(1), Li Y, Sarkar FH.

Author information:
(1)Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, MI 48201, USA.

MicroRNAs (miRNAs) are small single-strand non-coding endogenous RNAs that
regulate gene expression by multiple mechanisms. Recent evidence suggests that
miRNAs are critically involved in the pathogenesis, evolution, and progression of
cancer. The miRNAs are also crucial for the regulation of cancer stem cells
(CSCs). In addition, miRNAs are known to control the processes of
Epithelial-to-Mesenchymal Transition (EMT) of cancer cells. This evidence
suggests that miRNAs could serve as targets in cancer treatment, and as such
manipulating miRNAs could be useful for the killing CSCs or reversal of EMT
phenotype of cancer cells. Hence, targeting miRNAs, which are deregulated in
cancer, could be a promising strategy for cancer therapy. Recently, the
regulation of miRNAs by natural, nontoxic chemopreventive agents including
curcumin, resveratrol, isoflavones, (-)-epigallocatechin-3-gallate (EGCG),
lycopene, 3,3'- diindolylmethane (DIM), and indole-3-carbinol (I3C) has been
described. Therefore, natural agents could inhibit cancer progression, increase
drug sensitivity, reverse EMT, and prevent metastasis though modulation of
miRNAs, which will provide a newer therapeutic approach for cancer treatment
especially when combined with conventional therapeutics.

PMCID: PMC3899647
PMID: 23834152  [Indexed for MEDLINE]

Invest New Drugs. 2013 May 25. [Epub ahead of print]

Ring-substituted analogs of 3,3'-diindolylmethane (DIM) induce apoptosis and
necrosis in androgen-dependent and -independent prostate cancer cells.

Goldberg AA, Titorenko VI, Beach A, Abdelbaqi K, Safe S, Sanderson JT.

INRS - Institut Armand-Frappier, Université du Québec, 531 boulevard des
Prairies, Laval, QC, Canada.

We recently reported that novel ring-substituted analogs of 3,3'-diindolylmethane
(ring-DIMs) have anti-androgenic and growth inhibitory effects in
androgen-dependent prostate cancer cells. The objectives of this study were to
confirm the ability of 4,4'- and 7,7'-dibromo- and dichloro-substituted ring-DIMs
to inhibit androgen-stimulated proliferation of androgen-dependent LNCaP human
prostate cancer cells using a non-invasive, real-time monitoring technique. In
addition, their ability to induce apoptotic and necrotic cell death in
androgen-dependent as well as -independent (PC-3) prostate cancer cells was
studied. Prostate cancer cells were treated with increasing concentrations of DIM
and ring-DIMs (0.3-30 μM) and effects on cell proliferation were measured in
real-time using an xCELLigence cellular analysis system. Chromatin condensation
and loss of membrane integrity were determined by Hoechst and propidium iodide
staining, respectively. Apoptotic protein markers were measured by immunoblotting
and activation of caspases determined using selective fluorogenic substrates.
Intra- and extracellular concentrations of DIM and ring-DIMs were assessed by
electrospray ionization tandem mass spectrometry. Ring-DIMs inhibited
androgen-stimulated LNCaP cell proliferation and induced apoptosis and necrosis
in LNCaP and PC-3 cells with 2-4 fold greater potencies than DIM. DIM and the
ring-DIMs increased caspases -3, -8 and -9 activity, elevated expression of Fas,
FasL, DR4 and DR5 protein, and induced PARP cleavage in both cell lines. The
cytotoxicity of the most potent ring-DIM, 4,4'-dibromoDIM, but not the other
compounds was decreased by an inhibitor of caspase -3. The 4,4'-dibromoDIM was
primarily found in the extracellular medium, whereas all other compounds were
present to a much larger extent in the cell. In conclusion, ring-DIMs inhibited
prostate cancer cell growth and induced cell death in LNCaP and PC-3 cells with
greater potencies than DIM; they also structure-dependently activated different
cell death pathways suggesting that these compounds have clinical potential as
chemopreventive and chemotherapeutic agents in prostate cancer, regardless of
hormone-dependency.

PMID: 23709189  [PubMed - as supplied by publisher]

AAPS J. 2013 May 9. [Epub ahead of print]

Epigenetic Modifications of Nrf2 by 3,3'-diindolylmethane In Vitro in TRAMP C1
Cell Line and In Vivo TRAMP Prostate Tumors.

Wu TY, Khor TO, Su ZY, Saw CL, Shu L, Cheung KL, Huang Y, Yu S, Kong AN.

Center for Cancer Prevention Research, Department of Pharmaceutics, Ernest Mario
School of Pharmacy, Rutgers, The State University of New Jersey, Room 228, 160
Frelinghuysen Road, Piscataway, New Jersey, 08854, USA.

3,3'-diindolylmethane (DIM) is currently being investigated in many clinical
trials including prostate, breast, and cervical cancers and has been shown to
possess anticancer effects in several in vivo and in vitro models. Previously,
DIM has been reported to possess cancer chemopreventive effects in prostate
carcinogenesis in TRAMP mice; however, the in vivo mechanism is unclear. The
present study aims to investigate the in vitro and in vivo epigenetics modulation
of DIM in TRAMP-C1 cells and in TRAMP mouse model. In vitro study utilizing
TRAMP-C1 cells showed that DIM suppressed DNMT expression and reversed CpG
methylation status of Nrf2 resulting in enhanced expression of Nrf2 and
Nrf2-target gene NQO1. In vivo study, TRAMP mice fed with DIM-supplemented diet
showed much lower incidence of tumorigenesis and metastasis than the untreated
control group similar to what was reported previously. DIM increased apoptosis,
decreased cell proliferation and enhanced Nrf2 and Nrf2-target gene NQO1
expression in prostate tissues. Importantly, immunohistochemical analysis showed
that DIM reduced the global CpG 5-methylcytosine methylation. Focusing on one of
the early cancer chemopreventive target gene Nrf2, bisulfite genomic sequencing
showed that DIM decreased the methylation status of the first five CpGs of the
Nrf2 promoter region, corroborating with the results of in vitro TRAMP-C1 cells.
In summary, our current study shows that DIM is a potent cancer chemopreventive
agent for prostate cancer and epigenetic modifications of the CpG including Nrf2
could be a potential mechanism by which DIM exerts its chemopreventive effects.

PMID: 23658110

Hum Exp Toxicol. 2013 Apr;32(4):344-53. doi: 10.1177/0960327112462727.

Effect of diindolylmethane on Ca(2+) homeostasis and viability in MDCK renal
tubular cells.

Fang YC, Chou CT, Chi CC, Lin KL, Li YD, Cheng HH, Lu YC, Cheng JS, Kuo CC, Jan
CR.

Department of Laboratory Medicine, Zuoying Armed Forces General Hospital,
Kaohsiung, Taiwan.

The effect of the natural product diindolylmethane (DIM) on cytosolic Ca(2+)
concentrations ([Ca(2+)]i) and viability in MDCK renal tubular cells was
explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure
[Ca(2+)]i. DIM at concentrations 1-50 μM induced a [Ca(2+)]i rise in a
concentration-dependent manner. The response was reduced partly by removing
Ca(2+). DIM induced Mn(2+) influx leading to quenching of fura-2 fluorescence.
DIM-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365 and
protein kinase C modulators. In the absence of extracellular Ca(2+), incubation
with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or
2,5-di-tert-butylhydroquinone (BHQ) greatly inhibited DIM-induced [Ca(2+)]i rise.
Incubation with DIM abolished TG or BHQ-induced [Ca(2+)]i rise. Inhibition of
phospholipase C with U73122 reduced DIM-induced [Ca(2+)]i rise by 50%. At 1, 10,
40 and 50 μM, DIM slightly enhanced cell proliferation. The effect of 50 μM DIM
was reversed by chelating cytosolic Ca(2+) with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In sum, in MDCK cells,
DIM induced a [Ca(2+)]i rise by evoking phospholipase C-dependent Ca(2+) release
from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive
store-operated Ca(2+) channels. DIM did not induce cell death.

PMID: 23613483  [PubMed - in process]

In Vivo. 2013 May-Jun;27(3):299-304.

Evaluation of 3,3'-diindolylmethane with gardasil quadrivalent HPV vaccine in
K14-HPV16-transgenic mice cervical histology.

Sepkovic DW, Pagan DV, Stein J, Carlisle AD, Ksieski HB, Auborn K, Nyirenda T,
Bradlow HL.

Ph.D. The David and Alice Jurist Institute for Research, Hackensack University
Medical Center. Hackensack, NJ, U.S.A. [email protected].

BACKGROUND: The effects of 3,3'-diindolylmethane (DIM) together with the Gardasil
vaccine on cervical histology were evaluated using the K14-HPV16-transgenic mouse
model. The possibility that DIM could enhance the efficacy of this preventive
vaccine in this model was explored.
MATERIALS AND METHODS: Transgenic mice were given 1000 mg/kg of DIM in the diet
for 28 weeks. The mice were injected with Gardasil Quadrivalent HPV vaccine. Some
mice were sacrificed at 28 weeks. Other groups were removed from the DIM diet
after 28 weeks to a diet with no DIM for either 4 or 8 weeks.
RESULTS: Cervical histology indicated that a high percentage of transgenic mice
fed DIM and vaccinated with Gardasil manifested normal cervical epitheliums at 4
weeks after DIM discontinuation.
CONCLUSION: Vaccination pre-supplemented with DIM may provide with a window of
protection of at least four weeks in this transgenic model. However,
extrapolation to the effect in humans is beyond the limited scope of the
histological data presented here.

PMID: 23606684 

Br J Pharmacol. 2013 Apr 16. doi: 10.1111/bph.12205. [Epub ahead of print]

Indoles mitigate the development of experimental autoimmune encephalomyelitis by
induction of reciprocal differentiation of regulatory T cells and Th17 cells.

Rouse M, Singh NP, Nagarkatti PS, Nagarkatti M.

Department of Pathology, Microbiology and Immunology, University of South
Carolina School of Medicine, Columbia, SC 29208, USA.

BACKGROUND AND PURPOSE: Dietary indole derivatives, indole-3-carbinol (I3C), and
diindolylmethane (DIM), possess anti-cancer properties and exhibit the
characteristics of aryl hydrocarbon receptor (AhR) ligands. Because AhR
activation has recently been shown to regulate T cell differentiation, we tested
the hypothesis that I3C and DIM may mediate anti-inflammatory properties by
promoting regulatory T cell (T-regs) differentiation while inhibiting Th17 cells.
EXPERIMENTAL APPROACH: We investigated the therapeutic efficacy of I3C and DIM
against experimental autoimmune encephalomyelitis (EAE), a murine model of
multiple sclerosis (MS). The efficacy was evaluated based on clinical scores of
paralysis, histopathology, serum cytokines, and infiltration of T cells in the
CNS. We next studied the mechanism of induction of T cells against myelin
oligodendrocyte glycoprotein (MOG35-55 ) peptide, both in vivo and in vitro,
specifically investigating the differentiation of T-regs and Th17 cells, and
determined if indoles were acting through AhR. KEY RESULTS: Pre-treatment of EAE
mice with I3C or DIM completely prevented the clinical symptoms and cellular
infiltration into the CNS. Also, post-treatment of EAE with I3C or DIM also
proved highly effective in curtailing the overall severity of the disease. In
addition, I3C or DIM promoted the generation of T-regs, while downregulating the
induction of MOG-specific Th17 cells. The regulation of FoxP3 induction and
suppression of Th17 cells by indoles in vivo and in vitro were found to be
AhR-dependent. CONCLUSIONS AND IMPLICATIONS: Together, our studies demonstrate
for the first time that I3C and DIM may serve as novel therapeutics to suppress
neuroinflammation seen during MS through activation of AhR.

PMID: 23586923 

Cancer Prev Res (Phila). 2013 Jun;6(6):519-29. doi:
10.1158/1940-6207.CAPR-12-0419. Epub 2013 Apr 12.

Indole-3-Carbinol and 3',3'-Diindolylmethane Modulate Androgen's Effect on C-C
Chemokine Ligand 2 and Monocyte Attraction to Prostate Cancer Cells.

Kim EK, Kim YS, Milner JA, Wang TT.

U.S. Department of Agriculture, Building 307C, Room 132, BARC-EAST, 10300
Baltimore Ave., Beltsville, MD 20705. [email protected].

Inflammation has a role in prostate tumorigenesis. Recruitment of inflammatory
monocytes to the tumor site is mediated by C-C chemokine ligand 2 (CCL2) through
binding to its receptor CCR2. We hypothesized that androgen could modulate CCL2
expression in hormone-responsive prostate cancer cells and thereby promote
recruitment of monocytes. Given the inhibitory effect of broccoli-derived
compounds indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) on
androgen-dependent pathways, we also reasoned that I3C and DIM could modulate the
effect of androgen on CCL2-mediated pathways. Dihydrotestosterone was found to
induce a time-dependent (0-72 hours) and concentration-dependent (0-1 nmol/L)
increase in CCL2 mRNA levels in androgen-responsive human prostate cancer cells
(LNCaP). This increase in CCL2 mRNA corresponded with increased secretion of CCL2
protein. The effect of dihydrotestosterone was mediated through an androgen
receptor (AR)-dependent pathway as small inhibitor RNA against AR negated the
induction of CCL2. Although dihydrotestosterone also induced TWIST1 mRNA, an
epithelial-mesenchymal transition-related factor, and purported inducer of CCL2,
blocking its expression with small inhibitor RNA did not inhibit
dihydrotestosterone induction of CCL2 mRNA. Moreover, conditioned media from
androgen-treated cells promoted human monocyte THP-1 cell migration and this
effect was blocked by antibody against CCL-2. Both I3C and DIM inhibited
promotional effects of dihydrotestosterone on CCL2 and migration. These results
show that androgen may regulate CCL2 and promote inflammatory microenvironment in
prostate tumors and that this process can be blocked by broccoli-derived
compounds. Cancer Prev Res; 6(6); 519-29. ©2013 AACR.

PMID: 23585426  [PubMed - in process]

Toxicol Appl Pharmacol. 2013 Jul 15;270(2):139-48. doi:
10.1016/j.taap.2013.03.029. Epub 2013 Apr 11.

The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate
nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast
cancer cells.

Lo R, Matthews J.

Department of Pharmacology and Toxicology, University of Toronto, Toronto,
Ontario, Canada.

Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important
role in mediating cellular protection against reactive oxygen species. NRF2
signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but
inhibited by estrogen receptor alpha (ERα). In this study we investigated the
crosstalk among NRF2, AHR and ERα in MCF-7 breast cancer cells treated with the
NRF2 activator sulforaphane (SFN), a dual AHR and ERα activator,
3,3'-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or
17β-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1
(NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after
co-treatment with E2. E2-dependent repression of NQO1 and HMOX1 was associated
with increased ERα but reduced p300 recruitment and reduced histone H3
acetylation at both genes. In contrast, DIM+SFN or TCDD+SFN induced NQO1 and
HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by
RNAi-mediated knockdown of AHR. DIM+SFN but not TCDD+SFN also induced recruitment
of ERα to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored
p300 recruitment and histone H3 acetylation, thereby reversing the ERα-dependent
repression of NRF2. Taken together, our study provides further evidence of
functional interplay among NRF2, AHR and ERα signaling pathways through altered
p300 recruitment to NRF2-regulated target genes.

PMID: 23583297  [PubMed - in process]

Carcinogenesis. 2013 Apr 8. [Epub ahead of print]

In vivo and in vitro study on the role of 3, 3'-Diindolylmethane (DIM) in
treatment and prevention of nasopharyngeal carcinoma.

Chen C, Chen SM, Xu B, Chen Z, Wang F, Ren J, Xu Y, Wang Y, Xiao BK, Tao ZZ.

Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan
University, Wuhan 430060, China.

Nasopharyngeal carcinoma (NPC) is characterized by insidious progression and
atypical early symptoms, and mostly diagnosed in middle or late stages. The
long-term prognosis is not very good after conventional radiotherapy or
chemotherapy, and the therapy often has strong toxic effects on normal tissue and
organs. There were in-vitro and in-vivo pre-clinical evidences to support
chemotherapeutic and chemopreventive effect of 3, 3'-Diindolylmethane (DIM),
which was a natural compound extracted from cruciferous plants. The in-vitro
experiments showed that 100µM DIM DIM could induce remarkable apoptosis of NPC
cells, and no obvious damage was observed in normal human bronchial epithelial
cells and human liver cells(P <0.01). DIM could simultaneously regulate several
signaling pathways directly related to NPC, such as PI3K, MAPK, Akt and NF-κB. In
animal model of nasopharyngeal heterotopic transplanted tumors, the volume of
transplanted tumor in animal raised by feed containing DIM was significantly less
than that of control group (P<0.01). The animals with 2 weeks of preventive
feeding with feed containing DIM before inoculation had the smallest volume of
transplanted tumor (P<0.01). Intake of DIM had no toxic effects on vital organs
such as heart, liver and kidney of experimental animals. In summary, the results
of this study confirmed that the DIM effectively induced apoptosis of
nasopharyngeal carcinoma cells, and had a preventive and curative role in the
development and progression of nasopharyngeal carcinoma. The drug was safe and
had no toxic effects on normal tissues and organs.

PMID: 23568953  [PubMed - as supplied by publisher]

PLoS One. 2013;8(3):e59798. doi: 10.1371/journal.pone.0059798. Epub 2013 Mar 28.

2,2'-diphenyl-3,3'-diindolylmethane: a potent compound induces apoptosis in
breast cancer cells by inhibiting EGFR pathway.

Bhowmik A, Das N, Pal U, Mandal M, Bhattacharya S, Sarkar M, Jaisankar P, Maiti
NC, Ghosh MK.

Division of Cancer Biology and Inflammatory Disorder, Council of Scientific and
Industrial Research (CSIR)-Indian Institute of Chemical Biology (IICB), Kolkata,
West Bengal, India.

Despite recent advances in medicine, 30-40% of patients with breast cancer show
recurrence underscoring the need for improved effective therapy. In this study,
by in vitro screening we have selected a novel synthetic indole derivative
2,2'-diphenyl-3,3'-diindolylmethane (DPDIM) as a potential anti- breast cancer
agent. DPDIM induces apoptosis both in vitro in breast cancer cells MCF7, MDA-MB
231 and MDA-MB 468 and in vivo in 7,12-dimethylbenz[α]anthracene (DMBA) induced
Sprague-Dawley (SD) rat mammary tumor. Our in vitro studies show that DPDIM
exerts apoptotic effect by negatively regulating the activity of EGFR and its
downstream molecules like STAT3, AKT and ERK1/2 which are involved in the
proliferation and survival of these cancer cells. In silico predictions also
suggest that DPDIM may bind to EGFR at its ATP binding site. DPDIM furthermore
inhibits EGF induced increased cell viability. We have also shown decreased
expression of pro-survival factor Bcl-XL as well as increase in the level of
pro-apoptotic proteins like Bax, Bad, Bim in DPDIM treated cells in vitro and in
vivo. Our results further indicate that the DPDIM induced apoptosis is mediated
through mitochondrial apoptotic pathway involving the caspase-cascade. To the
best of our knowledge this is the first report of DPDIM for its anticancer
activity. Altogether this report suggests that DPDIM could be an effective
therapeutic agent for breast cancer.

PMCID: PMC3610887
PMID: 23555785  [PubMed - in process]

Aging Cell. 2013 Mar 23. doi: 10.1111/acel.12077. [Epub ahead of print]

Chemical genetic screen in fission yeast reveals roles for vacuolar
acidification, mitochondrial fission, and cellular GMP levels in lifespan
extension.

Stephan J, Franke J, Ehrenhofer-Murray AE.

Zentrum für Medizinische Biotechnologie, Universität Duisburg-Essen, Essen,
Germany.

The discovery that genetic mutations in several cellular pathways can increase
lifespan has lent support to the notion that pharmacological inhibition of aging
pathways can be used to extend lifespan and to slow the onset of age-related
diseases. However, so far, only few compounds with such activities have been
described. Here, we have conducted a chemical genetic screen for compounds that
cause the extension of chronological lifespan of Schizosaccharomyces pombe. We
have characterized eight natural products with such activities, which has allowed
us to uncover so far unknown anti-aging pathways in S. pombe. The ionophores
monensin and nigericin extended lifespan by affecting vacuolar acidification, and
this effect depended on the presence of the vacuolar ATPase (V-ATPase) subunits
Vma1 and Vma3. Furthermore, prostaglandin J2 displayed anti-aging properties due
to the inhibition of mitochondrial fission, and its effect on longevity required
the mitochondrial fission protein Dnm1 as well as the G-protein-coupled glucose
receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP)
synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension,
indicating that an imbalance in guanine nucleotide levels impinges upon
longevity. We furthermore have identified diindolylmethane (DIM), tschimganine,
and the compound mixture mangosteen as inhibiting aging. Taken together, these
results reveal unanticipated anti-aging activities for several phytochemicals and
open up opportunities for the development of novel anti-aging therapies.

PMID: 23521895  [PubMed - as supplied by publisher]

Eur J Med Chem. 2013 May;63:435-43. doi: 10.1016/j.ejmech.2013.02.024. Epub 2013
Feb 28.

Discovery of 3,3'-diindolylmethanes as potent antileishmanial agents.

Bharate SB, Bharate JB, Khan SI, Tekwani BL, Jacob MR, Mudududdla R, Yadav RR,
Singh B, Sharma PR, Maity S, Singh B, Khan IA, Vishwakarma RA.

Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR),
Canal Road, Jammu 180001, India. Electronic address: [email protected].

An efficient protocol for synthesis of 3,3'-diindolylmethanes using recyclable
Fe-pillared interlayered clay (Fe-PILC) catalyst under aqueous medium has been
developed. All synthesized 3,3'-diindolylmethanes showed promising
antileishmanial activity against Leishmania donovani promastigotes as well as
axenic amastigotes. Structure-activity relationship analysis revealed that
nitroaryl substituted diindolylmethanes showed potent antileishmanial activity.
The 4-nitrophenyl linked 3,3'-diindolylmethane 8g was found to be the most potent
antileishmanial analog showing IC50 values of 7.88 and 8.37 μM against both
L. donovani promastigotes and amastigotes, respectively. Further, a pharmacophore
based QSAR model was established to understand the crucial molecular features of
3,3'-diindolylmethanes essential for potent antileishmanial activity. These
compounds also exhibited promising antifungal activity against Cryptococcus
neoformans, wherein fluorophenyl substituted 3,3'-diindolylmethanes were found to
be most potent antifungal agents. Developed synthetic protocol will be useful for
economical and eco-friendly synthesis of potent antileishmanial and antifungal
3,3'-diindolylmethane class of compounds.

PMCID: PMC3674182 [Available on 2014/5/1]
PMID: 23517732  [PubMed - in process]

Oncol Lett. 2013 Feb;5(2):655-662. Epub 2012 Dec 5.

Anticancer effects of 3,3'-diindolylmethane are associated with G1 arrest and
mitochondria-dependent apoptosis in human nasopharyngeal carcinoma cells.

Xu Y, Zhang J, Shi W, Liu Y.

Departments of Otolaryngology, Wuhan University, Wuhan 430060, Hubei, P.R. China.

The antitumor effects of 3,3'-diindolylmethane (DIM) are exhibited in a number of
human cancer cells. However, there have been few studies performed concerning the
effect of DIM on nasopharyngeal cancer (NPC) cells. In the present study, we
examined the in vitro antitumor activity of DIM on the poorly differentiated NPC
cell line CNE-2. The potential molecular mechanisms of the activity were also
explored. CNE-2 cells were treated with varying concentrations of DIM for
different times. Cell proliferation and apoptosis were detected and the molecular
mechanisms involved in these effects were characterized. The results demonstrated
that DIM at concentrations of 15-100 μM caused dose- and time-dependent
inhibition of CNE-2 cell proliferation. Flow cytometry analysis revealed a high
sub-G1 cell peak following treatment with DIM, and the rate of apoptosis
increased. DIM may elevate the levels of cleaved Bid and Bax and enhance
mitochondrial membrane depolarization, allowing the efflux of cytochrome c, Smac
and Omi into the cytosol. The levels of caspases-3, -8 and -9 and cleaved poly
(ADP-ribose) polymerase (PARP) were upregulated following DIM treatment in a
dose-dependent manner. DIM also inhibits the phosphorylation of IκB-α, and showed
dose-dependent inhibition of Bcl-2, XIAP and NF-κB in CNE-2 cells in vitro. These
results indicate that DIM inhibits cell proliferation by inducing cell cycle
arrest at G0/G1 phase and induces the apoptosis of CNE-2 cells by regulating
multiple molecules in a mitochondria-dependent pathway. DIM may be a preventive
and therapeutic agent against NPC.

PMCID: PMC3573071
PMID: 23420395  [PubMed]

Antioxid Redox Signal. 2013 Mar 14. [Epub ahead of print]

Antioxidant Function of Isoflavone and 3,3'-Diindolylmethane: Are They Important
for Cancer Prevention and Therapy?

Li Y, Kong D, Ahmad A, Bao B, Sarkar FH.

1 Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine , Detroit, Michigan.

Abstract Significance: Oxidative stress has been mechanistically linked with
aging and chronic diseases, including cancer. In fact, oxidative stress status,
chronic disease-related inflammation, and cancer occurred in the aging population
are tightly correlated. It is well known that the activation of nuclear factor
kappa B (NF-κB) plays important roles in oxidative stress, inflammation, and
carcinogenesis. Therefore, targeting NF-κB is an important preventive or
therapeutic strategy against oxidative stress, inflammation, and cancer. Recent
Advances: A variety of natural compounds has been found to reduce oxidative
stress through their antioxidant activity. Among them, isoflavone,
indole-3-carbinol (I3C), and its in vivo dimeric compound 3,3'-diindolylmethane
(DIM) have shown their promising effects on the inhibition of NF-κB with
corresponding reduction of oxidative stress. Critical Issues: It has been found
that isoflavone, I3C, and DIM could inhibit cancer development and progression by
regulating multiple cellular signaling pathways that are related to oxidative
stress and significantly deregulated in cancer. Future Directions: The
antioxidative and anticancer effects of these natural agents make them strong
candidates for chemoprevention and/or therapy against human malignancies.

PMID: 23391445 

PLoS One. 2013;8(1):e53427. doi: 10.1371/journal.pone.0053427. Epub 2013 Jan 9.

3,3'-Diindolylmethane protects against cardiac hypertrophy via 5'-adenosine
monophosphate-activated protein kinase-α2.

Zong J, Deng W, Zhou H, Bian ZY, Dai J, Yuan Y, Zhang JY, Zhang R, Zhang Y, Wu
QQ, Guo HP, Li HL, Tang QZ.

Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan, China.

PURPOSE: 3,3'-Diindolylmethane (DIM) is a natural component of cruciferous
plants. It has strong antioxidant and anti-angiogenic effects and promotes the
apoptosis of a variety of tumor cells. However, little is known about the
critical role of DIM on cardiac hypertrophy. In the present study, we
investigated the effects of DIM on cardiac hypertrophy.
METHODS: Multiple molecular techniques such as Western blot analysis, real-time
PCR to determine RNA expression levels of hypertrophic, fibrotic and oxidative
stress markers, and histological analysis including H&E for histopathology, PSR
for collagen deposition, WGA for myocyte cross-sectional area, and
immunohistochemical staining for protein expression were used.
RESULTS: In pre-treatment and reverse experiments, C57/BL6 mouse chow containing
0.05% DIM (dose 100 mg/kg/d DIM) was administered one week prior to surgery or
one week after surgery, respectively, and continued for 8 weeks after surgery. In
both experiments, DIM reduced to cardiac hypertrophy and fibrosis induced by
aortic banding through the activation of 5'-adenosine monophosphate-activated
protein kinase-α2 (AMPKα2) and inhibition of mammalian target of the rapamycin
(mTOR) signaling pathway. Furthermore, DIM protected against cardiac oxidative
stress by regulating expression of estrogen-related receptor-alpha (ERRα) and
NRF2 etc. The cardioprotective effects of DIM were ablated in mice lacking
functional AMPKα2.
CONCLUSION: DIM significantly improves left ventricular function via the
activation of AMPKα2 in a murine model of cardiac hypertrophy.

PMCID: PMC3541184
PMID: 23326427  [PubMed - in process]

J Pharmacol Exp Ther. 2013 Apr;345(1):125-38. doi: 10.1124/jpet.112.201558. Epub
2013 Jan 14.

Neuroprotective efficacy and pharmacokinetic behavior of novel anti-inflammatory
para-phenyl substituted diindolylmethanes in a mouse model of Parkinson's
disease.

De Miranda BR, Miller JA, Hansen RJ, Lunghofer PJ, Safe S, Gustafson DL,
Colagiovanni D, Tjalkens RB.

Center for Environmental Medicine, Department of Environmental and Radiological
Health Sciences, Animal Cancer Center, Colorado State University, Fort Collins,
Colorado 80523-1680, USA.

There are currently no registered drugs that slow the progression of
neurodegenerative diseases, in part because translation from animal models to the
clinic has been hampered by poor distribution to the brain. The present studies
examined a selected series of para-phenyl-substituted diindolylmethane (C-DIM)
compounds that display anti-inflammatory and neuroprotective efficacy in vitro.
We postulated that the pharmacokinetic behavior of C-DIM compounds after oral
administration would correlate with neuroprotective efficacy in vivo in a mouse
model of Parkinson's disease. Pharmacokinetics and metabolism of
1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (C-DIM5),
1,1-bis(3'-indolyl)-1-(phenyl)methane,
1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (C-DIM8), and
1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12) were determined in plasma
and brain of C57Bl/6 mice after oral and intravenous administration at 10 and 1
mg/Kg, respectively. Putative metabolites were measured in plasma, liver, and
urine. C-DIM compounds given orally displayed the highest area under the curve,
Cmax, and Tmax levels, and C-DIM12 exhibited the most favorable pharmacokinetics
of the compounds tested. Oral bioavailability of each compound ranged from 6%
(C-DIM8) to 42% (C-DIM12). After pharmacokinetic studies, the neuroprotective
efficacy of C-DIM5, C-DIM8, and C-DIM12 (50 mg/Kg per oral) was examined in mice
exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid for
14 days, a model of progressive neurodegeneration with a strong neuroinflammatory
component. C-DIM5 and C-DIM12 given orally once daily after one week of exposure
to MPTP and probenecid prevented further loss of dopaminergic neurons in the
substantia nigra pars compacta and striatal dopamine terminals, indicating that
these compounds could be effective therapeutic agents to prevent
neurodegeneration.

PMID: 23318470 

Chemistry. 2013 Feb 4;19(6):2180-4. doi: 10.1002/chem.201202647. Epub 2012 Dec
20.

Carbophilic 3-component cascades: access to complex bioactive cyclopropyl
diindolylmethanes.

Groome NM, Elboray EE, Inman MW, Dondas HA, Phillips RM, Kilner C, Grigg R.

School of Chemistry, University of Leeds, Leeds, LS2 9 JT, UK.

Naturally occurring indole-3-carbinol and 3,3-diindolylmethane show bioactivity
in a number of disparate disease areas, including cancer, prompting substantial
synthetic analogue activity. We describe a new approach to highly functionalised
derivatives that starts from allene gas and proceeds via the combination of a
three-component Pd(0)-catalysed cascade with a one-pot, three-component
carbophilic Pt(II) cascade linked to a stereoselective acid-catalysed
Mannich-Michael reaction that generates complex cyclopropyl diindolylmethanes
which show selective activity against prostate cancer cell lines.

PMID: 23280956  [PubMed - in process]

Anticancer Agents Med Chem. 2012 Dec 11. [Epub ahead of print]

Targeted Regulation of PI3K/Akt/mTOR/NF-κB Signaling by Indole Compounds and
their Derivatives: Mechanistic Details and Biological Implications for Cancer
Therapy.

Ahmad A, Biersack B, Li Y, Kong D, Bao B, Schobert R, Padhye SB, Sarkar FH.

Department of Pathology and Oncology, Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, MI 48201, USA. [email protected].

Indole compounds, found in cruciferous vegetables, are potent anti-cancer agents.
Studies with indole-3-carbinol (I3C) and its dimeric product,
3,3'-diindolylmethane (DIM) suggest that these compounds have the ability to
deregulate multiple cellular signaling pathways, including PI3K/Akt/mTOR
signaling pathway. These natural compounds are also effective modulators of
downstream transcription factor NF-κB signaling which might help explain their
ability to inhibit invasion and angiogenesis, and the reversal of
epithelial-to-mesenchymal transition (EMT) phenotype and drug resistance.
Signaling through PI3K/Akt/mTOR and NF-κB pathway is increasingly being realized
to play important role in EMT through the regulation of novel miRNAs which
further validates the importance of this signaling network and its regulations by
indole compounds. Here we will review the available literature on the modulation
of PI3K/Akt/mTOR/NF-κB signaling by both parental I3C and DIM, as well as their
analogs/derivatives, in an attempt to catalog their anticancer activity.

PMID: 23272910  [PubMed - as supplied by publisher]

Carcinogenesis. 2013 Apr;34(4):841-9. doi: 10.1093/carcin/bgs390. Epub 2012 Dec
13.

Chemoprevention of lung tumorigenesis by intranasally administered
diindolylmethane in A/J mice.

Qian X, Song JM, Melkamu T, Upadhyaya P, Kassie F.

Department of Veterinary Clinical Sciences Masonic Cancer Center, University of
Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455, USA.

The main reasons for the failure of most chemopreventive agents during clinical
trials are poor in vivo bioavailability and dose-limiting side effects. One
potential approach to surmount these problems in lung cancer chemoprevention
trials could be direct delivery of agents into the pulmonary tissue. In this
study, we assessed the efficacy of intranasally delivered Diindolylmethane (DIM) against
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
(NNK)-induced lung tumorigenesis in mice. Mice treated with NNK (two doses of
50mg/kg at an interval of a week, intraperitoneal) developed 16.3±2.9 lung tumors
per mouse. Post-carcinogen administration of DIM, via intranasal instillation,
for 24 weeks, twice a week, at a dose of 2mg per mouse (0.6mg pure
diindolylmethane per mouse) reduced the lung tumor multiplicity to 4.6±2.2 tumors
per mouse (72% reduction). Likewise, large tumors (>1mm) were almost completely
abolished and multiplicities of tumors with a size of 0.5-1mm were reduced by
74%. Tumor volume was also reduced by 82%. Further studies using an in vitro
model of lung tumorigenesis showed that DIM exhibited pronounced
antiproliferative and apoptotic effects in premalignant and malignant bronchial
cells but only minimal effects in parental immortalized cells through, at least
in part, suppression of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling
pathway. These results showed the potent lung tumor inhibitory activities of low
doses of DIM given via intranasal instillation and, therefore, intranasal
delivery of DIM holds a great promise for lung cancer chemoprevention in subjects
at high risk to develop lung cancer.

PMCID: PMC3616664 [Available on 2014/4/1]
PMID: 23239747  [PubMed - in process]

EMBO Mol Med. 2012 Dec;4(12):1308-19. doi: 10.1002/emmm.201201298. Epub 2012 Nov
9.

MicroRNA-146a-mediated downregulation of IRAK1 protects mouse and human small
intestine against ischemia/reperfusion injury.

Chassin C, Hempel C, Stockinger S, Dupont A, Kübler JF, Wedemeyer J, Vandewalle
A, Hornef MW.

Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical
School, Hannover, Germany. [email protected]

Intestinal ischemia/reperfusion (I/R) injury causes inflammation and tissue
damage and is associated with high morbidity and mortality. Uncontrolled
activation of the innate immune system through toll-like receptors (Tlr) plays a
key role in I/R-mediated tissue damage but the underlying mechanisms have not
been fully resolved. Here, we identify post-transcriptional upregulation of the
essential Tlr signalling molecule interleukin 1 receptor-associated kinase (Irak)
1 as the causative mechanism for post-ischemic immune hyper-responsiveness of
intestinal epithelial cells. Increased Irak1 protein levels enhanced epithelial
ligand responsiveness, chemokine secretion, apoptosis and mucosal barrier
disruption in an experimental intestinal I/R model using wild-type, Irak1(-/-)
and Tlr4(-/-) mice and ischemic human intestinal tissue. Irak1 accumulation under
hypoxic conditions was associated with reduced K48 ubiquitination and enhanced
Senp1-mediated deSUMOylation of Irak1. Importantly, administration of microRNA
(miR)-146a or induction of miR-146a by the phytochemical diindolylmethane
controlled Irak1 upregulation and prevented immune hyper-responsiveness in mouse
and human tissue. These findings indicate that Irak1 accumulation triggers
I/R-induced epithelial immune hyper-responsiveness and suggest that the induction
of miR-146a offers a promising strategy to prevent I/R tissue injury.

PMCID: PMC3531605
PMID: 23143987  [PubMed - indexed for MEDLINE]

Curr Drug Targets. 2012 Dec;13(14):1869-75.

DIMming ovarian cancer growth.

Kandala PK, Srivastava SK.

Department of Biomedical Sciences and Cancer Biology Center, Texas Tech
University Health Sciences Center, Amarillo, Texas 79106, USA.

Ovarian cancer is the leading gynecologic malignancy with more than 22,000 new
cases and 15,000 deaths estimated each year. It is usually detected in late
stages with poor prognosis due to lack of sufficiently accurate screening tests.
Epidemiological studies continue to support the notion that consumption of
cruciferous vegetables reduces the risk of ovarian cancer. In the present review
article, we describe the anti-cancer effects of 3, 3'-diindolylmethane (DIM), a
compound present in cruciferous vegetables, against ovarian cancer. DIM targets
multiple aspects of cancer such as cellcycle regulation and survival, including
EGFR-JAK2-STAT3 signaling, checkpoint activation, caspase activation, endoplasmic
reticulum stress, autophagy and anoikis. This broad spectrum of anti-cancer
activities in conjunction with low systemic toxicity accentuates the
translational value of DIM in cancer therapy. Together, our pre-clinical studies
demonstrate that DIM has activity against ovarian cancer and hence should be
further investigated in clinical setting to exploit its therapeutic potential.

PMID: 23140296  [PubMed - in process]

Curr Drug Targets. 2012 Dec;13(14):1705-19.

Indole compounds against breast cancer: recent developments.

Biersack B, Schobert R.

Organic Chemistry Laboratory, University of Bayreuth, Universitaetsstrasse 30,
D-95440 Bayreuth, Germany. [email protected]

Breast cancer is still the leading cause of cancer deaths among women worldwide
and new therapies and drugs are continuously being conceived and explored to
better control or even cure this disease. Among the most efficacious
low-molecular drugs for the treatment of breast cancer are indole derivatives
such as 3,3'-diindolylmethane (DIM), the structurally complex antimitotic vinca
alkaloids, and the synthetic receptor tyrosine kinase inhibitor sunitinib. This
review is to give an insight into the latest developments in the field of indole
based drugs against breast cancers with a focus on those derived from natural
products and on their targets and modes of action. Pertinent literature is
covered from 2007 up to 2012.

PMID: 23140282  [PubMed - in process]

Eur J Nutr. 2012 Oct 23. [Epub ahead of print]

Modulation of CYP19 expression by cabbage juices and their active components:
indole-3-carbinol and 3,3'-diindolylmethene in human breast epithelial cell
lines.

Licznerska BE, Szaefer H, Murias M, Bartoszek A, Baer-Dubowska W.

Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences,
Poznan, Poland.

PURPOSE: The aim of this study was to evaluate the effect of white cabbage and
sauerkraut juices of different origin and indole-3-carbinol (I3C) and
diindolylmethane (DIM) on expression of CYP19 gene encoding aromatase, the key
enzyme of estrogen synthesis. METHODS: Human breast cell lines (MCF7, MDA-MB-231
and MCF10A) were examined to compare the action of cabbage juices versus their
active components (I3C, DIM). Real-time PCR and Western blot were used in order
to analyse CYP19 mRNA and protein, respectively. RESULTS: Remarkable differences
in the effect on CYP19 transcript and protein level were found between the
cabbage juices (in 2.5-25 mL/L concentrations) and indoles (in 2.5-50 μM doses)
in the three cell lines. While cabbage juices at the lower doses diminished the
aromatase expression in nontumorigenic/immortalized MCF10A breast cells
(0.25-0.86-fold change, P < 0.05), I3C and DIM were more efficient in decreasing
the aromatase expression in estrogen-dependant MCF7 breast cancer cells
(0.24-0.82-fold change, P < 0.05). Inhibition of aromatase by juice obtained from
cabbage grown on industrial farm was correlated with the induction of apoptosis
(1.7-1.8-fold change, P < 0.01) in MCF10A cells. In estrogen-independent
MDA-MB-231 cells, up-regulation of CYP19 expression by I3C and DIM (1.5-2.0-fold
change, P < 0.05) was observed. Similarly, in MCF7 cells juices increased
aromatase expression (1.1-2.2-fold change, P < 0.05). CONCLUSION: These results,
particularly that obtained in nontumorigenic/immortalized MCF10A cells, suggest
that chemopreventive activity of cabbage against breast cancer observed in
epidemiological studies may be partly explained by inhibition of the aromatase
expression.

PMID: 23090135  [PubMed - as supplied by publisher]

Mol Immunol. 2013 Apr;53(4):335-44. doi: 10.1016/j.molimm.2012.09.007. Epub 2012
Oct 17.

3,3'-Diindolylmethane alleviates oxazolone-induced colitis through Th2/Th17
suppression and Treg induction.

Huang Z, Jiang Y, Yang Y, Shao J, Sun X, Chen J, Dong L, Zhang J.

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing 210093, China.

The T cell is pivotal in orchestrating and promoting an immune response during
ulcerative colitis (UC). The aryl hydrocarbon receptor (AhR) is involved in the
regulation of T cell responses, and 3,3'-diindolylmethane (DIM) is a known ligand
of AhR. The aim of this study was to examine the therapeutic effects of DIM in
experimental colitis and to investigate the possible mechanisms underlying its
effects on mucosal T cell responses. The therapeutic effects of DIM were studied
in an oxazolone-induced colitis model. The pathologic markers of colitis were
measured, moreover, T-helper cell (Th)- and regulatory T cell (Treg)-related
transcription factor expression and associated colonic cytokine production were
determined. The impact of DIM on T cell differentiation was further investigated
in cultures of naive Th cells that were stimulated with anti-CD3/CD28 monoclonal
antibodies (mAbs). The administration of DIM attenuated experimental colitis, as
determined by pathological indices. DIM may affect signaling pathways downstream
of AhR, leading to decreased Th2/Th17 cells and increased Tregs. Ultimately, this
could result in the alleviation of experimental colitis. DIM has shown anti-UC
activity in animal models via inhibition of Th2/Th17 cells and promotion of Tregs
and may thus offer potential treatments for UC patients.

PMID: 23085552  [PubMed - indexed for MEDLINE]

PLoS One. 2012;7(10):e47186. doi: 10.1371/journal.pone.0047186. Epub 2012 Oct 9.

Activation of AMP-activated protein kinase by 3,3'-Diindolylmethane (DIM) is
associated with human prostate cancer cell death in vitro and in vivo.

Chen D, Banerjee S, Cui QC, Kong D, Sarkar FH, Dou QP.

Department of Oncology, Barbara Ann Karmanos Cancer Institute, Wayne State
University, School of Medicine, Detroit, Michigan, United States of America.

There is a large body of scientific evidence suggesting that
3,3'-Diindolylmethane (DIM), a compound derived from the digestion of
indole-3-carbinol, which is abundant in cruciferous vegetables, harbors
anti-tumor activity in vitro and in vivo. Accumulating evidence suggests that
AMP-activated protein kinase (AMPK) plays an essential role in cellular energy
homeostasis and tumor development and that targeting AMPK may be a promising
therapeutic option for cancer treatment in the clinic. We previously reported
that a formulated DIM (BR-DIM; hereafter referred as B-DIM) with higher
bioavailability was able to induce apoptosis and inhibit cell growth,
angiogenesis, and invasion of prostate cancer cells. However, the precise
molecular mechanism(s) for the anti-cancer effects of B-DIM have not been fully
elucidated. In the present study, we investigated whether AMP-activated protein
kinase (AMPK) is a molecular target of B-DIM in human prostate cancer cells. Our
results showed, for the first time, that B-DIM could activate the AMPK signaling
pathway, associated with suppression of the mammalian target of rapamycin (mTOR),
down-regulation of androgen receptor (AR) expression, and induction of apoptosis
in both androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer
cells. B-DIM also activates AMPK and down-regulates AR in androgen-independent
C4-2B prostate tumor xenografts in SCID mice. These results suggest that B-DIM
could be used as a potential anti-cancer agent in the clinic for prevention
and/or treatment of prostate cancer regardless of androgen responsiveness,
although functional AR may be required.

PMCID: PMC3467201
PMID: 23056607 

Top Curr Chem. 2012 Sep 6.

 

Cancer Chemoprevention and Nutri-Epigenetics: State of the Art and Future

Challenges.

 

Gerhauser C.

 

Division Epigenomics and Cancer Risk Factors, German Cancer Research Center, Im

Neuenheimer Feld 280, 69120, Heidelberg, Germany, [email protected].

 

The term "epigenetics" refers to modifications in gene expression caused by

heritable, but potentially reversible, changes in DNA methylation and chromatin

structure. Epigenetic alterations have been identified as promising new targets

for cancer prevention strategies as they occur early during carcinogenesis and

represent potentially initiating events for cancer development. Over the past few

years, nutri-epigenetics - the influence of dietary components on mechanisms

influencing the epigenome - has emerged as an exciting new field in current

epigenetic research. During carcinogenesis, major cellular functions and

pathways, including drug metabolism, cell cycle regulation, potential to repair

DNA damage or to induce apoptosis, response to inflammatory stimuli, cell

signalling, and cell growth control and differentiation become deregulated.

Recent evidence now indicates that epigenetic alterations contribute to these

cellular defects, for example epigenetic silencing of detoxifying enzymes, tumor

suppressor genes, cell cycle regulators, apoptosis-inducing and DNA repair genes,

nuclear receptors, signal transducers and transcription factors by promoter

methylation, and modifications of histones and non-histone proteins such as p53,

NF-κB, and the chaperone HSP90 by acetylation or methylation.The present review

will summarize the potential of natural chemopreventive agents to counteract

these cancer-related epigenetic alterations by influencing the activity or

expression of DNA methyltransferases and histone modifying enzymes.

Chemopreventive agents that target the epigenome include micronutrients (folate,

retinoic acid, and selenium compounds), butyrate, polyphenols from green tea,

apples, coffee, black raspberries, and other dietary sources, genistein and soy

isoflavones, curcumin, resveratrol, dihydrocoumarin, nordihydroguaiaretic acid

(NDGA), lycopene, anacardic acid, garcinol, constituents of Allium species and

cruciferous vegetables, including indol-3-carbinol (I3C), diindolylmethane (DIM),

sulforaphane, phenylethyl isothiocyanate (PEITC), phenylhexyl isothiocyanate

(PHI), diallyldisulfide (DADS) and its metabolite allyl mercaptan (AM), cambinol,

and relatively unexplored modulators of histone lysine methylation (chaetocin,

polyamine analogs). So far, data are still mainly derived from in vitro

investigations, and results of animal models or human intervention studies are

limited that demonstrate the functional relevance of epigenetic mechanisms for

health promoting or cancer preventive efficacy of natural products. Also, most

studies have focused on single candidate genes or mechanisms. With the emergence

of novel technologies such as next-generation sequencing, future research has the

potential to explore nutri-epigenomics at a genome-wide level to understand

better the importance of epigenetic mechanisms for gene regulation in cancer

chemoprevention.

 

PMID: 22955508 

 

Curr Cancer Drug Targets. 2012 Aug 16. [Epub ahead of print]

 

Inhibition of STAT Signalling in Bladder Cancer by Diindolylmethane - Relevance

to Cell Adhesion, Migration and Proliferation.

 

Sun Y, Cheng MK, Griffiths TR, Mellon JK, Kai B, Kriajevska M, Manson MM.

 

Department of Cancer Studies and Molecular Medicine, Biocentre, University of

Leicester, LE1 7RH, UK.

 

Effective treatments to prevent recurrence or progression of non-muscle-invasive

bladder cancer, or to inhibit metastasis of muscle-invasive forms of the disease,

would deliver significant patient benefit. Here the involvement of STAT

signalling and the chemopreventive potential of diindolylmethane (DIM) in human

bladder cancer were investigated. Muscle-invasive bladder cancer tissueswere

characterised by nuclear expression of phosphorylated STAT1, 3 and 5. In

E-cadherin positive tumour cell lines (RT112, RT4, HT1376), STAT5 was

constitutively phosphorylated, while E-cadherin negative lines (J82, T24, UMUC3)

contained phosphoSTAT3. Knockdown of STAT3induced G0/G1 arrest and inhibited

adhesion in J82cells, Knockdown of STAT1inhibited migration in J82 and RT112

lines. No significant increase in apoptosis was observed. In response to the

Janus kinase inhibitor, AG490, RT112 and J82 cells initially underwent G0/G1

arrest,with RT112 cells subsequently exhibiting S phase arrest. Phosphorylation

of STAT1Tyr701, STAT3Tyr705 and Ser727 and STAT5Tyr694 was inhibited by DIM, as

was adhesion of J82cells to collagen, an effect that was enhanced when STAT1 or 3

was reduced by siRNA. However, over-expression of STAT3C partially rescued the

DIM inhibitory effect on collagen-mediated adhesion. Migration of both lines was

inhibited by DIM, while transfection of constitutively active STAT3C enhanced

migration of RT112 cells. DIM induced cell cycle arrest and apoptosisin three

cell lines with different degrees of radioresistance. Taken together, these

results suggest that inhibition of STAT signalling and/or treatment with DIM may

decrease invasiveness of bladder cancer. DIM can induce apoptosis in cell lines

which are radioresistant, so in combination with radiotherapy may be useful in

overcomingsuch resistance.

 

PMID: 22920439 

 

J Recept Signal Transduct Res. 2012 Oct;32(5):271-8. Epub 2012 Jul 31.

 

Effect of diindolylmethane on Ca(2+) homeostasis and viability in PC3 human

prostate cancer cells.

 

Tsai JY, Chou CT, Liu SI, Liang WZ, Kuo CC, Liao WC, Lin KL, Hsu SS, Lu YC, Huang

JK, Jan CR.

 

Department of Surgery, Kaohsiung Veterans General Hospital , Kaohsiung , Taiwan.

 

The effect of the natural product diindolylmethane on cytosolic Ca(2+)

concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was

explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure

[Ca(2+)](i). Diindolylmethane at concentrations of 20-50 µM induced [Ca(2+)](i)

rise in a concentration-dependent manner. The response was reduced partly by

removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by

nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic

acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic

reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone

(BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise.

Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced

[Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced

diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 µM,

diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic

effect was not altered by chelating cytosolic Ca(2+) with

1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI

staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis

in a concentration-dependent manner. In conclusion, diindolylmethane induced a

[Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release

from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive

store-operated Ca(2+) channels. Diindolylmethane caused cell death in which

apoptosis may participate.

 

PMID: 22845469 

 

J Nutr Biochem. 2012 Jul 20.

 

3,3'-Diindolylmethane induces activating transcription factor 3 (ATF3) via ATF4

in human colorectal cancer cells.

 

Lee SH, Min KW, Zhang X, Baek SJ.

 

Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine,

University of Tennessee, Knoxville, TN 37996-4542, USA.

 

3,3'-Diindolylmethane (DIM) is a major in vivo condensation product of

indole-3-carbinol, which is present in cruciferous vegetables. Although these

compounds have been widely implicated in antitumorigenic and proapoptotic

properties in animal as well as in vitro models of cancer, the underlying

cellular mechanisms regulated by DIM are only partially understood. Activating

transcription factor 3 (ATF3) is a member of the ATF/c-AMP response

element-binding (CREB) subfamily of the basic-region leucine zipper family and

has been known to induce apoptosis in human colorectal cancer (CRC) cells. The

present study was performed to elucidate the molecular mechanism of ATF3

induction by DIM in human CRC cells. The DIM treatment induced apoptosis and

induced ATF3 gene expression at protein and messenger RNA levels. DIM increased

ATF3 promoter activity, and the region of -84 to +34 within ATF3 promoter was

responsible for promoter activation by DIM. This region contained an ATF binding

site. Deletion and point mutation of the ATF binding site (-23 to -16) abolished

ATF3 promoter activation by DIM, and overexpression of ATF4 enhanced ATF3

transactivation. Chromatin immunoprecipitation assay confirmed the binding of

ATF4 in the ATF3 promoter. Inhibition of ATF4 expression by small interference

RNA results in repression of DIM-induced ATF3 expression. The current study

demonstrates that DIM stimulates ATF3 expression through ATF4-mediated pathway

and subsequently induces apoptosis in human CRC cells.

 

PMID: 22819556 

 

Toxicol Appl Pharmacol. 2012 Sep 15;263(3):345-51. Epub 2012 Jul 16.

 

3,3'-Diindolylmethane, but not indole-3-carbinol, inhibits histone deacetylase

activity in prostate cancer cells.

 

Beaver LM, Yu TW, Sokolowski EI, Williams DE, Dashwood RH, Ho E.

 

Linus Pauling Institute, Oregon State University, 307 Linus Pauling Science

Center, Corvallis, OR 97331, USA; School of Biological and Population Health

Sciences, Oregon State University, 103 Milam Hall, Corvallis, OR 97331, USA.

 

Increased consumption of cruciferous vegetables is associated with a reduced risk

of developing prostate cancer. Indole-3-carbinol (I3C) and 3,3'-diindolylmethane

(DIM) are phytochemicals derived from cruciferous vegetables that have shown

promise in inhibiting prostate cancer in experimental models. Histone deacetylase

(HDAC) inhibition is an emerging target for cancer prevention and therapy. We

sought to examine the effects of I3C and DIM on HDACs in human prostate cancer

cell lines: androgen insensitive PC-3 cells and androgen sensitive LNCaP cells.

I3C modestly inhibited HDAC activity in LNCaP cells by 25% but no inhibition of

HDAC activity was detected in PC-3 cells. In contrast, DIM significantly

inhibited HDAC activity in both cell lines by as much as 66%. Decreases in HDAC

activity correlated with increased expression of p21, a known target of HDAC

inhibitors. DIM treatment caused a significant decrease in the expression of

HDAC2 protein in both cancer cell lines but no significant change in the protein

levels of HDAC1, HDAC3, HDAC4, HDAC6 or HDAC8 was detected. Taken together, these

results show that inhibition of HDAC activity by DIM may contribute to the

phytochemicals' anti-proliferative effects in the prostate. The ability of DIM to

target aberrant epigenetic patterns, in addition to its effects on detoxification

of carcinogens, may make it an effective chemopreventive agent by targeting

multiple stages of prostate carcinogenesis.

 

PMID: 22800507 

 

J Biol Chem. 2012 Aug 17;287(34):28745-54. Epub 2012 Jul 6.

 

Diindolylmethane-mediated Gli1 Protein Suppression Induces Anoikis in Ovarian

Cancer Cells in Vitro and Blocks Tumor Formation Ability in Vivo.

 

Kandala PK, Srivastava SK.

 

From the Department of Biomedical Sciences and Cancer Biology Center, Texas Tech

University Health Sciences Center, Amarillo, Texas 79106.

 

Anoikis is a cell death that occurs due to detachment of a cell from the

extracellular matrix (ECM). Resistance to anoikis is a primary feature of a cell

that undergoes metastasis. In this study for the first time, we demonstrated the

potential role of Gli1 in anoikis resistance. Treatment of various ovarian cancer

cells by different concentrations of diindolylmethane (DIM), an active ingredient

of cruciferous vegetables, reduced the anoikis resistance in a

concentration-dependent manner. Reduction in anoikis resistance was associated

with a decrease in the expression of Gli1 and an increase in the cleavage of

poly(ADP-ribose) polymerase (PARP). Sonic hedgehog (Shh) treatment not only

increased the expression of Gli1, but also blocked anoikis induced by DIM and

abrogated the change in the expression of Gli1 and cleaved PARP by DIM. To

confirm the role of Gli1, hedgehog inhibitor cyclopamine, Gli1 siRNA and

Gli1(-/-) mouse embryonic fibroblasts (MEFs) were used. Cyclopamine treatment

alone significantly reduced anoikis resistance in A2780 and OVCAR-429 cells.

Cyclopamine-mediated reduction in anoikis resistance was associated with reduced

expression of Gli1 and induction of cleaved PARP. Shh treatment blocked

cyclopamine-induced anoikis. Silencing Gli1 expression induced anoikis and

cleavage of PARP in A2780 and OVCAR-429 cells. Furthermore, Gli1(-/-) MEFs were

more sensitive to anoikis compared with Gli1(+/+) MEFs. Our in vivo studies

established that DIM- or cyclopamine-treated ovarian cancer cells under

suspension culture conditions drastically lost their ability of tumor formation

in vivo in mice. Taken together, our results establish that Gli1 is a critical

player in anoikis resistance in ovarian cancer.

 

PMID: 22773833  [PubMed - in process]

 

Oncol Rep. 2012 Sep;28(3):1063-8. doi: 10.3892/or.2012.1877. Epub 2012 Jun 20.

 

Anti-proliferative and pro-apoptotic effects of 3,3'-diindolylmethane in human

cervical cancer cells.

 

Zhu J, Li Y, Guan C, Chen Z.

 

Department of Gynecology, Renmin Hospital of Wuhan University, Wuchang, Wuhan

430060, PR China.

 

The antitumor effects of Indo-3-carbinol (I3C) have been proven in many human

carcinoma cells. However, the roles of 3,3-diindolylmethane (DIM), an important

polymer converted from I3C under pH 5.0-7.0, on the growth and proliferation of

cervical cancer HeLa and SiHa cells still remain unrevealed. In the present

study, we investigated the potential anti-proliferative and pro-apoptotic effects

of DIM on HeLa and SiHa cells. Cell proliferation was detected by Cell Counting

kit-8 and apoptosis was analyzed by flow cytometry. In addition, morphological

changes accompanying cell apoptosis were observed using an inverted microscope

after Hoechst 33258 staining. In addition, expression changes of proteins

involved in the MAPK and PI3K pathways were determined by western blotting. DIM

treatment inhibited the proliferation and induced apoptosis of HeLa and SiHa

cells significantly in a time- and dose-dependent manner. Moreover, SiHa cells

were more sensitive to DIM treatment than HeLa cells (P<0.05). In addition, the

expression of ERK, p38 and p-p38, which are involved in the MAPK pathway, was

downregulated by DIM treatment. Another protein involved in the MAPK pathway,

JNK, was upregulated. Furthermore, DIM treatment significantly suppressed the

expression of Akt, p-Akt, PI3K p110α, PI3K p110β, PI3K class III, GSK3-β, p-PDK1

and p-c-Raf which are involved in the PI3K pathway. These results demonstrate

that DIM exerts antitumor effects on HeLa and SiHa cells through its

anti-proliferative and pro-apoptotic roles, especially for SiHa cells. The

molecular mechanism for these effects may be related to its regulatory effects on

MAPK and PI3K pathway and apoptosis proteins. DIM may be a preventive and

therapeutic agent against cervical cancer.

 

PMID: 22736073

 

Biochem Biophys Res Commun. 2012 Jul 20;424(1):45-51. Epub 2012 Jun 19.

 

Diindolilmethane (DIM) selectively inhibits cancer stem cells.

 

Semov A, Iourtchenco L, Lin Fang L, Shengmin L, Xu Y, Su X, Muyjnek E, Kiselev V,

Alakhov V.

 

Supratek Pharma Inc., Montreal, Quebec, Canada. [email protected]

 

Epidemiologic studies repeatedly have shown chemopreventive effects of

cruciferous vegetables. Indole-3-carbinol (I3C) and its metabolite

diindolylmethane (DIM) were identified in these plants as active ingredients and

theirs anti-tumor activities were confirmed in multiple in vitro and in vivo

experiments. Here, we demonstrate that DIM is a selective and potent inhibitor of

cancer stem cells (CSCs). In several cancer cell lines, DIM inhibited tumor

sphere formation at the concentrations 30-300 times lower than concentrations

required for growth inhibition of parental cells cultured as adherent culture. We

also found that treatment with DIM overcomes chemoresistance of CSCs to

cytotoxics, such as paclitaxel, doxorubicin, and SN-38. Pre-treatment of tumor

spheres with DIM before implantation to mice significantly retarded the growth of

primary tumors compared to tumors formed by untreated tumor spheres. The

concentrations of DIM required to suppress CSCs formation are in the close range

to those achievable in human plasma after oral dosing of the compound. Therefore,

DIM can potentially be used in cancer patients, either alone, or in combinations

with existing drugs.

 

PMID: 22727906 

 

Nutr Cancer. 2012 Aug;64(6):879-88. Epub 2012 Jun 20.

 

Modulation of CYP1A1, CYP1A2 and CYP1B1 expression by cabbage juices and indoles

in human breast cell lines.

 

Szaefer H, Licznerska B, Krajka-Kuźniak V, Bartoszek A, Baer-Dubowska W.

 

Department of Pharmaceutical Biochemistry, Poznań University of Medical Sciences,

Poznań, Poland.

 

Epidemiological studies have shown that consumption of cabbage and sauerkraut is

connected with significant reduction of breast cancer incidences. Estrogens are

considered a major breast cancer risk factor and their metabolism by P450 enzymes

substantially contributes to carcinogenic activity. The aim of this study was to

investigate the effect of cabbage and sauerkraut juices of different origin on

the expression profile of the estrogen metabolism key enzymes (CYP1A1, CYP1A2,

CYP1B1) in breast cell lines MCF7, MDA-MB-231, and MCF10A. The effects of cabbage

juices were compared with that exerted by indole-3-carbinol (I3C) and

3,3'-diindolylmethane (DIM). The treatment with cabbage juices or indoles for

72 h affected the expression of CYP1 family genes in cell-type dependent manner.

Their induction was found in all cell lines, but the ratio of CYP1A1 to CYP1B1

was 1.22- to 10.6-fold in favor to CYP1A1 in MCF7 and MCF10A cells. Increased

levels of CYP1A2 in comparison with CYP1B1 were also observed in MCF7 cells. In

contrast, in MDA-MB-231 cells CYP1B1 was preferentially induced. Since the cell

lines investigated differ in invasion capacity, these results support

epidemiological observations and partly explain the mechanism of the

chemopreventive activity of white cabbage products.

 

PMID: 22716309 

 

Drug Discov Ther. 2012 Apr;6(2):94-101.

 

Regulation of Janus-activated kinase-2 (JAK2) by diindolylmethane in ovarian

cancer in vitro and in vivo.

 

Kandala PK, Srivastava SK.

 

Department of Biomedical Sciences and Cancer Biology Center, Texas Tech

University Health Sciences Center, Amarillo, TX, USA.

 

Janus-activated kinase-2 (JAK2) plays an important role in the activation of

signal transducer and activation of transcription 3 (STAT3), which is over

expressed in majority of ovarian tumors. We have reported previously that

diindolylmethane (DIM) induces apoptosis in ovarian cancer cells by inhibiting

STAT3. However, the role of JAK2 in our model was not yet understood and hence

evaluated in this report. SKOV-3 human ovarian cancer cells were used to evaluate

concentration and time dependent effects of DIM. Interleukin 3 (IL-3) and

epidermal growth factor (EGF) were used to activate JAK2. Tumor xenograft studies

were used to determine modulation of JAK2 in vivo. DIM treatment blocked the

phosphorylation of JAK2 at Tyr-1007 in a concentration-dependent manner. In a

time-dependent study, inhibition of JAK2 by DIM was as early as 1 h, which was

followed by the inhibition of STAT3 and survivin. IL-3-induced phosphorylation of

JAK2 and STAT3 was significantly blocked by DIM. IL-3 treatment blocked

DIM-induced apoptosis. EGF treatment resulted in the activation of JAK2 and STAT3

but suppressed by DIM. These results indicate the involvement of cytokines and

growth factors in the activation of JAK2/STAT3 and that DIM suppress their

activation. Furthermore, DIM in combination with cisplatin drastically reduced

the phosphorylation of JAK2 when compared to cisplatin alone. Western blot

analysis of tumors from DIM treated mice showed significant inhibition of JAK2

activation as compared with controls. These findings provide a rationale for

further clinical investigation of DIM for its potential use alone or in

combination with chemotherapy of ovarian cancer.

 

PMID: 22622019 

 

Neuro Endocrinol Lett. 2012;33(2):133-7.

 

An active product of cruciferous vegetables, 3,3'-diindolylmethane, inhibits

invasive properties of extravillous cytotrophoblastic cells.

 

Ribaux P, Irion O, Cohen M.

 

Laboratoire d'Hormonologie, Department of Gynaecology and Obstetrics, HUG,

Maternité, Geneva, Switzerland.

 

OBJECTIVES: During implantation, human trophoblastic cells have to proliferate,

migrate and invade pregnant uterus. A natural product of cruciferous vegetables,

3,3'-diindolylmethane (DIM), is known to induce some stress response genes (such

as glucose-regulated protein 78 kDa (GRP78)) and to have anti-invasive and

pro-apoptotic effects on tumor cells. Therefore, we have investigated the

potential effect of DIM on invasive extravillous cytotrophoblasts (evCTBs) cells.

MATERIALS AND METHODS: evCTBs were purified from first trimester trophoblasts and cultured in presence or not of DIM for 48h. In order to evaluate invasive

properties of cells, they were seeded on collagen-coated insert following boyden

chamber principle and matrix metalloproteinases (MMPs) and GRP78 expression was

evaluated by qPCR.

RESULTS: We showed that DIM decreases (p=0.013) invasive properties of evCTBs. In

parallel, we determined that MMP-2, -7 and -9 which are involved in evCTBs

invasion and known to be regulated by DIM, are not affected by DIM in evCTBs. In

contrast, MMP-1 mRNA is induced (p=0.03) and MMP-12 is decreased (p=0.01) in DIM

treated cells. Moreover, DIM treatment does not affect GRP78 mRNA expression in

evCTBs.

CONCLUSIONS: Collectively, the present results provide evidence that DIM does not

impact evenly on evCTBs and cancer cells.

 

PMID: 22592193

 

J Exp Clin Cancer Res. 2012 May 16;31:46.

 

A selective aryl hydrocarbon receptor modulator 3,3'-Diindolylmethane inhibits

gastric cancer cell growth.

 

Yin XF, Chen J, Mao W, Wang YH, Chen MH.

 

Department of Gastroenterology, the First Affiliated Hospital of Sun Yat-Sen

University, Guangzhou, People's Republic of China.

 

BACKGROUND: Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription

factor associated with gastric carcinogenesis. 3,3'-Diindolylmethane (DIM) is a

relatively non-toxic selective AhR modulator. This study was to detect the

effects of DIM on gastric cancer cell growth.

METHODS: Gastric cancer cell SGC7901 was treated with DIM at different

concentrations (0,10,20,30,40,50 μmol/L) with or without an AhR antagonist,

resveratrol. The expression of AhR and Cytochrome P4501A1 (CYP1A1), a classic

target gene of AhR pathway, were detected by RT-PCR and Western blot; cell

viability was measured by MTT assay, and the changes in cell cycle and apoptosis

were analyzed by flow cytometry.

RESULTS: RT-PCR and western-blot showed that with the increase of the

concentration of DIM, AhR protein gradually decreased and CYP1A1 expression

increased, suggesting that DIM activated the AhR pathway and caused the

translocation of AhR from cytoplasm to nucleus. MTT assay indicated that the

viability of SGC7901 cells was significantly decreased in a concentration- and

time-dependent manner after DIM treatment and this could be partially reversed by

resveratrol. Flow cytometry analysis showed that DIM arrested cell cycle in G1

phase and induced cell apoptosis.

CONCLUSION: Selective aryl hydrocarbon receptor modulator 3,3'-Diindolylmethane

inhibits SGC7901 cell proliferation by inducing apoptosis and delaying cell cycle

progression. AhR may be a potential therapeutic target for gastric cancer

treatment.

 

PMID: 22592002

 

Oncotarget. 2012 Apr;3(4):435-49.

 

Regulation of macroautophagy in ovarian cancer cells in vitro and in vivo by

controlling glucose regulatory protein 78 and AMPK.

 

Kandala PK, Srivastava SK.

 

Department of Biomedical Sciences and Cancer Biology Center, Texas Tech

University Health Sciences Center, Amarillo, TX, USA.

 

In this study we show that diindolylmethane (DIM) induces autophagy in ovarian

cancer cells by regulating endoplasmic reticulum (ER) stress and AMPK. Treatment

of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations

of DIM for 24 hours resulted in a concentration dependent induction of autophagy

as measured by flowcytometry. Electron microscopy confirmed the presence of

autophagosomes in DIM treated cells. Western blot analysis showed that DIM

treatment increased the expression of LC3B, a hall mark of autophagy as well as

p62 and Atg 12 proteins that are accumulated during autophagy. Autophagy

inhibitors bafilomycin or chloroquine inhibited DIM induced autophagy.

Furthermore, DIM treatment significantly increased the expression of ER stress

regulators such as Grp78, IRE1 and GADD153. Cycloheximide or ER stress inhibitor

mithramycin not only blocked ER stress proteins that were activated by DIM but

also autophagy. Silencing Grp78 or GADD 153 significantly blocked the expression

of LC3B and p62 indicating that autophagy in our model is mediated by ER stress.

Knocking out LC3B inhibited DIM induced autophagy. DIM treatment increased the

cytosolic calcium levels which lead to the activation of AMPK in our model.

Chelating cytosolic calcium with BAPT-AM abrogated not only the phosphorylation

of AMPK but also prevented DIM induced autophagy. Inhibiting AMPK by a chemical

inhibitor or siRNA blocked the induction of LC3B or p62, indicating that DIM

mediated autophagy requires activation of AMPK. Oral administration of DIM

significantly suppressed SKOV-3 tumor xenografts in nude mice. Activation of ER

stress and autophagy were observed in the tumors of DIM treated mice. Taken

together, these results suggest that induction of autophagy by DIM in ovarian

cancer cells was associated with ER stress and AMPK activation.

 

PMID: 22564965 

 

J Transl Med. 2012 May 1;10:81.

 

Molecular target based combinational therapeutic approaches in thyroid cancer.

 

Rajoria S, Suriano R, George AL, Kamat A, Schantz SP, Geliebter J, Tiwari RK.

 

Department of Microbiology and Immunology, New York Medical College, Valhalla,

New York, 10595, USA. [email protected].

 

ABSTRACT:BACKGROUND: Thyroid cancer, as with other types of cancer, is dependent

on angiogenesis for its continued growth and development. Interestingly, estrogen

has been shown to contribute to thyroid cancer aggressiveness in vitro, which is

in full support of the observed increased incidence of thyroid cancer in women

over men. Provided that estrogen has been observed to contribute to increased

angiogenesis of estrogen responsive breast cancer, it is conceivable to speculate

that estrogen also contributes to angiogenesis of estrogen responsive thyroid

cancer.

METHODS: In this study, three human thyroid cancer cells (B-CPAP, CGTH-W-1, ML-1)

were treated with estrogen alone or estrogen and anti-estrogens (fulvestrant and

3,3'-diindolylmethane, a natural dietary compound) for 24 hours. The cell culture

media was then added to human umbilical vein endothelial cell (HUVECs) and

assayed for angiogenesis associated events. Vascular endothelial growth factor

(VEGF) levels were also quantified in the conditioned media so as to evaluate if

it is a key player involved in these observations.

RESULTS: Conditioned medium from estrogen treated thyroid cancer cells enhanced

phenotypical changes (proliferation, migration and tubulogenesis) of endothelial

cells typically observed during angiogenesis. These phenotypic changes observed

in HUVECs were determined to be modulated by estrogen induced secretion of VEGF

by the cancer cells. Lastly, we show that VEGF secretion was inhibited by the

anti-estrogens, fulvestrant and 3,3'-diindolylmethane, which resulted in

diminished angiogenesis associated events in HUVECs.

CONCLUSION: Our data establishes estrogen as being a key regulator of VEGF

secretion/expression in thyroid cells which enhances the process of angiogenesis

in thyroid cancer. These findings also suggest the clinical utility of

anti-estrogens as anti-angiogenic compounds to be used as a therapeutic means to

treat thyroid cancer. We also observed that 3,3'-diindolylmethane is a promising

naturally occurring anti-estrogen which can be used as a part of therapeutic

regimen to treat thyroid cancer.

 

PMID: 22548798 

 

PLoS One. 2012;7(4):e34975. Epub 2012 Apr 13.

 

3,3'-Diindolylmethane induces G1 arrest and apoptosis in human acute T-cell

lymphoblastic leukemia cells.

 

Shorey LE, Hagman AM, Williams DE, Ho E, Dashwood RH, Benninghoff AD.

 

Department of Environmental and Molecular Toxicology, Oregon State University,

Corvallis, Oregon, United States of America.

 

Certain bioactive food components, including indole-3-carbinol (I3C) and

3,3'-diindolylmethane (DIM) from cruciferous vegetables, have been shown to

target cellular pathways regulating carcinogenesis. Previously, our laboratory

showed that dietary I3C is an effective transplacental chemopreventive agent in a

dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic

lymphoma. The primary objective of the present study was to extend our

chemoprevention studies in mice to an analogous human neoplasm in cell culture.

Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in

human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL

cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly

reduced cell proliferation and viability at concentrations 8- to 25-fold lower

than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the

G(1) phase of the cell cycle and 15 µM DIM significantly increased the percentage

of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein

expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3

(CCND3); DIM also significantly altered expression of eight transcripts related

to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8,

TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure

to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts

in immunodeficient SCID mice, reduced final tumor size by 44% and increased the

apoptotic index compared to control-fed mice. Taken together, our results

demonstrate a potential for therapeutic application of DIM in T-ALL.

 

PMID: 22514694 

 

Mol Carcinog. 2012 Apr 11. doi: 10.1002/mc.21906.

 

Pleiotropic effects of the sirtuin inhibitor sirtinol involves

concentration-dependent modulation of multiple nuclear receptor-mediated pathways

in androgen-responsive prostate cancer cell LNCaP.

 

Wang TT, Schoene NW, Kim EK, Kim YS.

 

Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research

Center, Agricultural Research Service, U.S. Department of Agriculture,

Beltsville, Maryland.

 

Sirtinol is a purported specific inhibitor of the nicotinamide adenine

dinucleotide (NAD)-dependent type III histone deacetylase (also known as

sirtuin). Sirtinol has been used extensively to identify

chemopreventive/chemotherapeutic agents that modulate the sirtuins. However, the

molecular effect of sirtinol other than serving as sirtuin inhibitor in cells is

less clear. The present study addressed this deficiency in the literature. Based

on structural similarity with plant-derived cancer preventive/therapeutic

compounds such as 3', 3'-diindolylmethane, resveratrol, and genistein, we

hypothesized that sirtinol may act on pathways similar to that affected by these

compounds in the human prostate cancer cell LNCaP. We found that treatment of

LNCaP cells with sirtinol led to concentration-dependent effects on multiple

pathways. Sirtinol inhibited LNCaP cell cycle and growth that was correlated with

up-regulation of cyclin-dependent kinase inhibitor 1A mRNA and protein levels.

This effect of sirtinol may due in part to modulation of androgen, estrogen, and

insulin-like growth factor-1 mediated pathways as sirtinol treatment led to

inhibition of mRNA and protein expression of marker genes involved in these

pathways. We also found sirtinol activates aryl hydrocarbon-dependent pathways in

LNCaP cells. The effects of sirtinol were observed at 25 µM, a concentration

lower than Ki (38 µM) for sirtuin activity. Based on these results we reasoned

that sirtinol exerts pleiotropic effects in cells and that biological effects of

sirtinol may not be due solely to inhibition of sirtuin. © 2012 Wiley

Periodicals, Inc.

 

PMID: 22495798 

 

PLoS One. 2012;7(3):e33729. Epub 2012 Mar 19.

 

Loss of let-7 up-regulates EZH2 in prostate cancer consistent with the

acquisition of cancer stem cell signatures that are attenuated by Diindolylmethane (DIM).

 

Kong D, Heath E, Chen W, Cher ML, Powell I, Heilbrun L, Li Y, Ali S, Sethi S,

Hassan O, Hwang C, Gupta N, Chitale D, Sakr WA, Menon M, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, Michigan, United States of America.

 

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the

high mortality of patients diagnosed with prostate cancer (PCa), which in part

could be attributed to the existence and the emergence of cancer stem cells

(CSCs). Recent studies have shown that deregulated expression of microRNAs

(miRNAs) contributes to the initiation and progression of PCa. Among several

known miRNAs, let-7 family appears to play a key role in the recurrence and

progression of PCa by regulating CSCs; however, the mechanism by which let-7

family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2

(EZH2), a putative target of let-7 family, was demonstrated to control stem cell

function. In this study, we found loss of let-7 family with corresponding

over-expression of EZH2 in human PCa tissue specimens, especially in higher

Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors

decreased EZH2 expression and repressed clonogenic ability and sphere-forming

capacity of PCa cells, which was consistent with inhibition of EZH2 3'UTR

luciferase activity. We also found that the treatment of PCa cells with DIM

up-regulated let-7 and down-regulated EZH2 expression, consistent with

inhibition of self-renewal and clonogenic capacity. Moreover, DIM intervention

in our on-going phase II clinical trial in patients prior to radical

prostatectomy showed upregulation of let-7 consistent with down-regulation of

EZH2 expression in PCa tissue specimens after DIM intervention. These results

suggest that the loss of let-7 mediated increased expression of EZH2 contributes

to PCa aggressiveness, which could be attenuated by DIM treatment, and thus

DIM is likely to have clinical impact.

 

PMID: 22442719 

 

 PLoS One. 2012;7(3):e33011. Epub 2012 Mar 7.

 

Targeting bone remodeling by isoflavone and 3,3'-diindolylmethane in the context

of prostate cancer bone metastasis.

 

Li Y, Kong D, Ahmad A, Bao B, Sarkar FH.

 

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State

University School of Medicine, Detroit, Michigan, United States of America.

 

Prostate cancer (PCa) bone metastases have long been believed to be osteoblastic

because of bone remodeling leading to the formation of new bone. However, recent

studies have shown increased osteolytic activity in the beginning stages of PCa

bone metastases, suggesting that targeting both osteolytic and osteoblastic

mediators would likely inhibit bone remodeling and PCa bone metastasis. In this

study, we found that PCa cells could stimulate differentiation of osteoclasts and

osteoblasts through the up-regulation of RANKL, RUNX2 and osteopontin, promoting

bone remodeling. Interestingly, we found that formulated isoflavone and

3,3'-diindolylmethane (DIM) were able to inhibit the differentiation of

osteoclasts and osteoblasts through the inhibition of cell signal transduction in

RANKL, osteoblastic, and PCa cell signaling. Moreover, we found that isoflavone

and DIM down-regulated the expression of miR-92a, which is known to be

associated with RANKL signaling, EMT and cancer progression. By pathway and

network analysis, we also observed the regulatory effects of isoflavone and

DIM on multiple signaling pathways such as AR/PSA, NKX3-1/Akt/p27, MITF, etc.

Therefore, isoflavone and DIM with their multi-targeted effects could be

useful for the prevention of PCa progression, especially by attenuating bone

metastasis mechanisms.

 

PMID: 22412975

 

PLoS One. 2012;7(2):e31783. Epub 2012 Feb 21.

 

3,3'-Diindolylmethane exhibits antileukemic activity in vitro and in vivo through

a Akt-dependent process.

 

Gao N, Cheng S, Budhraja A, Liu EH, Chen J, Chen D, Yang Z, Luo J, Shi X, Zhang

Z.

 

3,3'-Diindolylmethane (DIM), one of the active products derived from Brassica

plants, is a promising antitumor agent. The present study indicated that DIM

significantly induced apoptosis in U937 human leukemia cells in dose- and

time-dependent manners. These events were also noted in other human leukemia

cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal

bone marrow mononuclear cells. We also found that DIM-induced lethality is

associated with caspases activation, myeloid cell leukemia-1 (Mcl-1)

down-regulation, p21(cip1/waf1) up-regulation, and Akt inactivation accompanied

by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a

constitutively active Akt construct prevented DIM-mediated caspase activation,

Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality

was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway

by pharmacologic or genetic approaches attenuated DIM-induced caspases

activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor

growth of mouse U937 xenograft, which was related to induction of apoptosis and

inactivation of Akt, as well as activation of JNK. Collectively, these findings

suggest that DIM induces apoptosis in human leukemia cell lines and primary human

leukemia cells, and exhibits antileukemic activity in vivo through Akt

inactivation and JNK activation.

 

PMID: 22363731

 

In Vivo. 2012 Mar-Apr;26(2):207-11.

 

3,3'-Diindolylmethane increases serum interferon-γ levels in the K14-HPV16

transgenic mouse model for cervical cancer.

 

Sepkovic DW, Raucci L, Stein J, Carlisle AD, Auborn K, Ksieski HB, Nyirenda T,

Bradlow HL.

 

The David and Alice Jurist Institute for Research, Hackensack University Medical

Center, Hackensack, NJ, USA. [email protected]

 

While cervical cancer incidence and mortality rates have declined in the United

States, this cancer represents a worldwide threat. Human papilloma viral

infection causes cervical neoplasia (CIN). 3,3'-Diindolylmethane (DIM) prevents

or inhibits the progression from cervical dysplasia to cancer. The aim of this

study is to determine the most effective dose of DIM given continuously in food,

that significantly increases serum interferon gamma levels (IFN-γ) in the

K14-HPV16 transgenic mouse model for cervical cancer.MATERIALS AND METHODS: Five

doses of DIM in food were administered to the mouse model for 20 weeks. Serum

Interferon gamma (IFN-γ) levels and estrogen metabolite levels were quantified.

RESULTS: At 1000 ppm DIM, serum IFN-γ concentrations were significantly increased

(p<0.0396). The estrogen metabolites were unchanged. IFN-γ concentrations in CIN

free mice and the percentage of CIN free transgenic mice were well correlated

(r=0.88).

DISCUSSION: Significant increases in IFN-γ serum concentrations that correlate

with the percentage of CIN free mice in each group indicate that 1000 ppm of DIM

in food may be the most effective dose for future studies. These results may

eventually lead to new and effective vaccination strategies in women already

infected with the human papilloma virus.

 

PMID: 22351660 

 

Oncol Rep. 2012 May;27(5):1669-73. doi: 10.3892/or.2012.1662. Epub 2012 Jan 27.

 

3,3'-Diindolylmethane suppresses growth of human esophageal squamous cancer cells

by G1 cell cycle arrest.

 

Kim SJ, Lee JS, Kim SM.

 

Department of Physiology, Institute for Medical Sciences, Chonbuk National

University Medical School, Jeonju 561-180, Republic of Korea.

 

3,3'-Diindolylmethane (DIM), an active metabolite of indole-3-carbinol, is

thought to have antitumor effects in experimental animals and induce apoptosis in

various cancer cells. However, the biological functions of DIM in human

esophageal cancer cells are unknown. Thus, the purpose of this study was to

investigate the cytotoxic effects of DIM in human esophageal squamous cell

carcinoma (ESCC) cells to elucidate the molecular mechanism of cell death. Three

human ESCC cell lines (TT, TE-8 and TE-12) were used to test the response to DIM.

MTT, cell cycle and western blot analyses were conducted. DIM significantly

inhibited the proliferation of ESCC cells in a dose- and time-dependent manner.

The percentage of G1 phase cells increased 48 h after being treated with DIM. DIM

also reduced cyclin D1, cyclin E2, cyclin-dependent kinase (CDK) 4 and CDK 6

activities, and increased p15 and p27 levels. Additionally, DIM diminished

pro-caspase-9 protein expression levels and induced increased cleaved poly

(ADP-ribose) polymerase levels. These results indicate that DIM leads to G1 phase

cell cycle arrest and induces apoptosis by activating caspase-9 in ESCC cells.

 

PMID: 22293900

 

Chem Biol Interact. 2012 Feb 5;195(3):224-30. Epub 2012 Jan 24.

 

The dietary phytochemical 3,3'-diindolylmethane induces G2/M arrest and apoptosis

in oral squamous cell carcinoma by modulating Akt-NF-κB, MAPK, and p53 signaling.

 

Weng JR, Bai LY, Chiu CF, Wang YC, Tsai MH.

 

Department of Biological Science and Technology, China Medical University, 91

Hsueh-Shih Road, Taichung 40402, Taiwan. [email protected]

 

In light of the growing incidence of oral cancer in Taiwan, this study is aimed

at investigating the antitumor activity of 3,3'-diindolylmethane (DIM), an active

metabolite of the phytochemical indole-3-carbinol (I3C), in oral squamous cell

carcinoma (OSCC). DIM exhibited substantially higher antiproliferative potency

than I3C in three OSCC cell lines with IC(50) values in SCC2095, SCC9, and SCC15

cells, respectively, of 22 versus 168μM, 25 versus 176μM, and 29versus 300μM.

Flow cytometric analysis and Comet assay indicated that DIM suppressed the

viability of SCC2095 cells by inducing apoptosis and G2/M arrest. Western blot

analysis of various signaling markers revealed the ability of DIM to target

pathways mediated by Akt, mitogen-activated protein (MAP) kinases, nuclear factor

(NF)-κB, and p53, of which the concerted action underlined its antitumor

efficacy. The concomitant inactivation of Akt and MAP kinases in response to DIM

facilitated the dephosphorylation of the proapoptotic protein Bad at Ser-136 and

Ser-112, respectively. Through endoplasmic reticulum (ER) stress, DIM stimulated

the activation of p53 via Ser-15 phosphorylation, leading to increased expression

of the BH3-only proapoptotic Bcl-2 members Puma and Noxa. Together, these changes

decreased the mitochondrial threshold for apoptosis. G2/M arrest might be

attributable to the suppressive effect of DIM on the expression of cyclin B1 and

cdc25c. As many downstream effectors of the Akt-NF-κB pathway, including glycogen

synthase kinase 3β, IκB kinase α, and cyclooxygenase-2, have been shown to

promote oral tumorigenesis, the ability of DIM to inhibit this signaling axis

underscores its chemopreventive potential in oral cancer.

 

PMID: 22290291

 

BMC Med. 2012 Jan 26;10:9.

 

Diindolylmethane suppresses ovarian cancer growth and potentiates the effect of

cisplatin in tumor mouse model by targeting signal transducer and activator of

transcription 3 (STAT3).

 

Kandala PK, Srivastava SK.

 

Department of Biomedical Sciences and Cancer Biology Center, Texas Tech

University Health Sciences Center, Amarillo, TX 79106, USA.

[email protected].

 

ABSTRACT:BACKGROUND: Signal transducer and activator of transcription 3 (STAT3)

is activated in majority of ovarian tumors and confers resistance to cisplatin

treatment in patients with ovarian cancer. We have reported previously that

diindolylmethane (DIM) inhibits the growth of ovarian cancer cells. However, to

date the exact mechanism by which DIM induces growth suppressive effects has not

been clear. In this report the mode of action of DIM is investigated.

METHODS: Six human ovarian cancer cell lines and an ovarian tumor xenograft

animal model were used to study the effect of diindolylmethane alone or in

combination with cisplatin.

RESULTS: Diindolylmethane treatment induced apoptosis in all six ovarian cancer

cell lines. Phosphorylation of STAT3 at Tyr-705 and Ser-727 was reduced by DIM in

a concentration-dependent manner. In addition, diindolylmethane treatment

inhibited nuclear translocation, DNA binding, and transcriptional activity of

STAT3. Interleukin (IL)-6-induced phosphorylation of STAT3 at Tyr-705 was

significantly blocked by DIM. Overexpression of STAT3 by gene transfection

blocked DIM-induced apoptosis. In addition, DIM treatment reduced the levels of

IL-6 in ovarian cancer cells and in the tumors. DIM treatment also inhibited cell

invasion and angiogenesis by suppressing hypoxia-inducible factor 1α (HIF-1α) and

vascular epithelial growth factor (VEGF). Importantly, diindolylmethane treatment

potentiated the effects of cisplatin in SKOV-3 cells by targeting STAT3. Oral

administration of 3 mg diindolylmethane per day and subsequent administration of

cisplatin substantially inhibited in vivo tumor growth. Western blotting analysis

of tumor lysates indicated increased apoptosis and reduced STAT3 activation.

CONCLUSIONS: These findings provide a rationale for further clinical

investigation of DIM alone or in combination for chemoprevention and/or

chemotherapy of ovarian cancer.

 

PMID: 22280969

 

J Pharmacol Exp Ther. 2012 Apr;341(1):24-32. Epub 2011 Dec 28.

 

Blocking epidermal growth factor receptor activation by 3,3'-diindolylmethane

suppresses ovarian tumor growth in vitro and in vivo.

 

Kandala PK, Wright SE, Srivastava SK.

  

Genetic alterations, including the overexpression of epidermal growth factor

receptor (EGFR) (in approximately 70% of ovarian tumors), play a crucial role in

the signal transduction pathways that regulate key cellular functions, such as

cell survival and proliferation, and are responsible for compromising traditional

chemotherapy. 3,3'-Diindolylmethane (DIM) is an indole compound present in

Brassica vegetables. In our previous studies, we demonstrated that BR-DIM, a

formulated version of DIM, suppressed the growth of ovarian cancer cells by

causing cell cycle arrest and apoptosis. In the present study, we delineated the

mechanism by which DIM suppressed the growth of SKOV-3, OVCAR-3, and TOV-21G

human ovarian cancer cells. DIM treatment caused significant down-regulation of

the constitutive EGFR protein level as well as phosphorylation of EGFR at

Tyr1068, Tyr992, Tyr845, and Tyr1173 in various ovarian cancer cells. To

determine whether DIM suppressed the activation of EGFR by activating

phosphorylation, cells were treated with epidermal growth factor. Epidermal

growth factor treatment significantly blocked the DIM-mediated inhibition of EGFR

activation and apoptosis in both SKOV-3 and OVCAR-3 cells. In addition, DIM

treatment drastically reduced the phosphorylation of mitogen-activated protein

kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), which are

downstream to EGFR, without affecting their protein levels. DIM treatment also

inhibited the kinase activity of ERK, as observed by the down-regulation of

phospho-E twenty-six like transcription factor 1 (p-ELK1) in all three ovarian

cancer cell lines. DIM significantly suppressed the growth of ovarian tumors in

vivo. Tumor growth suppressive effects of DIM in SKOV-3 tumor xenografts were

associated with reduced phosphorylation of EGFR, MEK, and ERK. These results

indicate that DIM induces apoptosis in ovarian cancer cells by inhibiting the

EGFR-ERK pathway in vitro and in vivo.

 

PMID: 22205686

 

Front Biosci (Elite Ed). 2012 Jan 1;4:410-25.

 

Novel targets for detection of cancer and their modulation by chemopreventive

natural compounds.

 

Ahmad A, Sakr WA, Rahman KM.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, MI 48201, USA.

 

Cancer affects the lives of millions of people. Several signaling pathways have

been proposed as therapeutic targets for cancer therapy, and many more continue

to be validated. With the identification and validation of therapeutic targets

comes the question of designing novel strategies to effectively counter such

targets. Natural compounds from dietary sources form the basis of many ancient

medicinal systems. They are pleiotropic i.e. they act on multiple targets, and,

therefore, are often the first agents to be tested against a novel therapeutic

target. This review article summarizes the knowledge so far on some actively

pursued targets - Notch, CXCR4, Wnt and sonic hedgehog (shh) pathways, the

process of epithelial-mesenchymal transition (EMT) as well as molecular markers

such as uPA-uPAR, survivin, FoxM1, and the microRNAs. We have performed an

extensive survey of literature to list modulation of these targets by natural

agents such as curcumin, indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM),

resveratrol, epigallocatechin-3-gallate (EGCG), genistein etc. We believe that

this review will stimulate further research for elucidating and appreciating the

value of these wonderful gifts from nature.

 

PMID: 22201883

 

J Biomed Biotechnol. 2012;2012:256178. Epub 2011 Nov 13.

 

The indolic diet-derivative, 3,3'-diindolylmethane, induced apoptosis in human

colon cancer cells through upregulation of NDRG1.

 

Lerner A, Grafi-Cohen M, Napso T, Azzam N, Fares F.

 

Pediatric Gastroenterology and Nutrition Unit, Carmel Medical Center, Haifa

34362, Israel.

 

N-myc downstream regulated gene-1 participates in carcinogenesis, angiogenesis,

metastases, and anticancer drug resistance. In the present study, we analyzed the

expression pattern of N-myc downstream regulated gene-1 following treatment of

human colonic cancer cell lines; HCT-116 (well differentiated with wild-type p53

gene) and Colo-320 (poorly differentiated with mutant p53 gene), with

3,3'-diindolylmethane, a well-established proapoptotic agent product derived from

indole-3-carbinol. Treatment of Colo-320 and HCT-116 with 3,3'-diindolylmethane

disclosed inhibition of cell viability in a dose-dependent manner, mediated

through apoptosis induction. The increased expression of N-myc downstream

regulated gene-1 was detected only in poorly differentiated colon cancer cells,

Colo-320 cell line. Our results suggest that N-myc downstream regulated gene-1

expression is enhanced by 3,3'-diindolylmethane in poorly differentiated cells

and followed by induction of apoptosis. 3,3'-diindolylmethane induced apoptosis

may represent a new regulator of N-myc downstream regulated gene-1 in poorly

differentiated colonic cancer cells.

 

PMID: 22187533

 

Cancer Lett. 2012 May 1;318(1):86-92. Epub 2011 Dec 9.

 

DIM impairs radiation-induced survival pathways independently of androgen

receptor expression and augments radiation efficacy in prostate cancer.

 

Singh-Gupta V, Banerjee S, Yunker CK, Rakowski JT, Joiner MC, Konski AA, Sarkar

FH, Hillman GG.

 

Department of Radiation Oncology, Barbara Ann Karmanos Cancer Institute, Wayne

State University School of Medicine, Detroit, MI 48201, USA.

 

Increased consumption of cruciferous vegetables is associated with decreased risk

in prostate cancer (PCa). The active compound in cruciferous vegetables appears

to be the self dimerized product [3,3'-diindolylmethane (DIM)] of

indole-3-carbinol (I3C). Nutritional grade DIM has proven

safe in a Phase I trial in PCa. We investigated the anti-cancer activity of DIM

as a new biological approach to improve the effects of radiotherapy for hormone

refractory prostate cancer cells, which were either positive or negative for

androgen receptor (AR) expression. DIM inhibited cell growth in a

dose-dependent manner in both PC-3 (AR-) and C4-2B (AR+) cell lines. DIM was

effective at increasing radiation-induced cell killing in both cell lines,

independently of AR expression. DIM inhibited NF-κB and HIF-1α DNA activities

and blocked radiation-induced activation of these transcription factors in both

PC-3 and C4-2B cells. In C4-2B (AR+) cells, AR expression and nuclear

localization were significantly increased by radiation. However, DIM abrogated

the radiation-induced AR increased expression and trafficking to the nucleus,

which was consistent with decreased PSA secretion. In vivo, treatment of PC-3

prostate tumors in nude mice with DIM and radiation resulted in significant

primary tumor growth inhibition and control of metastasis to para-aortic lymph

nodes. These studies demonstrate that DIM augments radiation-induced cell

killing and tumor growth inhibition. DIM impairs critical survival signaling

pathways activated by radiation, leading to enhanced cell killing. These novel

observations suggest that DIM could be used as a safe compound to enhance the

efficacy of radiotherapy for castrate-resistant PCa.

 

PMID: 22155105

  

Toxicol Lett. 2011 Oct 10;206(2):218-28. Epub 2011 Jul 27.

 

3,3'-Diindolylmethane induces immunotoxicity via splenocyte apoptosis in neonatal

mice.

 

Roh YS, Cho A, Islam MR, Cho SD, Kim J, Kim JH, Lee JW, Lim CW, Kim B.

 

Biosafety Research Institute and Department of Pathology, College of Veterinary

Medicine, Chonbuk National University, Jeonju, South Korea.

 

3,3'-Diindolylmethane (DIM), a major product of indole-3-carbinol derived from

vegetables of the genus Brassica, exhibits chemotherapeutic activity and various

immune modulatory effects in animal models and in vitro studies. Although

extensive studies have only focused on DIM's beneficial effects, the toxic

effects of DIM on the immune systems have not been clearly elucidated. The aim of

this study was to explore the immunotoxic effects of DIM in a neonatal mouse and

to further evaluate whether DIM administration affects rotavirus (RV)-induced

gastroenteritis. Interestingly, multiple immunotoxic effects were observed in the

DIM treated group, including decreases in various immune cells (F4/80(+),

CD11c(+), CD19(+), and CD3(+) cells) in the spleen, induction of splenic white

pulp atrophy, an increase in immune cell apoptosis, and decreased expression of

various toll-like receptors (TLRs) in the spleen and small intestine. Apoptosis

was notably promoted by up-regulating caspase-3 activity and by the change in the

ratio of Bcl-2/Bax activities. Finally, oral administration of DIM led to

deterioration of RV-induced intestinal disease and delayed viral clearance in the

intestine and MLNs. Our results indicate that oral administration of DIM in

neonatal mice induces immunotoxicity and hampers efficient RV clearance in the

intestine. This new information about the immunotoxic roles of DIM in a newborn

mouse model may provide valuable clues for the development of a safe supplement,

especially one designed for human infants.

 

PMID: 21820497

 

Nutr Cancer. 2011;63(5):790-4. Epub 2011 Jun 22.

 

Inhibition of fatty acid synthase and Sp1 expression by 3,3'-diindolylmethane in

human breast cancer cells.

 

Saati GE, Archer MC.

 

Department of Nutritional Sciences, Faculty of Medicine, University of Toronto,

Toronto, Ontario, Canada.

 

The putative cancer preventive agent 3,3'-diindolylmethane (DIM) is formed in the

acidic environment of the stomach following consumption of indole-3-carbinol

(I3C), which is present in vegetables of the Brassica genus. We have recently

shown that the transcription factor Sp1 is involved in the regulation of both

proliferation and de novo lipogenesis in cancer cells. Here we show that DIM

inhibits the proliferation of 3 human breast cancer cell lines, MCF-7,

MDA-MB-231, and SKBr-3, and concomitantly inhibits the expression of Sp1 and

fatty acid synthase (FAS). There were no DIM-related effects on the proliferation

or expression of Sp1 or FAS in the nontumorigenic human breast epithelial cell

line MCF-10A. These results suggest that inhibition of Sp1 and/or FAS expression

could contribute to the anticancer properties of the dietary indoles.

 

PMID: 21767081 

 

Mol Cell Biochem. 2011 Dec;358(1-2):345-54. Epub 2011 Jul 15.

 

3,3'-Diindolylmethane inhibits breast cancer cell growth via miR-21-mediated

Cdc25A degradation.

 

Jin Y.

 

Laboratory of Cell Cycle and Cancer, College of Life Sciences, Capital Normal

University, HaiDian District, Beijing 100048, China. [email protected]

 

3,3'-Diindolylmethane (DIM) is a potential cancer preventive phytochemical

derived from Brassica vegetables. The effects of DIM on cell-cycle regulation in

both estrogen-dependent MCF-7 and estrogen receptor negative p53 mutant

MDA-MB-468 human breast cancer cells were assessed in this study. DIM inhibited

the breast cancer cell growth in vitro and in vivo, and caused cell-cycle arrest

by down-regulating protein levels of cell-cycle related kinases CDK1, CDK2, CDK4,

and CDK6, as well as Cyclin B1 and Cdc25A. Meanwhile, it was revealed that

Ser(124) phosphorylation of Cdc25A is primarily responsible for the DIM-induced

Cdc25A degradation. Furthermore, treatment of MCF-7 cells with DIM increased

miR-21 expression and down-regulated Cdc25A, resulting in an inhibition of breast

cancer cell proliferation. These observations collectively suggest that by

differentially modulating cellular signaling pathways DIM is able to arrest the

cell-cycle progression of human breast cancer cells.

 

PMID: 21761201 

 

Mutat Res. 2011 Jul-Oct;728(1-2):47-66. Epub 2011 Jun 15.

 

Attenuation of multi-targeted proliferation-linked signaling by

3,3'-diindolylmethane (DIM): from bench to clinic.

 

Banerjee S, Kong D, Wang Z, Bao B, Hillman GG, Sarkar FH.

  

Emerging evidence provide credible support in favor of the potential role of

bioactive products derived from ingesting cruciferous vegetables such as

broccoli, brussel sprouts, cauliflower and cabbage. Among many compounds,

3,3'-diindolylmethane (DIM) is generated in the acidic environment of the stomach

following dimerization of indole-3-carbinol (I3C) monomers present in these

classes of vegetables. Both I3C and DIM have been investigated for their use in

preventing, inhibiting, and reversing the progression of cancer - as a

chemopreventive agent. In this review, we summarize an updated, wide-ranging

pleiotropic anti-tumor and biological effects elicited by DIM against tumor

cells. It is unfeasible to point one single target as basis of cellular target of

action of DIM. We emphasize key cellular and molecular events that are

effectively modulated in the direction of inducing apoptosis and suppressing cell

proliferation. Collectively, DIM orchestrates signaling through Ah receptor,

NF-κB/Wnt/Akt/mTOR pathways impinging on cell cycle arrest, modulation of key

cytochrome P450 enzymes, altering angiogenesis, invasion, metastasis and

epigenetic behavior of cancer cells. The ability of DIM to selectively induce

tumor cells to undergo apoptosis has been observed in preclinical models, and

thus it has been speculated in improving the therapeutic efficacy of other

anticancer agents that have diverse molecular targets. Consequently, DIM has

moved through preclinical development into Phase I clinical trials, thereby

suggesting that DIM could be a promising and novel agent either alone or as an

adjunct to conventional therapeutics such as chemo-radio and targeted therapies.

An important development has been the availability of DIM formulation with

superior bioavailability for humans. Therefore, DIM appears to be a promising

chemopreventive agent or chemo-radio-sensitizer for the prevention of tumor

recurrence and/or for the treatment of human malignancies.

 

PMID: 21703360

 

Cancer Prev Res (Phila). 2011 Sep;4(9):1495-506. Epub 2011 Jun 16.

 

Inactivation of AR/TMPRSS2-ERG/Wnt signaling networks attenuates the aggressive

behavior of prostate cancer cells.

 

Li Y, Kong D, Wang Z, Ahmad A, Bao B, Padhye S, Sarkar FH.

 

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State

University School of Medicine, Detroit, MI 48201, USA.

 

The development of prostate cancer and its progression to castrate-resistant

prostate cancer (CRPC) after antiandrogen ablation therapy are driven by

persistent biological activity of androgen receptor (AR) signaling. Moreover,

studies have shown that more than 50% of human prostate cancers overexpress ERG

(v-ets avian erythroblastosis virus E26 oncogene related gene) due to

AR-regulated TMPRSS2-ERG fusion gene. However, the reported roles of TMPRSS2-ERG fusion in cancer progression are not clear. In this study, we investigated the

signal transduction in the AR/TMPRSS2-ERG/Wnt signaling network for studying the

aggressive behavior of prostate cancer cells and further assessed the effects of

DIM and CDF [natural agents-derived synthetic formulation and analogue of

3,3'-diindolylmethane (DIM) and curcumin, respectively, with improved

bioavailability] on the regulation of AR/TMPRSS2-ERG/Wnt signaling. We found that

activation of AR resulted in the induction of ERG expression through TMPRSS2-ERG

fusion. Moreover, we found that ERG overexpression and nuclear translocation

activated the activity of Wnt signaling. Furthermore, forced overexpression of

ERG promoted invasive capacity of prostate cancer cells. More important, we found

that DIM and CDF inhibited the signal transduction in the AR/TMPRSS2-ERG/Wnt

signaling network, leading to the inactivation of Wnt signaling consistent with

inhibition of prostate cancer cell invasion. In addition, DIM and CDF

inhibited proliferation of prostate cancer cells and induced apoptotic cell

death. On the basis of our findings, we conclude that because DIM and CDF

downregulate multiple signaling pathways including AR/TMPRSS2-ERG/Wnt signaling,

these agents could be useful for designing novel strategies for the prevention

and/or treatment of prostate cancer.

 

PMID: 21680704

 

Biopharm Drug Dispos. 2011 Jul;32(5):289-300. doi: 10.1002/bdd.759. Epub 2011 Jun 8.

 

Pharmacodynamics of dietary phytochemical indoles I3C and DIM: Induction of

Nrf2-mediated phase II drug metabolizing and antioxidant genes and synergism with

isothiocyanates.

 

Saw CL, Cintrón M, Wu TY, Guo Y, Huang Y, Jeong WS, Kong AN.

 

Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State

University of New Jersey, Piscataway, NJ 08854, USA.

 

The antioxidant response element (ARE) is a critical regulatory element for the

expression of many phase II drug metabolizing enzymes (DME), phase III

transporters and antioxidant enzymes, mediated by the transcription factor Nrf2.

The aim of this study was to examine the potential activation and synergism of

Nrf2-ARE-mediated transcriptional activity between four common phytochemicals

present in cruciferous vegetables; the indoles: indole-3-carbinol (I3C),

3,3'-diindolylmethane (DIM); and the isothiocyanates (ITCs): phenethyl

isothiocyanate (PEITC) and sulforaphane (SFN). The cytotoxicity of the compounds

was determined in a human liver hepatoma cell line (HepG2-C8). The combination

index was calculated to assess the synergistic effects on the induction of

ARE-mediated gene expressions. Quantitative real-time polymerase chain reaction

(qPCR) was employed to measure the mRNA expressions of Nrf2 and Nrf2-mediated

genes. I3C and DIM showed less cytotoxicity than SFN and PEITC. Compared with

I3C, DIM was found to be a stronger inducer of ARE. Synergism was observed after

combined treatments of 6.25 µm I3C + 1 µm SFN, 6.25 µm I3C + 1 µm PEITC and 6.25

µm DIM + 1 µm PEITC, while an additive effect was observed for 6.25 µm DIM + 1 µm

SFN. Induction of endogenous Nrf2, phase II genes (GSTm2, UGT1A1 and NQO1) and

antioxidant genes (HO-1 and SOD1) was also observed. In summary, the indole I3C

or DIM alone could induce or syngergistically induce in combination with the ITCs

SFN or PEITC, Nrf2-ARE-mediated gene expression, which could potentially enhance

cancer chemopreventive activity.

 

PMID: 21656528

 

J Phys Chem A. 2011 Jul 7;115(26):7700-8. Epub 2011 Jun 7.

 

Dimer radical cations of indole and indole-3-carbinol: localized and delocalized

radical cations of diindolylmethane.

 

Błoch-Mechkour A, Bally T, Marcinek A.

 

Department of Chemistry, University of Fribourg, CH-1700, Fribourg, Switzerland.

 

Extending our previous study on the title species (J. Phys. Chem. A2010, 114,

6787), we investigated the dimer cations that are formed on oxidation of the

glucobrassin derivatives indole-3-carbinol (I3C) and diindolylmethane (DIM) and

of parent indole (I). Radiolysis in ionic liquid and Ar matrices shows that, at

sufficiently high concentrations and/or on annealing the solid glasses, intense

intermolecular charge-resonance (CR) absorption bands in the NIR herald the

formation of sandwich-type dimer cations. The molecular and electronic structure

of these species is modeled by calculations with the double-hybrid B2-PLYP-D

density functional method which yields predictions in good accord with

experiment. The radical cation of DIM also shows a CR band, but unlike in the

case of I and I3C, its occurrence is not dependent on the concentration but

instead on the solvent: in ionic liquid the CR band is initially absent and

arises only on annealing, whereas in Ar matrices it is present from the outset

and undergoes blue shifting and sharpening on annealing. These puzzling findings

are rationalized on the basis of B2-PLYP-D calculations which predict that

neutral DIM exists in the form of two conformers, present in different relative

amounts in the two experiments, which on vertical ionization form distinct

radical cations, a nonsymmetric one where the odd electron is largely localized

on one of the two indole moieties and one with C(2) symmetry where charge and

spin are completely delocalized over both halves of the molecule, thus giving

rise to an intramolecular CR transition. On annealing, the nonsymmetric cation

relaxes to a similarly delocalized structure with C(s) symmetry, thus explaining

the observed increase and the shift of the CR band. We believe that DIM(•+)

represents the first example of a radical cation which can exist under the same

conditions as a localized and a delocalized complex cation.

 

PMID: 21648387

 

Oncol Rep. 2011 Sep;26(3):731-6. doi: 10.3892/or.2011.1316. Epub 2011 May 23.

 

Effects of 5,6-benzoflavone, indole-3-carbinol (I3C) and diindolylmethane (DIM)

on chemically-induced mammary carcinogenesis: is DIM a substitute for I3C?

 

Lubet RA, Heckman BM, De Flora SL, Steele VE, Crowell JA, Juliana MM, Grubbs CJ.

 

The abilities of 5,6-benzoflavone (5,6-BF, a synthetic flavonoid),

indole-3-carbinol (I3C, a plant derived product) or diindolylmethane (DIM, a

condensation product of I3C) to alter the induction of mammary cancers induced by

the carcinogens 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitrosourea

(MNU) were evaluated. Interestingly, the first two agents act as aryl hydrocarbon

receptor (AhR) agonists, while DIM does not. The agents were initially examined

for their ability to inhibit DMBA-induced mammary carcinogenesis. Agents were

administered for 14 days starting 7 days prior to a single dose of the

carcinogen. Evaluated over an extensive range of doses (165, 550 and 1650 ppm in

the diet), 5,6-BF caused a dose-dependent decrease of mammary cancers. In

addition, 5,6-BF at doses of 1650 and 165 ppm in the diet blocked the induction

of DMBA-induced DNA adducts in the mammary gland by approximately 85% and 45%,

respectively. In contrast, DIM (180 or 20 mg/kg BW/day) failed to block induction

of DMBA tumors. The effect of these agents on the promotion/progression phase of

carcinogenesis using the MNU mammary cancer model was also determined. 5,6-BF

(1650 or 165 ppm in the diet), I3C (180 or 60 mg/kg BW/day administered by

gavage), or DIM (180 or 60 mg/kg BW/day by gavage) were initiated 5 days after

the administration of MNU, and continually thereafter. 5,6-BF decreased MNU-

induced mammary tumor multiplicity by 40-60%. I3C reduced tumor multiplicity at

the high dose, while DIM at either dose had minimal effects on tumor

multiplicity. Thus, 5,6-BF and I3C were highly effective against initiation of

DMBA-induced mammary carcinogenesis, and were also effective against MNU-induced

tumors during the promotion/progression phase of carcinogenesis. In contrast, DIM

had minimal effects in either model; arguing that administration of DIM is not

analogous to administration of I3C.

 

PMID: 21617870  [PubMed - indexed for MEDLINE]

 

Free Radic Res. 2011 Aug;45(8):941-9. Epub 2011 May 26.

 

3,3'-Diindolylmethane but not indole-3-carbinol activates Nrf2 and induces Nrf2

target gene expression in cultured murine fibroblasts.

 

Ernst IM, Schuemann C, Wagner AE, Rimbach G.

 

Institute of Human Nutrition and Food Science, Christian-Albrechts-University

Kiel , Hermann-Rodewald-Strasse 6, 24118 Kiel, Germany.

 

There is increasing interest in the gene-regulatory activity of Brassica

vegetable derived phytochemicals such as 3,3'-diindolylmethane (DIM) and

indole-3-carbinol (I3C). DIM is formed under acidic conditions by dimerization of

I3C. This study compared the Nrf2 activating potential of DIM and I3C in murine

fibroblasts (NIH3T3). In contrast to its precursor I3C, DIM induces the

transactivation of Nrf2. Furthermore, Nrf2 targets such as HO-1, γGCS and NQO1

were increased on the mRNA and protein levels following DIM treatment. DIM was

less potent than sulforaphane (used as positive control) in inducing

Nrf2-dependent gene expression. The present data suggest that the dimerization of

I3C to DIM increases its Nrf2 inducing activity.

 

PMID: 21615272

 

Eur J Cancer Prev. 2011 Sep;20(5):417-25.

 

Chemopreventive effects of synthetic C-substituted diindolylmethanes originating

from cruciferous vegetables in human oral cancer cells.

 

Shin JA, Shim JH, Choi ES, Leem DH, Kwon KH, Lee SO, Safe S, Cho NP, Cho SD.

 

Department of Oral Pathology, School of Dentistry, Chonbuk National University,

Republic of Korea.

 

Diindolylmethane (DIM), an isothiocyanate found in cruciferous vegetables, has

been shown to have cancer chemopreventive effects. A series of synthetic

C-substituted DIMs (C-DIMs) analogs was developed, including DIM-C-pPhtBu and

DIM-C-pPhC6H5, which exhibited better inhibitory activity in cancer cells than

DIM. This study examined the effects of C-DIMs on the growth of human oral cancer

cells. DIM-C-pPhtBu and DIM-C-pPhC6H5 decreased the number of viable KB cells and

induced caspase-dependent apoptosis. The apoptotic cell death was accompanied by

a change in Bax/Bcl-2 ratio and damage to mitochondrial membrane potential

through the induction of death receptor 5 and the cleavage of Bid and caspase 8.

Studies on the mechanism of action showed that the apoptotic cell death induced

by DIM-C-pPhtBu and DIM-C-pPhC6H5 was mediated by endoplasmic reticulum stress.

In addition, C-DIMs inhibited cell proliferation and induced PARP cleavage

through death receptor 5 and CHOP in HEp-2 and HN22 cells. This provides the

first evidence that synthetic C-DIMs originating from cruciferous vegetables

induce apoptosis in human oral cancer cells through the endoplasmic reticulum

stress pathway.

 

PMID: 21597379

 

Mol Carcinog. 2011 Apr 22.

Broccoli-derived phytochemicals indole-3-carbinol and 3,3'-diindolylmethane
exerts concentration-dependent pleiotropic effects on prostate cancer cells:
Comparison with other cancer preventive phytochemicals.

Wang TT, Schoene NW, Milner JA, Kim YS.

Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research
Center, Agricultural Research Service, U.S. Department of Agriculture,
Beltsville, Maryland.

In the present studies, we utilized prostate cancer cell culture models to
elucidate the mechanisms of action of broccoli-derived phytochemicals
3,3'-diindolylmethane (DIM) and indole-3-carbinol (I3C). We found DIM and I3C at
1-5 µM inhibited androgen and estrogen-mediated pathways and induced xenobiotic
metabolism pathway. By contrast, DIM and I3C induced cyclin inhibitors,
indicators of stress/DNA damage, only at ≥25 µM. We also demonstrated that an
inhibitory effect of DIM and I3C on cell growth involves inhibition of
insulin-like growth factor-1 receptor expression. More importantly, we showed
that differences in efficacies and mechanisms existed between DIM and I3C. These
included differences in effective concentrations, a differential effect on
androgen receptor binding, and a differential effect on xenobiotic metabolic
pathway through aryl hydrocarbon receptor-dependent and -independent mechanism.
Furthermore we determined that several other diet-derived cancer protective
compounds, similar to DIM and I3C, exhibited pleiotrophic effects on signaling
pathways that included proliferation, cell cycle, and nuclear receptors-mediated
pathways. However, the efficacies and mechanisms of these compounds vary. We also
showed that some cellular pathways are not likely to be affected by DIM or I3C
when circulating concentration of orally ingested DIM or I3C is considered. Based
on our results, a model for cancer protective effects of DIM and I3C was
proposed.

PMID: 21520295

Cancer Prev Res (Phila). 2011 Mar 7.

Results from a dose response study using 3, 3'-diindolylmethane in the K14-HPV16
transgenic mouse model: Cervical histology.

Sepkovic DW, Stein J, Carlisle AD, Ksieski HB, Auborn K, Raucci L, Nyirenda T,
Bradlow HL.

1Research, Hackensack University Medical Center.

The Human Papilloma Virus is the major cause of cervical cancer. Viral infection
initiates cervical intraepithelial neoplasia which progresses through several
stages to cervical cancer. The objective of this study is to identify the minimum
effective dose of diindolylmethane that prevents the progression from cervical
dysplasia to carcinoma in situ. We document cervical histology in K14-HPV16 mice
receiving different doses of diindolylmethane. Urinary diindolylmethane
concentrations are reported. Diindolylmethane could enhance the efficacy of human
papilloma virus vaccines, creating a new therapeutic use for these vaccines in
women already infected with the virus. Five doses (0 to 2500ppm) of
diindolylmethane were incorporated into each mouse diet. The reproductive tract
was serially sectioned and urine was obtained for analysis of urinary
diindolylmethane. The results indicate that 62% of mice receiving 1,000ppm
diindolylmethane, remained dysplasia free after 20 weeks compared to 16% of mice
receiving no diindolylmethane and 18% receiving 500ppm. 1000ppm of
3,3'-diindolylmethane in the diet completely suppressed the development of
cervical cancer. Urinary diindolylmethane levels increased significantly as
diindolylmethane in food increased. These findings imply usefulness for
diindolylmethane in the search to prevent cervical cancer when used in
combination with prophylactic or therapeutic vaccines.

PMID: 21383027

Carcinogenesis. 2011 Mar 1.

Activation of Nuclear TR3 (Nr4a1) by A Diindolylmethane Analog Induces Apoptosis
and Proapoptotic Genes in Pancreatic Cancer Cells and Tumors.

Yoon K, Lee SO, Cho SD, Kim K, Khan S, Safe S.

Institute of Bioscience and Technology, Texas A&M Health Science Center, 2121 W.
Holcombe Blvd., Houston, TX 77030, USA.

NR4A1 (Nur77, TR3) is overexpressed in pancreatic tumors and activation of TR3 by
1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH(3)) inhibits cell
and tumor growth and induces apoptosis. Microarray analysis demonstrates that in
L3.6pL pancreatic cancer cells DIM-C-pPhOCH(3) induces genes associated with
metabolism, homeostasis, signal transduction, transcription, stress, transport,
immune responses, growth inhibition, and apoptosis. Among the most highly induced
growth inhibitory and proapoptotic genes including activating transcription
factor 3 (ATF3), p21, cystathionase, dual specificity phosphatase 1, and growth
differentiation factor 15, RNA interference studies demonstrated that induction
of all but the later gene by DIM-C-pPhOCH(3) were TR3-dependent. We also observed
that DIM-C-pPhOCH(3) induced Fas ligand and tumor necrosis factor-related
apopotosis-inducing ligand (TRAIL) and induction of TRAIL was ATF3-dependent.
Results of this and previous studies demonstrate that TR3 is unique among nuclear
receptors since nuclear TR3 is activated or deactivated by diindolylmethane
derivatives to induce different apoptotic and growth inhibitory pathways that
inhibit pancreatic cancer cell and tumor growth.

PMID: 21362629

Prostate. 2011 Feb 14. doi: 10.1002/pros.21356.

Antiandrogenic and growth inhibitory effects of ring-substituted analogs of
3,3'-diindolylmethane (Ring-DIMs) in hormone-responsive LNCaP human prostate
cancer cells.

Abdelbaqi K, Lack N, Guns ET, Kotha L, Safe S, Sanderson JT.

INRS-Institut Armand-Frappier, Université du Québec, Laval, QC, Canada.

BACKGROUND: Cruciferous vegetables protect against prostate cancer.
Indole-3-carbinol (I3C) and its major metabolite 3,3'-diindolylmethane (DIM),
exhibit antitumor activities in vitro and in vivo. Several synthetic
ring-substituted dihaloDIMs (ring-DIMs) appear to have increased anticancer
activity. METHODS: Inhibition of LNCaP prostate cancer cell growth was measured
by a WST-1 cell viability assay. Cytoplasmic and nuclear proteins were analyzed
by immunoblotting and immunofluorescence. Androgen receptor (AR) activation was
assessed by measuring prostate-specific antigen (PSA) expression and using LNCaP
cells containing human AR and an AR-dependent probasin promoter-green fluorescent
protein (GFP) construct. RESULTS: Like DIM, several ring-substituted dihaloDIM
analogs, namely 4,4'-dibromo-, 4,4'-dichloro-, 7,7'-dibromo-, and
7,7'-dichloroDIM, significantly inhibited DHT-stimulated growth of LNCaP cells at
concentrations ≥1 µM. We observed structure-dependent differences for the effects
of the ring-DIMs on AR expression, nuclear AR accumulation and PSA levels in
LNCaP cells after 24 hr. Both 4,4'- and 7,7'-dibromoDIM decreased AR protein and
mRNA levels, whereas 4,4'- and 7,7'-dichloroDIM had minimal effect. All four
dihaloDIMs (10 and 30 µM) significantly decreased PSA protein and mRNA levels.
Immuofluorescence studies showed that only the dibromoDIMs increased nuclear
localization of AR. All ring-DIMs caused a concentration-dependent decrease in
fluorescence induced by the synthetic androgen R1881 in LNCaP cells transfected
with wild-type human AR and an androgen-responsive probasin promoter-GFP gene
construct, with potencies up to 10-fold greater than that of DIM. CONCLUSION: The
antiandrogenic effects of ring-DIMs suggest they may form the basis for the
development of novel agents against hormone-sensitive prostate cancer, alone or
in combination with other drugs.

PMID: 21321979

PLoS One. 2011 Jan 18;6(1):e15879.

Estrogen induced metastatic modulators MMP-2 and MMP-9 are targets of
3,3'-diindolylmethane in thyroid cancer.

Rajoria S, Suriano R, George A, Shanmugam A, Schantz SP, Geliebter J, Tiwari RK.

Department of Microbiology and Immunology, New York Medical College, Valhalla,
New York, United States of America.

BACKGROUND: Thyroid cancer is the most common endocrine related cancer with
increasing incidences during the past five years. Current treatments for thyroid
cancer, such as surgery or radioactive iodine therapy, often require patients to
be on lifelong thyroid hormone replacement therapy and given the significant
recurrence rates of thyroid cancer, new preventive modalities are needed. The
present study investigates the property of a natural dietary compound found in
cruciferous vegetables, 3,3'-diindolylmethane (DIM), to target the metastatic
phenotype of thyroid cancer cells through a functional estrogen receptor.
METHODOLOGY/PRINCIPAL FINDINGS: Thyroid cancer cell lines were treated with
estrogen and/or DIM and subjected to in vitro adhesion, migration and invasion
assays to investigate the anti-metastatic and anti-estrogenic effects of DIM. We
observed that DIM inhibits estrogen mediated increase in thyroid cell migration,
adhesion and invasion, which is also supported by ER-α downregulation (siRNA)
studies. Western blot and zymography analyses provided direct evidence for this
DIM mediated inhibition of E(2) enhanced metastasis associated events by virtue
of targeting essential proteolytic enzymes, namely MMP-2 and MMP-9.
CONCLUSION/SIGNIFICANCE: Our data reports for the first time that DIM displays
anti-estrogenic like activity by inhibiting estradiol enhanced thyroid cancer
cell proliferation and in vitro metastasis associated events, namely adhesion,
migration and invasion. Most significantly, MMP-2 and MMP-9, which are known to
promote and enhance metastasis, were determined to be targets of DIM. This
anti-estrogen like property of DIM may lead to the development of a novel
preventive and/or therapeutic dietary supplement for thyroid cancer patients by
targeting progression of the disease.

PMID: 21267453

Thyroid. 2011 Mar;21(3):299-304. Epub 2011 Jan 22.

3,3'-diindolylmethane modulates estrogen metabolism in patients with thyroid
proliferative disease: a pilot study.

Rajoria S, Suriano R, Parmar PS, Wilson YL, Megwalu U, Moscatello A, Bradlow HL,
Sepkovic DW, Geliebter J, Schantz SP, Tiwari RK.

Department of Microbiology and Immunology, New York Medical College, Valhalla,
New York 10595, USA.

BACKGROUND: The incidence of thyroid cancer is four to five times higher in women
than in men, suggesting a role for estrogen (E₂) in the pathogenesis of thyroid
proliferative disease (TPD) that comprises cancer and goiter. The objective of
this study was to investigate the antiestrogenic activity of
3,3'-diindolylmethane (DIM), a bioactive compound derived from cruciferous
vegetables, in patients with TPD.
METHODS: In this limited phase I clinical trial study, patients found to have TPD
were administered 300 mg of DIM per day for 14 days. Patients subsequently
underwent a total or partial thyroidectomy, and tissue, urine, and serum samples
were collected. Pre- and post-DIM serum and urine samples were analyzed for DIM
levels as well as estrogen metabolites. DIM levels were also determined in
thyroid tissue samples.
RESULTS: DIM was detectable in thyroid tissue, serum, and urine of patients after
14 days of supplementation. Urine analyses revealed that DIM modulated estrogen
metabolism in patients with TPD. There was an increase in the ratio of
2-hydroxyestrones (C-2) to 16α-hydroxyestrone (C-16), consistent with
antiestrogenic activity that results in more of C-2 product compared with C-16.
CONCLUSION: Our data suggest that DIM enhances estrogen metabolism in TPD
patients and can potentially serve as an antiestrogenic dietary supplement to
help reduce the risk of developing TPD. The fact that DIM is detected in thyroid
tissue implicates that it can manifest its antiestrogenic activity in situ to
modulate TPD.

PMID: 21254914

Mol Carcinog. 2011 Feb;50(2):100-12. doi: 10.1002/mc.20698. Epub 2010 Nov 8.

3,3'-Diindolylmethane inhibits prostate cancer development in the transgenic
adenocarcinoma mouse prostate model.

Cho HJ, Park SY, Kim EJ, Kim JK, Park JH.

Department of Food Science and Nutrition, Hallym University, Chuncheon, Korea.

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol,
which is present in cruciferous vegetables and has been reported to possess
anti-carcinogenic properties. In the present study, we examined whether DIM
inhibits the development of prostate cancer using the transgenic adenocarcinoma
mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in
TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen
and proliferating cell nuclear antigen, and increased the number of terminal dUTP
nick-end labeling-positive cells in the dorsolateral lobes of the prostate.
Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent
kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess
the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate
cancer cells were cultured with various concentrations of DIM. DIM induced a
substantial reduction in the numbers of viable cells and induced apoptosis in
LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly
(ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability
and the translocation of cytochrome c and Smac/Diablo from the mitochondria.
Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated
Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to
mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid.
These results indicate that DIM inhibits prostate carcinogenesis via induction of
apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in
prostate cancer cells via the mitochondria- and death receptor-mediated pathways.

PMID: 21229607

Expert Opin Ther Targets. 2011 Feb;15(2):195-206. Epub 2011 Jan 5.

Targeting NR4A1 (TR3) in cancer cells and tumors.

Lee SO, Li X, Khan S, Safe S.

Institute of Bioscience and Technology, Texas A&M Health Science Center, 2121 W.
Holcombe Boulevard, Houston, TX 77030, USA.

INTRODUCTION: Nuclear receptor 4A1(NR4A1) (testicular receptor 3 (TR3), nuclear
hormone receptor (Nur)77) is a member of the nuclear receptor superfamily of
transcription factors and is highly expressed in multiple tumor types. RNA
interference studies indicate that NR4A1 exhibits growth-promoting, angiogenic
and prosurvival activity in most cancers. AREAS COVERED: Studies on several
apoptosis-inducing agents that activate nuclear export of NR4A1, which
subsequently forms a mitochondrial NR4A1-bcl-2 complex that induces the intrinsic
pathway for apoptosis are discussed. Cytosporone B and related compounds that
induce NR4A1-dependent apoptosis in cancer cells through both modulation of
nuclear NR4A1 and nuclear export are discussed. A relatively new class of
diindolylmethane analogs (C-DIMs) including
1,1-bis(3'-indolyl)-1-(p-methoxyphenyl)methane (DIM-C-pPhOCH(3)) (NR4A1
activator) and 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH)
(NR4A1 deactivator) are discussed in more detail. These anticancer drugs (C-DIMs)
act strictly through nuclear NR4A1 and induce apoptosis in cancer cells and
tumors. EXPERT OPINION: It is clear that NR4A1 plays an important pro-oncogenic
role in cancer cells and tumors, and there is increasing evidence that this
receptor can be targeted by anticancer drugs that induce cell death via
NR4A1-dependent and -independent pathways. Since many of these compounds exhibit
relatively low toxicity, they represent an important class of mechanism-based
anticancer drugs with excellent potential for clinical applications.

PMID: 21204731

Curr Drug Targets. 2010 Dec 15. [Epub ahead of print]

Cancer Chemoprevention by Targeting the Epigenome.

Huang J, Plass C, Gerhäuser C.

Division Epigenomics and Cancer Risk Factors, German Cancer Research Center,
Heidelberg, Germany. [email protected].

The term "epigenetics" refers to modifications in gene expression caused by
heritable, but potentially reversible, changes in DNA methylation and chromatin
structure. Given the fact that epigenetic modifications occur early in
carcinogenesis and represent potentially initiating events in cancer development,
they have been identified as promising new targets for prevention strategies. The
present review will give a comprehensive overview of the current literature on
chemopreventive agents and their influence on major epigenetic mechanisms, that
is DNA methylation, histone acetylation and methylation, and microRNAs, both in
vitro and in rodent and human studies, taking into consideration specific
mechanisms of action, target sites, concentrations, methods used for analysis,
and outcome. Chemopreventive agents with reported mechanisms targeting the
epigenome include micronutrients (folate, selenium, retinoic acid, Vit. E),
butyrate, polyphenols (from green tea, apples, coffee, and other dietary
sources), genistein and soy isoflavones, parthenolide, curcumin, ellagitannin,
indol-3-carbinol (I3C) and diindolylmethane (DIM), mahanine, nordihydroguaiaretic
acid (NDGA), lycopene, sulfur-containing compounds from Allium and cruciferous
vegetables (sulforaphane, phenylethyl isothiocyanate (PEITC), phenylhexyl
isothiocyanate (PHI), diallyldisulfide (DADS), allyl mercaptan (AM)), antibiotics
(mithramycin A, apicidin), pharmacological agents (celecoxib, DFMO,
5-aza-2'-deoxycytidine and zebularine), compounds affecting sirtuin activity
(resveratrol, dihydrocoumarin, cambinol), inhibitors of histone acetyl
transferases (anacardic acid, garcinol, ursodeoxycholic acid), and relatively
unexplored modulators of histone lysine methylation (chaetocin, polyamine
analogues, n-3 polyunsaturated fatty acids). Their effects on global DNA
methylation, tumor suppressor genes silenced by promoter methylation, histone
modifications, and miRNAs deregulated during carcinogenesis have potential impact
on multiple mechanisms relevant for chemoprevention, including signal
transduction mediated by nuclear receptors and transcription factors such as
NF-κB, cell cycle progression, cellular differentiation, apoptosis induction,
senescence and others. In vivo studies that demonstrate the functional relevance
of epigenetic mechanisms for chemopreventive efficacy are still limited. Future
research will need to identify best strategies for chemopreventive intervention,
taking into account the importance of epigenetic mechanisms for gene regulation.

PMID: 21158707

Int J Cancer. 2010 Dec 10. [Epub ahead of print]

3, 3'-diindolylmethane enhances taxotere-induced growth inhibition of breast
cancer cells through down-regulation of FoxM1.

Ahmad A, Ali S, Wang Z, Ali AS, Sethi S, Sakr WA, Raz A, Rahman KW.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI.

Emerging evidence suggests that the transcription factor Forkhead Box M1 (FoxM1)
is associated with aggressive human carcinomas, including breast cancer. Since
elevated expression of FoxM1 has been observed in human breast cancers, FoxM1 has
attracted much attention in recent years as a potential target for the prevention
and/or therapeutic intervention in breast cancer. However, no information is
currently available regarding how down-regulation of FoxM1 could be achieved for
breast cancer prevention and therapy. Here we report for the first time that 3,
3'-diindolylmethane (DIM), a non-toxic dietary chemopreventive agent could
effectively down-regulate FoxM1 in various breast cancer cell lines. Using gene
transfection, real-time RT-PCR, Western blotting, invasion and MTT assays, we
found that DIM could enhance Taxotere-induced growth inhibition of breast cancer
cells, and decreased invasive capacity of breast cancer cells was observed after
either treatment alone or the combination. These effects were associated with
down-regulation of FoxM1. We also found that knock-down of FoxM1 expression by
siRNA transfection increased DIM-induced cell growth inhibition, whereas
over-expression of FoxM1 by cDNA transfection attenuated DIM-induced cell growth
inhibition, suggesting the mechanistic role of FoxM1. Most importantly, the
combination treatment significantly inhibited tumor growth in SCID mice, and the
results were correlated with the down-regulation of FoxM1 in tumor remnants. We
conclude that inactivation of FoxM1 and its target genes by DIM could enhance the
therapeutic efficacy of Taxotere in breast cancer, which could be a useful
strategy for the prevention and/or treatment of breast cancer.

PMID: 21154750

Oncol Rep. 2011 Feb;25(2):491-7. doi: 10.3892/or.2010.1076. Epub 2010 Dec 7.

3,3'-diindolylmethane inhibits migration and invasion of human cancer cells
through combined suppression of ERK and AKT pathways.

Rajoria S, Suriano R, Wilson YL, Schantz SP, Moscatello A, Geliebter J, Tiwari
RK.

Department of Microbiology and Immunology, New York Medical College, Valhalla,
New York, NY 10595, USA.

Metastasis of cancer is a multifactorial disease and is the main cause of death
in patients with malignancy. This disease demands treatments which may target
multiple dysregulated cellular pathways in cancer cells. The anti-tumor and
anti-metastatic properties of natural products in cancer prevention have been
confirmed by several epidemiological studies, with cruciferous vegetables being
especially protective against many cancers. In this study, we evaluated the
anti-carcinogenic effects of 3,3'-diindolylmethane (DIM), which is a bioactive
compound present in cruciferous vegetables and a widely used dietary supplement,
on events commonly observed during metastasis using in vitro adhesion, migration
and invasion assays. Our results indicate that DIM inhibits human cancer cell in
vitro proliferation, adhesion, migration and invasion. Western blot analyses show
that this inhibition of cell proliferation by DIM is exerted by combined
suppression of AKT and ERK pathways. Furthermore, DIM also leads to
down-regulation of G1-S cell cycle markers: cyclin D1, cdk6 and cdk4. These
findings may lead to development of a novel preventive and/or therapeutic dietary
supplement for patients diagnosed with cancer or predisposed to developing
certain cancers.

PMID: 21152869

Mol Carcinog. 2010 Nov 8. [Epub ahead of print]

3,3'-diindolylmethane inhibits prostate cancer development in the transgenic
adenocarcinoma mouse prostate model.

Cho HJ, Park SY, Kim EJ, Kim JK, Park JH.

Department of Food Science and Nutrition, Hallym University, Chuncheon, Korea.

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of indole-3-carbinol,
which is present in cruciferous vegetables and has been reported to possess
anti-carcinogenic properties. In the present study, we examined whether DIM
inhibits the development of prostate cancer using the transgenic adenocarcinoma
mouse prostate (TRAMP) model. DIM feeding inhibited prostate carcinogenesis in
TRAMP mice, reduced the number of cells expressing the SV40 large tumor antigen
and proliferating cell nuclear antigen, and increased the number of terminal dUTP
nick-end labeling-positive cells in the dorsolateral lobes of the prostate.
Additionally, DIM feeding reduced the expression of cyclin A, cyclin-dependent
kinase (CDK)2, CDK4, and Bcl-xL, and increased p27 and Bax expression. To assess
the mechanisms by which DIM induces apoptosis, LNCaP and DU145 human prostate
cancer cells were cultured with various concentrations of DIM. DIM induced a
substantial reduction in the numbers of viable cells and induced apoptosis in
LNCaP and DU145 cells. DIM increased the cleavage of caspase-9, -7, -3, and poly
(ADP-ribose) polymerase (PARP). DIM increased mitochondrial membrane permeability
and the translocation of cytochrome c and Smac/Diablo from the mitochondria.
Additionally, DIM induced increases in the levels of cleaved caspase-8, truncated
Bid, Fas, and Fas ligand, and the caspase-8 inhibitor Z-IETD-FMK was shown to
mitigate DIM-induced apoptosis and the cleavage of caspase-3, PARP, and Bid.
These results indicate that DIM inhibits prostate carcinogenesis via induction of
apoptosis and inhibition of cell cycle progression. DIM induces apoptosis in
prostate cancer cells via the mitochondria- and death receptor-mediated pathways.

PMID: 21061271

Free Radic Biol Med. 2011 Jan 15;50(2):228-36. Epub 2010 Oct 26.

3,3'-Diindolylmethane decreases VCAM-1 expression and alleviates experimental
colitis via a BRCA1-dependent antioxidant pathway.

Huang Z, Zuo L, Zhang Z, Liu J, Chen J, Dong L, Zhang J.

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing, People's Republic of China.

Reactive oxygen species (ROS) exhibit a key role in the pathogenesis of
inflammatory bowel disease (IBD). 3,3'-Diindolylmethane (DIM) can protect against
oxidative stress in a breast cancer susceptibility gene 1 (BRCA1)-dependent
manner. The aim of this study was to examine the therapeutic effects of DIM in
experimental colitis and investigate the possible mechanisms underlying its
effects on intestinal inflammation. The therapeutic effects of DIM were studied
in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Pathological
markers of colitis severity, antioxidant activity, and ROS generation in colonic
tissue were measured. The impact of DIM on ROS-induced endothelial vascular cell
adhesion molecule 1 (VCAM-1) expression and leukocyte-endothelial cell
interaction was further investigated in cultures of endothelial cells and in the
TNBS-induced colitis model. Administration of DIM was demonstrated to attenuate
experimental colitis, as judged by pathological indices. DIM could effectively
stimulate the expression of BRCA1 in vitro and in vivo and reduce ROS generation,
leading to the inhibition of VCAM-1 expression and leukocyte-endothelial cell
adhesion, and finally resulted in an alleviation of experimental colitis. DIM has
shown anti-IBD activity in animal models by inhibiting ROS-induced VCAM-1
expression and leukocyte recruitment via a BRCA1-dependent antioxidant pathway
and thus may offer potential treatments for IBD patients.

PMID: 21034812

Am J Transl Res. 2010 Jul 23;2(4):402-11.

A phase I dose-escalation study of oral DIM (3,3'-Diindolylmethane) in

castrate-resistant, non-metastatic prostate cancer.

Heath EI, Heilbrun LK, Li J, Vaishampayan U, Harper F, Pemberton P, Sarkar FH.

Karmanos Cancer Institute, Wayne State University Detroit, MI, USA.

3, 3'-diindolylmethane (DIM) modulates estrogen metabolism and acts as an
anti-androgen which down-regulates the androgen receptor and prostate specific
antigen (PSA). We conducted a dose-escalation, phase I study of DIM with

objectives to determine the maximum tolerated dose (MTD), toxicity
profile, and phar-macokinetics (PK) of DIM, and to assess its effects on serum
PSA and quality of life (QoL).PATIENTS AND METHODS: Cohorts of 3-6 patients
received escalating doses of twice daily oral DIM providing DIM at 75 mg, then
150 mg, 225 mg, and 300 mg. Toxicity was evaluated monthly. Serum PSA and QoL
were measured at baseline, monthly during treatment, and at end of study.
RESULTS: 12 patients with castrate-resistant, non-metastatic, PSA relapse
prostate cancer were treated over 4 dose cohorts; 2 patients (at 150 mg and 225
mg, respectively) underwent intra-patient dose escalation, by one dose level.
After oral administration of the first dose of DIM, the plasma exposure to DIM
appeared dose proportional at doses ranging from 75 to 300 mg, with the mean
C(max) and mean AUC(last) increasing from 41.6 to 236.4 ng/ml and from 192.0 to
899.0 ng/ml*h, respectively. Continued relatively stable systemic exposure to DIM
was achieved following twice daily oral administration of DIM. Minimal
toxicity was observed. Two of the four patients treated at 300 mg had grade 3
asymptomatic hyponatremia (AH) discovered on routine blood work. The other 2
patients at this dose had no AH. Therefore, the maximum tolerated dose (MTD) was
deemed to be 300 mgand the recommended phase II dose (RP2D) of DIM was 225 mg
twice daily. One patient without AH at 225 mg experienced a 50% PSA decline. One
patient with DIM dose of 225 mg had PSA stabilization. The other 10 patients
had an initial deceleration of their PSA rise (decrease in slope), but eventually
progressed based on continual PSA rise or evidence of metastatic disease. Ten
patients completed monthly QoL reports for a mean of 6 months (range: 1-13). QoL
measures emotional functioning may have held up somewhat better over time than
their physical functioning.

CONCLUSION: DIM was well tolerated. Increasing systemic exposure to DIM was
achieved with the increase of DIM dose. Modest efficacy was demonstrated.
Patients' QoL varied over time with length of treatment. Phase II studies are
recommended at the dose of 225 mg orally twice daily.

PMID: 20733950

Anticancer Drugs. 2010 Oct;21(9):814-22.

3,3'-Diindolylmethane negatively regulates Cdc25A and induces a G2/M arrest by
modulation of microRNA 21 in human breast cancer cells.

Jin Y, Zou X, Feng X.

Capital Normal University, Beijing, China. [email protected]

3,3'-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived
from Brassica vegetables. In this study, we assessed the effects of DIM on cell
cycle regulation in both estrogen-dependent MCF-7 and estrogen receptor negative
p53 mutant MDA-MB-468 human breast cancer cells. In-vitro culture studies showed
that DIM dose dependently inhibited the proliferation of both cells. In addition,
in-vivo xenograft model showed that DIM strongly inhibited the development of
human breast tumors. Fluorescence activated cell sorter analysis showed a
DIM-mediated G2/M cell cycle arrest in MCF-7 and MDA-MB-468 cells. Western blot
analysis showed that DIM downregulated the expression of cyclin-dependent kinases
2 and 4 and Cdc25A, which plays an important role in G2/M phase. Furthermore,
treatment of MCF-7 cells with DIM, which increased microRNA 21 expression, caused
a downregulation of Cdc25A, resulting in an inhibition of breast cancer cell
proliferation. Taken together, our data show that DIM is able to stop the cell
cycle progression of human breast cancer cells regardless of their
estrogen-dependence and p53 status, by differentially modulating cell cycle
regulatory pathways. The modulation of microRNA 21 mediates the DIM cell cycle
regulator effect in MCF-7 cells.

PMID: 20724916

Cancer Metastasis Rev. 2010 Sep;29(3):383-94.

The role of nutraceuticals in the regulation of Wnt and Hedgehog signaling in
cancer.

Sarkar FH, Li Y, Wang Z, Kong D.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, 740 HWCRC, 4100 John R Street, Detroit, MI 48201,
USA.

Multiple cellular signaling pathways have been involved in the processes of
cancer cell invasion and metastasis. Among many signaling pathways, Wnt and
Hedgehog (Hh) signaling pathways are critically involved in embryonic
development, in the biology of cancer stem cells (CSCs) and in the acquisition of
epithelial to mesenchymal transition (EMT), and thus this article will remain
focused on Wnt and Hh signaling. Since CSCs and EMT are also known to be
responsible for cancer cell invasion and metastasis, the Wnt and Hedgehog
signaling pathways are also intimately associated with cancer invasion and
metastasis. Emerging evidence suggests the beneficial role of chemopreventive
agents commonly known as nutraceutical in cancer. Among many such agents, soy
isoflavones, curcumin, green tea polyphenols, 3,3'-diindolylmethane, resveratrol,
lycopene, vitamin D, etc. have been found to prevent, reverse, or delay the
carcinogenic process. Interestingly, these agents have also shown to prevent or
delay the progression of cancer, which could in part be due to their ability to
attack CSCs or EMT-type cells by attenuating the Wnt and Hedgehog signaling
pathways. In this review, we summarize the current state of our knowledge on the
role of Wnt and Hedgehog signaling pathways, and their targeted inactivation by
chemopreventive agents (nutraceuticals) for the prevention of tumor progression
and/or treatment of human malignancies.

PMID: 20711635

 

Mol Carcinog. 2010 Jul;49(7):672-83.
 
 3,3'-diindolylmethane suppresses 12-O-tetradecanoylphorbol-13-acetate-induced
 inflammation and tumor promotion in mouse skin via the downregulation of
 inflammatory mediators.
 
 Kim EJ, Park H, Kim J, Park JH.
 
 Center for Efficacy Assessment and Development of Functional Foods and Drugs,
 Hallym University, Chuncheon, Korea.
 
 3,3'-Diindolylmethane (DIM) is a major acid-condensation product of
 indole-3-carbinol and is present in cruciferous vegetables. In this study, we
 evaluated the effects of DIM on antiinflammatory and antitumor promotion activity
 in mouse skin and explored the relevant mechanisms. When
 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied topically to the mouse ear
 to induce inflammation, DIM pretreatment effectively inhibited TPA-induced ear
 edema formation. To evaluate the mechanisms underlying DIM's antiinflammatory
 effects, DIM was topically treated to the shaved backs of mice 30 min before TPA
 treatment. DIM inhibited the TPA-induced increases in the expression of
 cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), chemokine (C-X-C
 motif) ligand (CXCL) 5, and interleukin (IL)-6 in mouse skin. DIM also inhibited
 nuclear factor-kappa B (NF-kappaB)'s DNA binding activity, the nuclear
 translocation of p65, and the degradation of inhibitor of kappaB (IkappaB) alpha
 in TPA-stimulated mouse skin. Furthermore, DIM reduced TPA-induced increases in
 the activity of extracellular signal regulated protein kinase (ERK)-1/2 and
 IkappaB kinase (IKK). When mouse skin papillomas were initiated via the topical
 application of 7,12-dimethylbenz[alpha]anthracene (DMBA) and promoted with
 repeated topical applications of TPA, repeated topical applications of DIM prior
 to each TPA treatment significantly suppressed the incidence and multiplicity of
 the papillomas. DIM also reduced the expression of COX-2 and iNOS, ERK
 phosphorylation, and the nuclear translocation of p65 in papillomas.
 Collectively, these results show that DIM exerts antiinflammatory and
 chemopreventive effects in mouse skin via the downregulation of COX-2, iNOS,
 CXCL5, and IL-6 expression, which may be mediated by reductions in NF-kappaB
 activation.
 
 PMID: 20564344

 

Pharm Res. 2010 Jun;27(6):1027-41.

Regulation of microRNAs by natural agents: an emerging field in chemoprevention
and chemotherapy research.

Li Y, Kong D, Wang Z, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

In recent years, microRNAs have received greater attention in cancer research.
These small, non-coding RNAs could inhibit target gene expression by binding to
the 3' untranslated region of target mRNA, resulting in either mRNA degradation
or inhibition of translation. miRNAs play important roles in many normal
biological processes; however, studies have also shown that aberrant miRNA
expression is correlated with the development and progression of cancers. The
miRNAs could have oncogenic or tumor suppressor activities. Moreover, some miRNAs
could regulate formation of cancer stem cells and epithelial-mesenchymal
transition phenotype of cancer cells which are typically drug resistant.
Furthermore, miRNAs could be used as biomarkers for diagnosis and prognosis, and
thus miRNAs are becoming emerging targets for cancer therapy. Recent studies have
shown that natural agents including curcumin, isoflavone, indole-3-carbinol,
3,3'-diindolylmethane, (-)-epigallocatechin-3-gallate, resveratrol, etc. could
alter miRNA expression profiles, leading to the inhibition of cancer cell growth,
induction of apoptosis, reversal of epithelial-mesenchymal transition, or
enhancement of efficacy of conventional cancer therapeutics. These emerging
results clearly suggest that specific targeting of miRNAs by natural agents could
open newer avenues for complete eradication of tumors by killing the
drug-resistant cells to improve survival outcome in patients diagnosed with
malignancies.

PMID: 20306121

 

Drug Resist Update. 2010 Jun;13(3):57-66.

Implication of microRNAs in drug resistance for designing novel cancer therapy.

Sarkar FH, Li Y, Wang Z, Kong D, Ali S.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, MI 48201, USA.

Recently, microRNAs (miRNAs) have received increasing attention in the field of
cancer research. miRNAs play important roles in many normal biological processes;
however, the aberrant miRNA expression and its correlation with the development
and progression of cancers is an emerging field. Therefore, miRNAs could be used
as biomarkers for diagnosis of cancer and prediction of prognosis. Importantly,
some miRNAs could regulate the formation of cancer stem cells and the acquisition
of epithelial-mesenchymal transition, which are critically associated with drug
resistance. Moreover, some miRNAs could target genes related to drug sensitivity,
resulting in the altered sensitivity of cancer cells to anti-cancer drugs.
Emerging evidences have also shown that knock-down or re-expression of specific
miRNAs by synthetic anti-sense oligonucleotides or pre-miRNAs could induce drug
sensitivity, leading to increased inhibition of cancer cell growth, invasion, and
metastasis. More importantly, recent studies have shown that natural agents
including isoflavone, 3,3'-diindolylmethane, and (-)-epigallocatechin-3-gallate
altered miRNA expression profiles, leading to an increased sensitivity of cancer
cells to conventional therapeutics. These emerging results suggest that specific
targeting of miRNAs by different approaches could open new avenues for cancer
treatment through overcoming drug resistance and thereby improve the outcome of
cancer therapy.

PMID: 20236855

 

Curr Drug Targets. 2010 Jun 1;11(6):652-66.

Anticancer properties of indole compounds: mechanism of apoptosis induction and
role in chemotherapy.

Ahmad A, Sakr WA, Rahman KM.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA.

Indole compounds, obtained from cruciferous vegetables, have been investigated
for their putative anti-cancer properties. Studies with indole-3-carbinol (I3C)
and its dimeric product, 3, 3' diindolylmethane (DIM), have indicated efficacy of
these compounds against a number of human cancers. Available as well as emerging
data suggests that these compounds act on a number of cellular signaling pathways
leading to their observed biological effects. Such pleiotropic effects of these
compounds are also considered crucial for their chemosensitization activity
wherein they help reduce the toxicity and resistance against conventional
chemotherapeutic drugs. These observations have major clinical implications
especially in chemotherapy. Through this review, we have attempted to update
current understanding on the state of anti-cancer research involving indole
compounds. We have also summarized the available literature on modulatory effects
of indoles on molecular targets such as survivin, uPA/uPAR and signaling pathways
such as the NF-kappaB pathway, which are important for the apoptosis-inducing and
chemosensitizing properties of these compounds.

PMID: 20298156

 

J Cell Biochem. 2010 May;110(1):171-81.

Concurrent inhibition of NF-kappaB, cyclooxygenase-2, and epidermal growth factor
receptor leads to greater anti-tumor activity in pancreatic cancer.

Ali S, Banerjee S, Schaffert JM, El-Rayes BF, Philip PA, Sarkar FH.

Division of Hematology/Oncology, Karmanos Cancer Institute, Wayne State
University, Detroit, Michigan 48201, USA.

Inactivation of survival pathways such as NF-kappaB, cyclooxygenase (COX-2), or
epidermal growth factor receptor (EGFR) signaling individually may not be
sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by
recent clinical trials. 3,3'-Diindolylmethane (DIM) is an inhibitor of
NF-kappaB and COX-2 and is a well-known chemopreventive agent. We hypothesized
that the inhibition of NF-kappaB and COX-2 by DIM concurrently with the
inhibition of EGFR by erlotinib will potentiate the anti-tumor effects of
cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo.
Inhibition of viable cells in seven PC cell lines treated with DIM, erlotinib,
or gemcitabine alone or their combinations was evaluated using
3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Significant inhibition in cell viability was observed in PC cells expressing high
levels of COX-2, EGFR, and NF-kappaB proteins. The observed inhibition was
associated with an increase in apoptosis as assessed by ELISA. A significant
down-regulation in the expression of COX-2, NF-kappaB, and EGFR in BxPC-3,
COLO-357, and HPAC cells was observed, suggesting that simultaneous targeting of
EGFR, NF-kappaB, and COX-2 is more effective than targeting either signaling
pathway separately. Our in vitro results were further supported by in vivo
studies showing that DIM in combination with erlotinib and gemcitabine was
significantly more effective than individual agents. Based on our preclinical in
vitro and in vivo results, we conclude that this multi-targeted combination could
be developed for the treatment of PC patients whose tumors express high levels of
COX-2, EGFR, and NF-kappaB.

PMID: 20213764

Mol Pharmacol. 2010 May 5.

Activation of Chk2 by 3,3' Diinolylmethane is required for causing G2/M cell
cycle arrest in human ovarian cancer cells.

Kandala PK, Srivastava SK.

Texas Tech University Health Sciences Center.

We evaluated the effect of 3,3'-diindolylmethane (DIM) in ovarian cancer cells.
DIM treatment inhibited the growth of SKOV-3, TOV-21G and OVCAR-3 ovarian cancer
cells in both dose and time-dependent manner with effective concentrations
ranging from 40muM to 100muM. Growth inhibitory effects of DIM were mediated by
cell cycle arrest in G2/M phase in all the three cell lines. G2/M arrest was
associated with DNA damage as indicated by phosphorylation of H(2)A.X at Ser 139
and activation of Chk2 in all the three cell lines. Other G2/M regulatory
molecules such as Cdc25C, Cdk1, Cyclin B1 were downregulated by DIM.
Cycloheximide or Chk2 inhibitor pretreatment abrogated not only activation of
Chk2 but also G2/M arrest and apoptosis mediated by DIM. To further establish the
involvement of Chk2 in DIM-mediated G2/M arrest, cells were transfected with
dominant negative Chk2 (DN-Chk2). Blocking Chk2 activation by DN-Chk2 completely
protected cells from DIM- mediated G2/M arrest. These results were further
confirmed in Chk2 knock out DT40 lymphoma cells where DIM failed to cause cell
cycle arrest. These results clearly indicate the requirement of Chk2 activation
to cause G2/M arrest by DIM in ovarian cancer cells. Moreover, blocking Chk2
activation also abrogates the apoptosis-inducing effects of DIM. Further our
results show that DIM treatment cause ROS generation. Blocking ROS generation by
NAC protect the cells from DIM- mediated G2/M arrest and apoptosis. Our results
establish Chk2 as a potent molecular target of DIM in ovarian cancer cells and
provide the rationale for further clinical investigation of DIM.

PMID: 20444961

Cancer Lett. 2010 Mar 16.

3,3'-Diindolylmethane (DIM) inhibits the growth and invasion of drug-resistant
human cancer cells expressing EGFR mutants.

Rahimi M, Huang KL, Tang CK.

Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown
University Medical Center, Washington, DC 20057, United States.

Epidermal Growth Factor Receptor (EGFR) mutants are associated with resistance to
chemotherapy, radiation, and targeted therapies. Here we found that the
phytochemical 3,3'-Diindolylmethane (DIM) can inhibit the growth and also the
invasion of breast cancer, glioma, and non-small cell lung cancer cells
regardless of which EGFR mutant is expressed and the drug-resistant phenotype.
DIM reduced an array of growth factor signaling pathways and altered cell cycle
regulators and apoptotic proteins favoring cell cycle arrest and apoptosis.
Therefore, DIM may be used in treatment regimens to inhibit cancer cell growth
and invasion, and potentially overcome EGFR mutant-associated drug resistance.

PMID: 20299148
 

Endocrinology. 2010 Apr;151(4):1662-7.

Selective activation of estrogen receptor-beta target genes by
3,3'-diindolylmethane.

Vivar OI, Saunier EF, Leitman DC, Firestone GL, Bjeldanes LF.

Department of Nutritional Science and Toxicology, University of California,
Berkeley, Berkeley, California 94720-3104, USA.

3,3'-Diindolylmethane (DIM) is a natural compound found in cruciferous vegetables
that has antiproliferative and estrogenic activity. However, it is not clear
whether the estrogenic effects are mediated through estrogen receptor (ER)alpha,
ERbeta, or both ER subtypes. We investigated whether DIM has ER subtype
selectivity on gene transcription. DIM stimulated ERbeta but not ERalpha
activation of an estrogen response element upstream of the luciferase reporter
gene. DIM also selectively activated multiple endogenous genes through ERbeta.
DIM did not bind to ERbeta, indicating that it activates genes by a
ligand-independent mechanism. DIM causes ERbeta to bind regulatory elements and
recruit the steroid receptor coactivator (SRC)-2 coactivator, which leads to the
activation of ER target genes. Silencing of SRC-2 inhibited the activation of ER
target genes, demonstrating that SRC-2 is required for transcriptional activation
by DIM. Our results demonstrate that DIM is a new class of ERbeta-selective
compounds, because it does not bind to ERbeta, but instead it selectively
recruits ERbeta and coactivators to target genes.

PMID: 20160136

Cancer Res. 2010 Feb 15;70(4):1486-95.

MiR-146a suppresses invasion of pancreatic cancer cells.

Li Y, Vandenboom TG 2nd, Wang Z, Kong D, Ali S, Philip PA, Sarkar FH.

Departments of Pathology and Internal Medicine, Barbara Ann Karmanos Cancer
Institute, Wayne State University School of Medicine, Detroit, Michigan 48201,
USA.

The aggressive course of pancreatic cancer is believed to reflect its unusually
invasive and metastatic nature, which is associated with epidermal growth factor
receptor (EGFR) overexpression and NF-kappaB activation. MicroRNAs (miRNA) have
been implicated in the regulation of various pathobiological processes in cancer,
including metastasis in pancreatic cancer and in other human malignancies. In
this study, we report lower expression of miR-146a in pancreatic cancer cells
compared with normal human pancreatic duct epithelial cells. Reexpression of
miR-146a inhibited the invasive capacity of pancreatic cancer cells with
concomitant downregulation of EGFR and the NF-kappaB regulatory kinase
interleukin 1 receptor-associated kinase 1 (IRAK-1). Cellular mechanism studies
revealed crosstalk between EGFR, IRAK-1, IkappaBalpha, NF-kappaB, and MTA-2, a
transcription factor that regulates metastasis. Treatment of pancreatic cancer
cells with the natural products 3,3'-diinodolylmethane (DIM) or isoflavone, which
increased miR-146a expression, caused a downregulation of EGFR, MTA-2, IRAK-1,
and NF-kappaB, resulting in an inhibition of pancreatic cancer cell invasion. Our
findings reveal DIM and isoflavone as nontoxic activators of a miRNA that can
block pancreatic cancer cell invasion and metastasis, offering starting points to
design novel anticancer agents.

PMID: 20124483

 

Cancer Res. 2010 Jan 12.

Chemopreventive Agent 3,3'-Diindolylmethane Selectively Induces Proteasomal
Degradation of Class I Histone Deacetylases.

Li Y, Li X, Guo B.

Authors' Affiliation: Department of Pharmaceutical Sciences, College of Pharmacy,
North Dakota State University, Fargo, North Dakota.

3,3'-Diindolylmethane (DIM) is an anticancer agent that induces cell cycle arrest

and apoptosis. Here, we report that DIM can selectively induce proteasome-mediated

degradation of class I histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC8)

without affecting the class II HDAC proteins. DIM induced downregulation of class I

HDACs in human colon cancer cells in vitro and in vivo in tumor xenografts. HDAC

depletion relieved HDAC-mediated transcriptional inhibition of the cyclin-dependent

kinase inhibitors p21WAF1 and p27KIP2, significantly increasing their expression and

triggering cell cycle arrest in the G(2) phase of the cell cycle. Additionally, HDAC

depletion was associated with an induction of DNA damage that triggered apoptosis. Our

findings indicate that DIM acts to selectively target the degradation of class I HDACs.
Cancer Res; 70(2); 646-54.

PMID: 20068155

Eur J Cancer Prev.

The potential efficacy of 3,3'-diindolylmethane in prevention of prostate cancer
development.

Fares F, Azzam N, Appel B, Fares B, Stein A.

 

Department of Biology, Faculty of Science and Science Education, University of
Haifa bDepartment of Molecular Genetics, Carmel Medical Center cDepartment of
Urology, Carmel Medical Center, Faculty of Medicine, Technion-Israel Institute of
Technology, Haifa, Israel.

The objective of this study was to examine the efficacy of 3,3'-diindolylmethane
(DIM) in prevention of prostate cancer tumor development in an animal model.
Mouse prostate cancer cells (TRAMP-C2, 2x10) were injected subcutaneously into
three groups of C57BL/6 mice (10 mice in each group). Two groups were treated
earlier with DIM; 2 or 10 mg/kg each, and an additional control group was
injected with medium. Animals were treated for five more weeks until sacrificed.
Tumor sizes were measured biweekly. At the end of the experiment, mice were
sacrificed, and tumors were excised, weighed, measured and tested using
immunohistochemical studies. In addition blood samples were collected for
biochemical analysis. The results indicated that DIM significantly reduced tumor
development in treated animals when compared with controls. Tumors developed in
80% of controls and 40% and 60% of animals treated with 10 or 2 mg/kg of DIM,
respectively. Moreover, tumors that developed in treated animals were
significantly (P<0.001) smaller than in controls. Additionally, our results
indicated that DIM has no effect on animal weight or liver and kidney functions.
These results indicated that the DIM agent is not toxic and has an in-vivo
preventive effect against the development of prostate cancer in a mouse model.

PMID: 20010430

Gynecol Oncol. 2009 Nov 24.

Oral diindolylmethane (DIM): Pilot evaluation of a nonsurgical treatment for
cervical dysplasia.

Del Priore G, Gudipudi DK, Montemarano N, Restivo AM, Malanowska-Stega J, Arslan
AA.

Indiana University School of Medicine, Dept of Ob-Gyn, Div Gyn Oncology,
Indianapolis, IN, USA; Research Department, New York Downtown Hospital-member
NY-Presbyterian Healthcare, New York, NY, USA.

OBJECTIVE: Standard surgical treatment for CIN may impair fertility generating a
need for alternative treatment options. We tested the efficacy and toxicity of
oral DIM in the treatment of CIN 2 or 3 lesions. METHODS: Patients with
biopsy-proven cervical intraepithelial neoplasia (CIN) 2 or 3 scheduled for loop
electrosurgical excision procedure (LEEP) were randomized 2:1 to receive
diindolylmethane (DIM) orally at approximately 2 mg/kg/day for 12 weeks or placebo.

Subjects were evaluated every 3-4 months for 1 year. Analysis of
data up to 1 year was assessed including Pap smear, HPV, colposcopy, biopsy and
physical examination were performed at follow-up. Central pathology review
confirmed all histology diagnoses. RESULTS: To date, 64 subjects (mean age 28
years, range 18-61) have been enrolled (45 in the DIM arm, 19 in the placebo
arm), with 60 available for analysis. Average follow-up was 6 months. At
enrollment, 58% were diagnosed with CIN 2 and 42% with CIN 3, 57% of subjects
were Caucasian, 15% African American, 12% Hispanic and 17% Asian. During
treatment 2 subjects (3%) complained of nausea (grade 2) at the 3- to 4-month
visit. No systemic toxicities were observed (normal CBC, LFTs, comprehensive
metabolic). Forty-six subjects had biopsies at first follow-up (77%). Twenty-one
subjects (47%) in the DIM group had improved CIN with a decrease by 1-2 grades or
a normal result. Median time to improvement was 5 months. Improved Pap smear was
seen in 49% (22/45) with either a less severe abnormality or normal result.
Colposcopy improved in twenty-five subjects in the DIM group (56%). Of these 25
subjects, 21 (84%) had improved colposcopic impression, 13 (52%) had a decrease
in involved quadrants and 18 (72%) had a decrease in lesion number. Complete
colposcopic response was observed in 4 subjects (9%). Stratifying by level of
dysplasia, age, race, HPV status, tobacco use, contraceptive used did not alter
the results. At median follow-up of 6 months, 85% of subjects have not required
LEEP based on routine clinical triage of improving global assessment.

CONCLUSION: Oral DIM at 2 mg/kg/day is well tolerated with no
significant toxicity. We observed a high rate of clinically significant
improvement in confirmed CIN 2 or 3 lesions among both treatment groups in this
randomized clinical trial.

PMID: 19939441

J Nutr. 2010 Jan;140(1):1-6. Epub 2009 Nov 4.

Antiangiogenic effects of indole-3-carbinol and 3,3'-diindolylmethane are
associated with their differential regulation of ERK1/2 and Akt in tube-forming
HUVEC.

Kunimasa K, Kobayashi T, Kaji K, Ohta T.

Department of Food and Nutritional Sciences, Graduate School of Nutritional and
Environmental Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.

We previously reported that indole-3-carbinol (I3C), found in cruciferous
vegetables, suppresses angiogenesis in vivo and in vitro. However, the underlying
molecular mechanisms still remain unclear. Antiangiogenic effects of its major
metabolite, 3,3'-diindolylmethane (DIM), also have not been fully elucidated. In
this study, we investigated the effects of these indoles on angiogenesis and
tested a hypothesis that I3C and DIM inhibit angiogenesis and induce apoptosis by
affecting angiogenic signal transduction in human umbilical vein endothelial
cells (HUVEC). We found that I3C and DIM at 25 micromol/L significantly inhibited
tube formation and only DIM induced a significant increase in apoptosis in
tube-forming HUVEC. DIM showed a stronger antiangiogenic activity than I3C. At
the molecular level, I3C and DIM markedly inactivated extracellular
signal-regulated kinase 1/2 (ERK1/2) and the inhibitory effect of DIM was
significantly greater than that of I3C. DIM treatment also resulted in activation
of the caspase pathway and inactivation of Akt, whereas I3C did not affect them.
These results indicate that I3C and DIM had a differential potential in the
regulation of the 2 principal survival signals, ERK1/2 and Akt, in endothelial
cells. We also demonstrated that pharmacological inhibition of ERK1/2 and/or Akt
was enough to inhibit tube formation and induce caspase-dependent apoptosis in
tube-forming HUVEC. We conclude that both I3C and DIM inhibit angiogenesis at
least in part via inactivation of ERK1/2 and that inactivation of Akt by DIM is
responsible for its stronger antiangiogenic effects than those of I3C.

PMID: 19889811

J Nutr. 2009 Dec;139(12):2373-9. Epub 2009 Oct 28.

Oral administration of 3,3'-diindolylmethane inhibits lung metastasis of 4T1
murine mammary carcinoma cells in BALB/c mice.

Kim EJ, Shin M, Park H, Hong JE, Shin HK, Kim J, Kwon DY, Park JH.

Center for Efficacy Assessment and Development of Functional Foods and Drugs,
Hallym University, Chuncheon, Korea.

3,3'-diindolylmethane (DIM) is the major in vivo product of the acid-catalyzed
oligomerization of indole-3-carbinol present in cruciferous vegetables, and it
has been shown to exhibit anticancer properties. In this study, we assessed the
effects of DIM on the metastasis of 4T1 mouse mammary carcinoma cells. In vitro
culture studies showed that DIM dose-dependently inhibited the migration,
invasion, and adhesion of 4T1 cells at concentrations of 0-10 micromol/L without
attendant changes in cell viability. In an in vivo lung metastasis model, 4T1
cells (2 x 10(5) cells/mouse) were injected into the tail veins of syngeneic
female BALB/c mice. Beginning on the second day, the mice were subjected to
gavage with 0-10 mg DIM/(kg body weight x d) for 13 d. Oral DIM administration
resulted in a marked reduction in the number of pulmonary tumor nodules. DIM
treatment significantly reduced the levels of matrix metalloproteinase (MMP)-2,
MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and vascular cell adhesion
molecule (VCAM)-1 and increased TIMP-2 levels in the sera and lungs of mice
injected with 4T1 cells. Additionally, DIM treatment reduced the serum
concentrations of interleukin (IL)-1beta, IL-6 and tumor necrosis factor
(TNF)alpha. We have demonstrated that DIM profoundly inhibits the lung metastasis
of 4T1 cells, which was accompanied by reduced levels of MMP, adhesion molecules,
and proinflammatory cytokines. These results indicate that DIM has potential as
an antimetastatic agent for the treatment of breast cancer.

PMID: 19864400

Cancer Epidemiol Biomarkers Prev. 2009 Nov;18(11):2957-64. Epub 2009 Oct 27.

Diindolylmethane inhibits cervical dysplasia, alters estrogen metabolism, and
enhances immune response in the K14-HPV16 transgenic mouse model.

Sepkovic DW, Stein J, Carlisle AD, Ksieski HB, Auborn K, Bradlow HL.

The David and Alice Jurist Institute for Research, Hackensack University Medical
Center, Hackensack, New Jersey 07601, USA. [email protected]

This study was designed to establish whether 3,3'-diindolylmethane (DIM) can
inhibit cervical lesions, alter estrogen metabolism in favor of C-2
hydroxylation, and enhance immune function in the K14-HPV16 transgenic mouse
model. Mice were bred, genotyped, implanted with E(2) pellets (0.25 mg/90-day
release) under anesthesia, and divided into groups. Wild-type and transgenic mice
were given either AIN76A diet alone or with 2,000 ppm DIM for 12 weeks. Blood and
reproductive tracts were obtained. Blood was analyzed for estrogen metabolites
and IFN-gamma. The cervical transformation zone was sectioned and stained for
histology. Estradiol C-2 hydroxylation and serum IFN-gamma levels were
significantly increased over controls in wild-type and transgenic mice receiving
DIM. In wild-type mice without DIM, hyperplasia of the squamous epithelium was
observed. Wild-type mice fed DIM displayed a normal thin epithelium. In
transgenic mice without DIM, epithelial cell projections into the stroma
(papillae) were present. An additional degree of nuclear anaplasia in the stratum
espinosum was observed. Dysplastic cells were present. Transgenic mice fed DIM
displayed some mild hyperplasia of the squamous epithelium. DIM increases
estrogen C-2 hydroxylation in this model. Serum INF-gamma was increased,
indicating increased immune response in the DIM-fed animals. Histopathology
showed a marked decrease in cervical dsyplasia in both wild-type and transgenic
mice, indicating that DIM delays or inhibits the progression from cervical
dysplasia to cervical cancer. Using the K14-HPV16 transgenic mouse model, we have
shown that DIM inhibits the development of E6/E7 oncogene-induced cervical
lesions.

PMID: 19861518

Biochem Pharmacol. 2009 Oct 23. [Epub ahead of print]

3,3'-Diindolylmethane attenuates experimental arthritis and osteoclastogenesis.

Dong L, Xia S, Gao F, Zhang D, Chen J, Zhang J.

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing 210093, PR China.

3,3'-Diindolylmethane (DIM) is a natural compound formed during the autolysis of
glucobrassicin present in Brassica food plants. This study aimed to investigate
the therapeutic efficacies of DIM on experimental arthritis. The effects of DIM
on experimental arthritis were examined on a rat model of adjuvant-induced
arthritis (AIA), with daily AIA paw swelling observation and
histological/radiographic analysis. To elucidate the possible mechanisms of its
action, serum cytokine levels as well as the expression of receptor activator for
nuclear factor kappa B ligand (RANKL) in infected tissues were subsequently
analyzed. The impact of DIM on osteoclastogenesis was further investigated on a
mouse model of endotoxin-induced bone resorption (EIBR) and in vitro cultures of
fibroblast-like cells and osteoblasts, with RANKL expression being evaluated with
great interest. The administration of DIM was demonstrated to attenuate AIA in
animal models, as judged by clinical and histologic indices of inflammation and
tissue damage. On the one hand, DIM could reduce the expression of several
inflammatory cytokines, which was, however, not adequate to prevent the
development of the arthritis. On the other hand, DIM was shown to effectively
inhibit the expression of RANKL, leading to the blockade of osteoclastogenesis
and consequently an alleviation of experimental arthritis. Further in vitro and
in vivo studies confirmed the inhibition of RANKL by DIM. DIM has shown
anti-arthritis activity in animal models via inhibiting the expression of RANKL,
and thus may offer potential treatments for arthritis and associated disorders.

PMID: 19854159

Mol Endocrinol. 2009 Dec;23(12):1940-7. Epub 2009 Oct 16.

Minireview: modulation of hormone receptor signaling by dietary anticancer
indoles.

Firestone GL, Sundar SN.

Department of Molecular and Cell Biology, The University of California at
Berkeley, 94720-3200, USA.

Indole-3-carbinol and its diindole condensation product 3-3'-diindolylmethane are
dietary phytochemicals that have striking anticarcinogenic properties in human
cancer cells. Molecular, cellular, physiological, and clinical studies have
documented that both indole-3-carbinol and 3-3'-diindolylmethane have potent
endocrine modulating activities through a myriad of mechanisms. The focus of this
review is to discuss the evidence that directly links the anticancer actions of
these two indole compounds to the control of steroid receptor and growth factor
receptor signaling.

PMID: 19837944

Int J Oncol. 2009 Nov;35(5):1191-9.

1,1-Bis(3'-indolyl)-1-(p-bromophenyl)methane and related compounds repress
survivin and decrease gamma-radiation-induced survivin in colon and pancreatic
cancer cells.

Sreevalsan S, Jutooru I, Chadalapaka G, Walker M, Safe S.

Department of Veterinary Physiology and Pharmacology, Texas A&M University,
College Station, TX 77843-4466, USA.

1,1-Bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr) and the 2,2'-dimethyl
analog (2,2'-diMeDIM-C-pPhBr) inhibit proliferation and induce apoptosis in SW480
colon and Panc28 pancreatic cancer cells. In this study, treatment with 10-20
microM concentrations of these compounds for 24 h induced cleaved PARP and
decreased survivin protein and mRNA expression in both cell lines. However,
results of time course studies show that DIM-C-pPhBr and 2,2'-diMeDIM-C-pPhBr
decrease survivin protein within 2 h after treatment, whereas survivin mRNA
levels were decreased only at later time-points indicating activation of
transcription-independent and -dependent pathways for downregulation of survivin.
In addition, we also observed that gamma-radiation inhibited pancreatic and colon
cancer cell growth and this was associated with enhanced expression of survivin
after 24 (SW480) or 24 and 48 h (Panc28) and correlated with previous studies on
the role of survivin in radiation-resistance. However, in cells co-treated with
gamma-radiation plus DIM-C-pPhBr or 2,2'-diMeDIM-C-pPhBr, induction of survivin
by gamma-radiation was inhibited after co-treatment with both compounds,
suggesting applications for these drugs in combination cancer chemotherapy with
gamma-radiation.

PMID: 19787275

J Cell Biochem. 2009 Nov 1;108(4):916-25.

Down-regulation of uPA and uPAR by 3,3'-diindolylmethane contributes to the
inhibition of cell growth and migration of breast cancer cells.

Ahmad A, Kong D, Wang Z, Sarkar SH, Banerjee S, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Center, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

3,3'-Diindolylmethane (DIM) is a known anti-tumor agent against breast and other
cancers; however, its exact mechanism of action remains unclear. The urokinase
plasminogen activator (uPA) and its receptor (uPAR) system are involved in the
degradation of basement membrane and extracellular matrix, leading to tumor cell
invasion and metastasis. Since uPA-uPAR system is highly activated in aggressive
breast cancer, we hypothesized that the biological activity of DIM could be
mediated via inactivation of uPA-uPAR system. We found that DIM treatment as
well as silencing of uPA-uPAR led to the inhibition of cell growth and motility
of MDA-MB-231 cells, which was in part due to inhibition of VEGF and MMP-9.
Moreover, silencing of uPA-uPAR led to decreased sensitivity of these cells to
DIM indicating an important role of uPA-uPAR in DIM-mediated inhibition of
cell growth and migration. We also found similar effects of DIM on MCF-7, cells
expressing low levels of uPA-uPAR, which was due to direct down-regulation of
MMP-9 and VEGF, independent of uPA-uPAR system. Interestingly, over-expression of
uPA-uPAR in MCF-7 cells attenuated the inhibitory effects of DIM. Our results,
therefore, suggest that DIM down-regulates uPA-uPAR in aggressive breast
cancers but in the absence of uPA-uPAR, DIM can directly inhibit VEGF and MMP-9
leading to the inhibition of cell growth and migration of breast cancer cells.
(c) 2009 Wiley-Liss, Inc.

PMID: 19693769

 

Biochem Pharmacol. 2009 Sep 1;78(5):469-76. Epub 2009 May 9.

3,3'-Diindolylmethane induces a G(1) arrest in human prostate cancer cells
irrespective of androgen receptor and p53 status.

Vivar OI, Lin CL, Firestone GL, Bjeldanes LF.

Department of Nutritional Sciences and Toxicology, 119 Morgan Hall, University of
California, Berkeley, CA 94720, USA.

3,3'-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived
from Brassica vegetables. In this study we characterized the effect of DIM on
cell cycle regulation in both androgen-dependent LNCaP and androgen receptor
negative p53 mutant DU145 human prostate cancer cells. DIM had an
anti-proliferative effect on both LNCaP and DU145 cells, as it significantly
inhibited [3H]-thymidine incorporation. FACS analysis revealed a DIM-mediated
G(1) cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4
protein and increased the expression of the cell cycle inhibitor p27(Kip1)
protein in LNCaP and DU145 cells. Promoter deletion studies with p27(Kip1)
reporter gene constructs showed that this DIM-mediated increase in p27(Kip1) was
dependent on the Sp1 transcription factor. Moreover, using a dominant negative
inhibitor of p38 MAPK, we showed that the induction of p27(Kip1) and subsequent
G(1) arrest by DIM involve activation of the p38 MAPK pathway in the DU145 cells.
Taken together, our results indicate that DIM is able to stop the cell cycle
progression of human prostate cancer cells regardless of their
androgen-dependence and p53 status, by differentially modulating cell cycle
regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle
regulatory effect in DU145 cells.

PMID: 19433067

Cancer Res. 2009 Aug 15;69(16):6704-12. Epub 2009 Aug 4.

Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of
epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer
cells.

Li Y, VandenBoom TG 2nd, Kong D, Wang Z, Ali S, Philip PA, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

Pancreatic cancer is the fourth most common cause of cancer death in the United
States, and the aggressiveness of pancreatic cancer is in part due to its
intrinsic and extrinsic drug resistance characteristics, which are also
associated with the acquisition of epithelial-to-mesenchymal transition (EMT).
Emerging evidence also suggests that the processes of EMT are regulated by the
expression status of many microRNAs (miRNA), which are believed to function as
key regulators of various biological and pathologic processes during tumor
development and progression. In the present study, we compared the expression of
miRNAs between gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer
cells and investigated whether the treatment of cells with "natural agents"
[3,3'-diindolylmethane (DIM) or isoflavone] could affect the expression of
miRNAs. We found that the expression of miR-200b, miR-200c, let-7b, let-7c,
let-7d, and let-7e was significantly down-regulated in gemcitabine-resistant
cells, which showed EMT characteristics such as elongated fibroblastoid
morphology, lower expression of epithelial marker E-cadherin, and higher
expression of mesenchymal markers such as vimentin and ZEB1. Moreover, we found
that reexpression of miR-200 by transfection studies or treatment of
gemcitabine-resistant cells with either DIM or isoflavone resulted in the
down-regulation of ZEB1, slug, and vimentin, which was consistent with
morphologic reversal of EMT phenotype leading to epithelial morphology. These
results provide experimental evidence, for the first time, that DIM and
isoflavone could function as miRNA regulators leading to the reversal of EMT
phenotype, which is likely to be important for designing novel therapies for
pancreatic cancer.

PMID: 19654291

Cancer Res. 2009 Aug;69(15):6083-91. Epub 2009 Jul 21.

Low concentrations of diindolylmethane, a metabolite of indole-3-carbinol,
protect against oxidative stress in a BRCA1-dependent manner.

Fan S, Meng Q, Saha T, Sarkar FH, Rosen EM.

Department of Oncology, Georgetown University, Washington, District of
Columbia200 57, USA.

The indole-3-carbinol (I3C) metabolite 3,3'-diindolylmethane (DIM) is a proposed
cancer prevention agent for various tumor types, including breast cancer. Here,
we show that DIM up-regulates expression of the tumor suppressor protein BRCA1 in
carcinoma and normal cell types. Up-regulation of BRCA1 was dose and time
dependent, and it was observed at physiologically relevant micromolar and
submicromolar DIM concentrations when cells were exposed for 72 hours. Treatment
with the parent compound (I3C) or DIM (1 micromol/L) protected against cell
killing due to H(2)O(2) and other oxidants, and the protection was abrogated by
knockdown of BRCA1. DIM stimulated signaling by the antioxidant transcription
factor NFE2L2 (NRF2) through the antioxidant response element in a
BRCA1-dependent manner. We further showed that DIM rapidly stimulated
phosphorylation of BRCA1 on Ser (1387) and Ser (1524) and that these
phosphorylations are required for protection against oxidative stress.
DIM-induced phosphorylation of BRCA1 on Ser (1387) was dependent on
ataxia-telangiectasia mutated. Finally, in our assay systems, H(2)O(2)-induced
cell death was not due to apoptosis. However, a significant component of cell
death was attributable to autophagy, and both DIM and BRCA1 inhibited
H(2)O(2)-induced autophagy. Our findings suggest that low concentrations of DIM
protect cells against oxidative stress via the tumor suppressor BRCA1 by several
distinct mechanisms.

PMID: 19622773

Cancer Res. 2009 Aug 4.

Up-regulation of miR-200 and let-7 by Natural Agents Leads to the Reversal of
Epithelial-to-Mesenchymal Transition in Gemcitabine-Resistant Pancreatic Cancer
Cells.

Li Y, Vandenboom TG 2nd, Kong D, Wang Z, Ali S, Philip PA, Sarkar FH.

Departments of Pathology and Internal Medicine, Barbara Ann Karmanos Cancer
Institute, Wayne State University School of Medicine, Detroit, Michigan.

Pancreatic cancer is the fourth most common cause of cancer death in the United
States, and the aggressiveness of pancreatic cancer is in part due to its
intrinsic and extrinsic drug resistance characteristics, which are also
associated with the acquisition of epithelial-to-mesenchymal transition (EMT).
Emerging evidence also suggests that the processes of EMT are regulated by the
expression status of many microRNAs (miRNA), which are believed to function as
key regulators of various biological and pathologic processes during tumor
development and progression. In the present study, we compared the expression of
miRNAs between gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer
cells and investigated whether the treatment of cells with "natural agents"
[3,3'-diindolylmethane (DIM) or isoflavone] could affect the expression of
miRNAs. We found that the expression of miR-200b, miR-200c, let-7b, let-7c,
let-7d, and let-7e was significantly down-regulated in gemcitabine-resistant
cells, which showed EMT characteristics such as elongated fibroblastoid
morphology, lower expression of epithelial marker E-cadherin, and higher
expression of mesenchymal markers such as vimentin and ZEB1. Moreover, we found
that reexpression of miR-200 by transfection studies or treatment of
gemcitabine-resistant cells with either DIM or isoflavone resulted in the
down-regulation of ZEB1, slug, and vimentin, which was consistent with
morphologic reversal of EMT phenotype leading to epithelial morphology. These
results provide experimental evidence, for the first time, that DIM and
isoflavone could function as miRNA regulators leading to the reversal of EMT
phenotype, which is likely to be important for designing novel therapies for
pancreatic cancer. [Cancer Res 2009;69(16):6704-12].

PMID: 19654291

Cancer Treat Rev. 2009 Aug 4.

Harnessing the fruits of nature for the development of multi-targeted cancer
therapeutics.

Sarkar FH, Li Y.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, MI, USA.

Cancer cells exhibit deregulation in multiple cellular signaling pathways.
Therefore, treatments using specific agents that target only one pathway usually
fail in cancer therapy. The combination treatments using chemotherapeutic agents
with distinct molecular mechanisms are considered more promising for higher
efficacy; however, using multiple agents contributes to added toxicity. Emerging
evidence has shown that some "natural products" such as isoflavones,
indole-3-carbinol (I3C) and its in vivo dimeric product 3,3'-diindolylmethane
(DIM), and curcumin among many others, have growth inhibitory and apoptosis
inducing effects on human and animal cancer cells mediated by targeting multiple
cellular signaling pathways in vitro without causing unwanted toxicity in normal
cells. Therefore, these non-toxic "natural products" from natural resources could
be useful in combination with conventional chemotherapeutic agents for the
treatment of human malignancies with lower toxicity and higher efficacy. In fact,
recently increasing evidence from pre-clinical in vivo studies and clinical
trials have shown some success in support of the use of rational design of
multi-targeted therapies for the treatment of cancers using conventional
chemotherapeutic agents in combination with "natural products". These studies
have provided promising results and further opened-up newer avenues for cancer
therapy. In this review article, we have succinctly summarized the known effects
of "natural products" especially by focusing on isoflavones, indole-3-carbinol
(I3C) and its in vivo dimeric product 3,3'-diindolylmethane (DIM), and curcumin,
and provided a comprehensive view on the molecular mechanisms underlying the
principle of cancer therapy using combination of "natural products" with
conventional therapeutics.

PMID: 19660870

Cancer Res. 2009 Aug 1;69(15):6083-91. Epub 2009 Jul 21.

Low concentrations of diindolylmethane, a metabolite of indole-3-carbinol,
protect against oxidative stress in a BRCA1-dependent manner.

Fan S, Meng Q, Saha T, Sarkar FH, Rosen EM.

Department of Oncology, Georgetown University, Washington, District of
Columbia200 57, USA.

The indole-3-carbinol (I3C) metabolite 3,3'-diindolylmethane (DIM) is a proposed
cancer prevention agent for various tumor types, including breast cancer. Here,
we show that DIM up-regulates expression of the tumor suppressor protein BRCA1 in
carcinoma and normal cell types. Up-regulation of BRCA1 was dose and time
dependent, and it was observed at physiologically relevant micromolar and
submicromolar DIM concentrations when cells were exposed for 72 hours. Treatment
with the parent compound (I3C) or DIM (1 micromol/L) protected against cell
killing due to H(2)O(2) and other oxidants, and the protection was abrogated by
knockdown of BRCA1. DIM stimulated signaling by the antioxidant transcription
factor NFE2L2 (NRF2) through the antioxidant response element in a
BRCA1-dependent manner. We further showed that DIM rapidly stimulated
phosphorylation of BRCA1 on Ser (1387) and Ser (1524) and that these
phosphorylations are required for protection against oxidative stress.
DIM-induced phosphorylation of BRCA1 on Ser (1387) was dependent on
ataxia-telangiectasia mutated. Finally, in our assay systems, H(2)O(2)-induced
cell death was not due to apoptosis. However, a significant component of cell
death was attributable to autophagy, and both DIM and BRCA1 inhibited
H(2)O(2)-induced autophagy. Our findings suggest that low concentrations of DIM
protect cells against oxidative stress via the tumor suppressor BRCA1 by several
distinct mechanisms.

PMID: 19622773

Inflamm Bowel Dis. 2009 Aug;15(8):1164-73.

3,3'-diindolylmethane attenuates colonic inflammation and tumorigenesis in mice.

Kim YH, Kwon HS, Kim DH, Shin EK, Kang YH, Park JH, Shin HK, Kim JK.

Center for Efficacy Assessment and Development of Functional Foods and Drugs,
Hallym University, Chuncheon, South Korea.

BACKGROUND: 3,3-Diindolylmethane (DIM) is a major in vivo product of
acid-catalyzed oligomerization of indole-3-carbinol (I3C) derived from Brassica
food plants. Although DIM is known as a chemopreventive and chemotherapeutic
phytochemical, the effects of DIM on inflammation in vivo are still unknown. In
the present study we investigated the antiinflammatory effects of DIM on
experimental colitis and colitis-associated colorectal carcinogenesis. METHODS:
To determine if DIM has an antiinflammatory effect in vivo, we examined the
therapeutic effects of DIM in dextran sodium sulfate (DSS)-induced experimental
colitis and colitis-associated colon carcinogenesis induced by azoxymethane
(AOM)/DSS in BALB/c mice. RESULTS: Treatment with DIM significantly attenuated
loss of body weight, shortening of the colon, and severe clinical signs in a
colitis model. This was associated with a remarkable amelioration of the
disruption of the colonic architecture and a significant reduction in colonic
myeloperoxidase activity and production of prostaglandin E(2), nitric oxide, and
proinflammatory cytokines. Further, DIM administration dramatically decreased the
number of colon tumors in AOM/DSS mice. CONCLUSIONS: These results suggest that
DIM-mediated antiinflammatory action at colorectal sites may be therapeutic in
the setting of inflammatory bowel disease and colitis-associated colon cancer.

PMID: 19334074

Cancer Res. 2009 Jul 1;69(13):5592-600. Epub 2009 Jun 16.

3,3'-Diindolylmethane enhances chemosensitivity of multiple chemotherapeutic
agents in pancreatic cancer.

Banerjee S, Wang Z, Kong D, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

Clinical management of pancreatic cancer is a major problem, which is in part due
to both de novo and acquired resistance to conventional therapeutics. Here, we
present in vitro and in vivo preclinical evidence in support of
chemosensitization of pancreatic cancer cells by 3,3-diindolylmethane (DIM), a
natural compound that can be easily obtained by consuming cruciferous vegetables.
DIM pretreatment of pancreatic cancer cells led to a significantly increased
apoptosis (P < 0.01) with suboptimal concentrations of chemotherapeutic agents
(cisplatin, gemcitabine, and oxaliplatin) compared with monotherapy. It is known
that resistance to chemotherapy in pancreatic cancer is associated with
constitutively activated nuclear factor-kappaB (NF-kappaB), which becomes further
activated by chemotherapeutic drugs. Our data provide mechanistic evidence for
the first time showing that DIM potentiates the killing of pancreatic cancer
cells by down-regulation of constitutive as well as drug-induced activation of
NF-kappaB and its downstream genes (Bcl-xL, XIAP, cIAP, and survivin). Most
importantly, using an orthotopic animal model, we found reduction in tumor size
(P < 0.001) when DIM was given in combination with oxaliplatin compared with
monotherapy. This was accompanied by loss of phospho-p65 and down-regulation of
NF-kappaB activity and its downstream genes (Bcl-xL, survivin, and XIAP), which
correlated with reduced cell proliferation (as assessed by Ki-67 immunostaining
of tumor specimens) and evidence of apoptosis [as assessed by poly(ADP-ribose)
polymerase cleavage and terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling staining]. These results provide strong in vivo evidence in support
of our hypothesis that DIM could abrogate chemotherapeutic drug (cisplatin,
gemcitabine, and/or oxaliplatin)-induced activation of NF-kappaB, resulting in
the chemosensitization of pancreatic tumors to conventional therapeutics.

PMID: 19531648

Cancer Lett. 2009 Jun 18;278(2):201-9. Epub 2009 Feb 23.

Sensitization of squamous cell carcinoma to cisplatin induced killing by natural
agents.

Ali S, Varghese L, Pereira L, Tulunay-Ugur OE, Kucuk O, Carey TE, Wolf GT, Sarkar
FH.

Division of Hematology/Oncology, Karmanos Cancer Center, Wayne State University,
Detroit, MI, USA.

Cisplatin resistance is a major problem in the successful treatment of squamous
cell carcinoma (SCC). In the present study we showed, for the first time, that
the constitutive activation of NF-kappaB partly contributes to cisplatin
resistance and that the inactivation of NF-kappaB by natural agents [G2535
(isoflavone mixture containing genistein and diadzein), 3,3'-diindolylmethane
(DIM)] could overcome this resistance, resulting in the inhibition of cell growth

and induction of apoptosis, which might be an useful strategy for achieving better

treatment outcome in patients diagnosed with cisplatin-resistant tumors of SCC.

PMID: 19231069

Laryngoscope. 2009 Jun 18.

A controlled safety study of diindolylmethane in the immature rat model.

Elackattu AP, Feng L, Wang Z.

Department of Otolaryngology-Head and Neck Surgery, Boston University School of
Medicine, Boston, Massachusetts, U.S.A.

OBJECTIVES/HYPOTHESIS:: Diindolylmethane (DIM), a natural product from
cruciferous vegetables, has been shown to be a dietary component that has
inhibitory effects on some tumors (e.g., laryngeal papilloma). However, current
evidence to support its safety is based on adult humans or mature animals. There
is little to show on its safety in children. This study is designed to assess
safety in the young rat model. STUDY DESIGN:: Prospective controlled animal
study. METHODS:: Forty rats were separated into four treatment groups of 10 rats
each, based on the amount of study drug they received in their daily food: 1)
immature rats fed a low dose of DIM, (0.6 mg/kg/day); 2) immature rats fed a high
dose of DIM (6.0 mg/kg/day); 3) immature rats fed no DIM (control); and 4) adult
rats fed a high dose of DIM (6.0 mg/kg/day). At the conclusion of the study we
collected blood to compare serum chemistries and vitamin D levels, and harvested
organs to observe for any gross or histological changes among the groups.
Statistical methods involved one-way analysis of variance and pairwise
comparisons with Tukey multiple comparison adjustment. RESULTS:: Although our
numbers do not allow for statistical significance, there was no appreciable
difference in rat weights among the immature groups, nor was there appreciable
difference in serum chemistries, or gross or histological examination of liver,
kidney, and bone. CONCLUSIONS:: Diindolylmethane seems to have no adverse affects
on the rat. This adds evidence to the safety of this drug in the pediatric population as a
treatment option for recurrent respiratory papilloma.

PMID: 19544380

Cancer Prev Res (Phila Pa). 2009 Jun;2(6):581-9. Epub 2009 May 26.

3,3'-diindolylmethane enhances the efficacy of butyrate in colon cancer
prevention through down-regulation of survivin.

Bhatnagar N, Li X, Chen Y, Zhou X, Garrett SH, Guo B.

Department of Pharmaceutical Sciences, College of Pharmacy, North Dakota State
University, Fargo, North Dakota 58105, USA.

Butyrate is an inhibitor of histone deacetylase (HDAC) and has been extensively
evaluated as a chemoprevention agent for colon cancer. We recently showed that
mutations in the adenomatous polyposis coli (APC) gene confer resistance to HDAC
inhibitor-induced apoptosis in colon cancers. Here, we show that APC mutation
rendered colon cancer cells resistant to butyrate-induced apoptosis due to the
failure of butyrate to down-regulate survivin in these cells. Another
cancer-preventive agent, 3,3'-diindolylmethane (DIM), was identified to be able
to down-regulate survivin in colon cancers expressing mutant APC. DIM inhibited
survivin mRNA expression and promoted survivin protein degradation through
inhibition of p34(cdc2)-cyclin B1-mediated survivin Thr(34) phosphorylation.
Pretreatment with DIM enhanced butyrate-induced apoptosis in colon cancer cells
expressing mutant APC. DIM/butyrate combination treatment induced the expression
of proapoptotic Bax and Bak proteins, triggered Bax dimerization/activation, and
caused release of cytochrome c and Smac proteins from mitochondria. Whereas
overexpression of survivin blocked DIM/butyrate-induced apoptosis, knocking down
of survivin by small interfering RNA increased butyrate-induced apoptosis in
colon cancer cells. We further showed that DIM was able to down-regulate survivin
and enhance the effects of butyrate in apoptosis induction and prevention of
familial adenomatous polyposis in APC(min/+) mice. Thus, the combination of DIM
and butyrate is potentially an effective strategy for the prevention of colon
cancer.

PMID: 19470789

Cancer Prev Res (Phila Pa). 2009 Jun;2(6):566-71. Epub 2009 May 26.

3,3'-diindolylmethane induction of p75NTR-dependent cell death via the p38
mitogen-activated protein kinase pathway in prostate cancer cells.

Khwaja FS, Wynne S, Posey I, Djakiew D.

Department of Biochemistry, the Vincent T Lombardi Comprehensive Cancer Center,
Georgetown University Medical, University of the District of Columbia,
Washington, DC 20057-1436, USA.

The p75(NTR) functions as a tumor suppressor in prostate epithelial cells, where
its expression declines with progression to malignant cancer. Previously, we
showed that treatment with the nonsteroidal anti-inflammatory drug, indomethacin,
induced p75(NTR) expression in the T24 cancer cell line leading to
p75(NTR)-mediated decreased survival. Utilizing the indole moiety of indomethacin
as a pharmacophore, we identified in rank-order with least efficacy, ketorolac,
etodolac, indomethacin, 5-methylindole-3-acetic acid, indole-3-carbinol, and
3,3'-diindolylmethane (DIM) exhibiting greatest activity for induction of
p75(NTR) levels and inhibition of cell survival. Prostate (PC-3, DU-145) and
bladder (T24) cancer cells were more sensitive to DIM induction of
p75(NTR)-associated loss of survival than breast (MCF7) and fibroblast (3T3)
cells. Transfection of the PC-3 prostate cell line with a dominant-negative form
of p75(NTR) before DIM treatment significantly rescued cell survival
demonstrating a cause and effect relationship between DIM induction of p75(NTR)
levels and inhibition of survival. Furthermore, siRNA knockdown of the p38
mitogen-activated protein kinase (MAPK) protein prevented induction of p75(NTR)
by DIM in the PC-3 prostate cell line. DIM treatment induced phosphorylation of
p38 MAPK as early as within 1 minute. Collectively, we identify DIM as an indole
capable of inducing p75(NTR)-dependent apoptosis via the p38 MAPK pathway in
prostate cancer cells.

PMID: 19470787

J Cell Biochem. 2009 Jun 1;107(3):516-27.

Inactivation of uPA and its receptor uPAR by 3,3'-diindolylmethane (DIM) leads to
the inhibition of prostate cancer cell growth and migration.

Ahmad A, Kong D, Sarkar SH, Wang Z, Banerjee S, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Center, Wayne State
University School of Medicine, Detroit, Michigan 48201, USA.

3,3'-Diindolylmethane (DIM) has been studied for its putative anti-cancer
properties, especially against prostate cancer; however, its exact mechanism of
action remains unclear. We recently provided preliminary data suggesting
down-regulation of uPA during DIM (a clinically active DIM)-induced inhibition
of invasion and angiogenesis in prostate cancer cells. Since the expression and
activation of uPA plays important role in tumorigenicity, and high endogenous
levels of uPA and uPAR are found in advanced metastatic cancers, we investigated
their role in DIM-mediated inhibition of prostate cancer cell growth and
motility. Using PC3 cells, we found that DIM treatment as well as the silencing
of uPA and uPAR by siRNAs led to the inhibition of cell growth and motility.
Conversely, over-expression of uPA/uPAR in LNCaP and C4-2B cells resulted in
increased cell growth and motility, which was effectively inhibited by DIM.
Moreover, we found that uPA as well as uPAR induced the production of VEGF and
MMP-9, and that the down-regulation of uPA/uPAR by siRNAs or DIM treatment
resulted in the inhibition of VEGF and MMP-9 secretion which could be responsible
for the observed inhibition of cell migration. Interestingly, silencing of
uPA/uPAR led to decreased sensitivity to DIM indicating important role of
uPA/uPAR in DIM-mediated regulation of prostate cancer cell growth and
migration. Our data suggest that chemopreventive and/or therapeutic activity of
DIM is in part due to down-regulation of uPA-uPAR leading to reduced production
of VEGF/MMP-9 which ultimately leads to the inhibition of cell growth and
migration of aggressive prostate cancer cells.

PMID: 19330806

Mol Cancer Res. 2009 Jun;7(6):977-86. Epub 2009 May 26.

Estrogen receptor subtype- and promoter-specific modulation of aryl hydrocarbon
receptor-dependent transcription.

Wihlén B, Ahmed S, Inzunza J, Matthews J.

Department of Biosciences and Nutrition at Novum, Karolinska Institutet,
Huddinge, Sweden.

In this study, we examined the role of estrogen receptors (ER) in aryl
hydrocarbon receptor (AHR)-dependent transactivation. Chromatin
immunoprecipitation assays showed that AHR agonists differentially induced
recruitment of ERalpha to the AHR target genes CYP1A1 and CYP1B1. Cotreatment
with 17beta-estradiol significantly increased beta-naphthoflavone (BNF)- and
2,3,7,8-tetrachlorodibenzo-p-dioxin-induced recruitment of ERalpha to CYP1A1,
whereas 3,3'-diindolylmethane induced promoter occupancy of ERalpha at CYP1A1
that was unaffected by cotreatment with 17beta-estradiol. Cyclical recruitment of
AHR and ERalpha to CYP1A1 was only observed in cells treated with BNF. Stable and
subtype-specific knockdown of ERalpha or ERbeta using shRNA showed that
suppression of ERalpha significantly reduced, whereas knockdown of ERbeta
significantly enhanced, AHR agonist-induced Cyp1a1 expression in HC11 mouse
mammary epithelial cells. AHR agonist-induced Cyp1b1 expression was reduced by
ERbeta knockdown but unaffected by ERalpha knockdown. The siRNA-mediated
knockdown of ERalpha in MCF-7 human breast cancer cells did not affect
2,3,7,8-tetrachlorodibenzo-p-dioxin-dependent regulation of CYP1A1 and CYP1B1
mRNA expression. In agreement with our in vitro findings in the HC11 cells,
ERalpha knockout mice exhibit reduced BNF-dependent induction of Cyp1a1 mRNA.
These results establish ligand- and promoter-specific influences on the cyclical
recruitment patterns for AHR and show ER species-, subtype-, and
promoter-specific modulation of AHR-dependent transcription.

PMID: 19470599

BMC Gastroenterol. 2009 May 29;9:39.

Induction of G1 and G2/M cell cycle arrests by the dietary compound
3,3'-diindolylmethane in HT-29 human colon cancer cells.

Choi HJ, Lim do Y, Park JH.

Department of Food Science and Nutrition, Hallym University, Chuncheon, Republic
of Korea.

BACKGROUND: 3,3'-Diindolylmethane (DIM), an indole derivative produced in the
stomach after the consumption of broccoli and other cruciferous vegetables, has
been demonstrated to exert anti-cancer effects in both in vivo and in vitro
models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the
growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In
this study, we evaluated the effects of DIM on cell cycle progression in HT-29
cells. METHODS: HT-29 cells were cultured with various concentrations of DIM (0 -
30 micromol/L) and the DNA was stained with propidium iodide, followed by flow
cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses,
immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK)
and cell division cycle (CDC)2 were conducted. RESULTS: The percentages of cells
in the G1 and G2/M phases were dose-dependently increased and the percentages of
cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced
DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and
the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also
increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of
cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors,
p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the
levels of CDC25C phosphatase and cyclin B1. CONCLUSION: Here, we have
demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells,
and this effect may be mediated by reduced CDK activity.

PMID: 19480695

Cancer Res. 2009 May 15;69(10):4468-75. Epub 2009 May 12.

3,3'-Diindolylmethane enhances taxotere-induced apoptosis in hormone-refractory
prostate cancer cells through survivin down-regulation.

Rahman KM, Banerjee S, Ali S, Ahmad A, Wang Z, Kong D, Sakr WA.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA. [email protected]

Survivin, a member of inhibitor of apoptosis family, is associated with both
prostate cancer progression and drug resistance. Therefore, we hypothesized that
survivin may play a potentially important role in hormone-refractory prostate
cancer (HRPC) and bone metastatic disease; thus, targeting of survivin signaling
could enhance therapeutic efficacy in prostate cancer. 3,3'-Diindolylmethane
(DIM) has been known to have cancer chemoprevention activity. However, no
information is available regarding the down-regulation of survivin by DIM, which
could result in the chemosensitization of HRPC cells to Taxotere-induced killing.
We investigated the effect of DIM alone or in combination with Taxotere using
LNCaP and C4-2B prostate cancer cells. We observed that DIM enhanced
Taxotere-induced apoptotic death in both cell lines. These enhancing effects were
related to a decrease in survivin expression as well as androgen receptor and
nuclear factor-kappaB (NF-kappaB) DNA-binding activity. We also found that
knockdown of survivin expression by small interfering RNA transfection increased
DIM-induced cell growth inhibition and apoptosis, whereas overexpression of
survivin by cDNA transfection abrogated DIM-induced cell growth inhibition and
apoptosis in both prostate cancer cells. Importantly, luciferase assays showed a
significant reduction of survivin-Luc and NF-kappaB-Luc activity in prostate
cancer cells exposed to DIM and Taxotere. Furthermore, combination treatment
significantly inhibited C4-2B bone tumor growth, and the results were correlated
with the down-regulation of survivin. From these results, we conclude that
inactivation of survivin by DIM enhanced the therapeutic efficacy of Taxotere in
prostate cancer in general, which could be useful for the treatment of HRPC and
metastatic prostate cancer.

PMID: 19435906

Chem Pharm Bull (Tokyo). 2009 May;57(5):536-40.

Enhancement of chemically-induced HL-60 cell differentiation by
3,3'-diindolylmethane derivatives.

Noguchi-Yachide T, Tetsuhashi M, Aoyama H, Hashimoto Y.

Institute of Molecular & Cellular Biosciences, The University of Tokyo, Tokyo,
Japan.

3,3'-Diindolylmethane (DIM, 1) and its derivatives have been prepared, and their
enhancing effects on chemically-induced HL-60 cell differentiation were analyzed.
Among the prepared compounds, IndDIM (12) showed the most potent enhancing effect
on HL-60 cell differentiation induced by chemicals, including retinoids,
1,25-dihydroxyvitamin D(3), 12-O-tetradecanoyl phorbol-13-acetate and dimethyl
sulfoxide.

PMID: 19420792

J Biol Chem. 2009 May 1;284(18):12328-38. Epub 2009 Mar 13.

Lipid G protein-coupled receptor ligand identification using beta-arrestin
PathHunter assay.

Yin H, Chu A, Li W, Wang B, Shelton F, Otero F, Nguyen DG, Caldwell JS, Chen YA.

GPCR Platform, Genomics Institute of the Novartis Research Foundation, San Diego,
California 92121, USA.

A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported
to be activated by lipid ligands, such as lysophosphatidic acid, sphingosine
1-phosphate (S1P), and cannabinoids, for which there are already well established
receptors. These new ligand claims are controversial due to either lack of
independent confirmations or conflicting reports. We used the beta-arrestin
PathHunter assay system, a newly developed, generic GPCR assay format that
measures beta-arrestin binding to GPCRs, to evaluate lipid receptor and ligand
pairing. This assay eliminates interference from endogenous receptors on the
parental cells because it measures a signal that is specifically generated by the
tagged receptor and is immediately downstream of receptor activation. We screened
a large number of newly "deorphaned" receptors (GPR23, GPR92, GPR55, G2A, GPR18,
GPR3, GPR6, GPR12, and GPR63) and control receptors against a collection of
approximately 400 lipid molecules to try to identify the receptor ligand in an
unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with
weaker responses to farnesyl pyrophosphate and geranylgeranyl diphosphate. The
putative cannabinoid receptor GPR55 responded strongly to AM251, rimonabant, and
lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor
was confirmed to be an oxidized free fatty acid receptor. In addition, we
discovered that 3,3'-diindolylmethane, a dietary molecule from cruciferous
vegetables, which has known anti-cancer properties, to be a CB(2) receptor
partial agonist, with binding affinity around 1 microm. The anti-inflammatory
effect of 3,3'-diindolylmethane in RAW264.7 cells was shown to be partially
mediated by CB(2).

PMID: 19286662

J Cell Physiol. 2009 Apr;219(1):94-9.

Cell cycle-dependent effects of 3,3'-diindolylmethane on proliferation and
apoptosis of prostate cancer cells.

Chinnakannu K, Chen D, Li Y, Wang Z, Dou QP, Reddy GP, Sarkar FH.

Vattikuti Urology Institute, Henry Ford Health System, Detroit, Michigan, USA.

Epidemiological studies have shown that a diet rich in fruits and cruciferous
vegetables is associated with a lower risk of prostate cancer. Indole-3-carbinol
(I3C) and its dimeric product 3,3'-diindolylmethane (DIM) have been shown to
exhibit anti-tumor activity both in vitro and in vivo. Recently, we have reported
that DIM induced apoptosis and inhibited growth, angiogenesis, and invasion of

prostate cancer cells by regulating Akt, NF-kappaB, VEGF and the androgen receptor

(AR) signaling pathway. However, the precise molecular mechanism(s) by which DIM

inhibits prostate cancer cell growth and induces apoptosis have not been fully elucidated.

Most importantly, it is not known how DIM affects cell cycle regulators and proteasome

activity, which are  critically involved in cell growth and apoptosis. In this study, we

investigated the effects of DIM on proteasome activity and AR transactivation with

respect to DIM-mediated cell cycle regulation and induction of apoptosis in both
androgen-sensitive LNCaP and androgen-insensitive C4-2B prostate cancer cells. We
believe that our results show for the first time the cell cycle-dependent effects
of DIM on proliferation and apoptosis of synchronized prostate cancer cells
progressing from G(1) to S phase. DIM inhibited this progression by induction
of p27(Kip1) and down-regulation of AR. We also show for the first time that
DIM inhibits proteasome activity in S phase, leading to the inactivation of
NF-kappaB signaling and induction of apoptosis in LNCaP and C4-2B cells. These
results suggest that DIM could be a potent agent for the prevention and/or
treatment of both hormone sensitive as well as hormone-refractory prostate
cancer.

PMID: 19062173

Cell Signal. 2009 Mar 16. [Epub ahead of print]

Cellular signaling perturbation by natural products.

Sarkar FH, Li Y, Wang Z, Kong D.

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State
University School of Medicine, 740 Hudson Webber Cancer Research Center, 4100
John R, Detroit, MI 48201, USA.

Cancer cells are known to have alterations in multiple cellular signaling
pathways and because of the complexities in the communication between multiple
signaling networks, the treatment and the cure for most human malignancies is
still an open question. Perhaps, this is the reason why specific inhibitors that
target only one pathway have been typically failed in cancer treatment. However,
the in vitro and in vivo studies have demonstrated that some natural products
such as isoflavones, indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM),
curcumin, (-)-epigallocatechin-3-gallate (EGCG), resveratrol, lycopene, etc, have
inhibitory effects on human and animal cancers through targeting multiple
cellular signaling pathways and thus these "natural agents" could be classified
as multi-targeted agents. This is also consistent with the epidemiological
studies showing that the consumption of fruits, soybean and vegetables is
associated with reduced risk of several types of cancers. By regulating multiple
important cellular signaling pathways including NF-kappaB, Akt, MAPK, Wnt, Notch,
p53, AR, ER, etc, these natural products are known to activate cell death signals
and induce apoptosis in pre-cancerous or cancer cells without affecting normal
cells. Therefore, non-toxic "natural agents" harvested from the bounties of
nature could be useful either alone or in combination with conventional
therapeutics for the prevention of tumor progression and/or treatment of human
malignancies.

PMID: 19298854

Cancer Prev Res (Phila Pa). 2009 Mar;2(3):251-6. Epub 2009 Feb 17.

Toxic and chemopreventive ligands preferentially activate distinct aryl
hydrocarbon receptor pathways: implications for cancer prevention.

Okino ST, Pookot D, Basak S, Dahiya R.

Department of Urology, San Francisco Veterans Affairs Medical Center and the
University of California at San Francisco, San Francisco, California 94121, USA.

The aryl hydrocarbon receptor (AhR) is a ligand-activated regulatory protein that
controls estrogen action through two distinct pathways. In one pathway, AhR acts
as a transcription factor that induces the expression of the CYP1 family of
estrogen-metabolizing genes; in the other pathway, AhR initiates the degradation
of the estrogen receptor and suppresses estrogen signaling. The AhR ligand
3,3'-diindolylmethane (DIM) is a beneficial dietary constituent that prevents
breast tumors in rodents and is associated with decreased breast cancer risk in
humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic AhR ligand that is
implicated in birth defects, infertility, and cancer. We analyzed MCF-7 cells to
gain insight into how two AhR ligands can exert such fundamentally different
health effects. We find that DIM and TCDD have differing abilities to activate
the distinct AhR-controlled pathways. TCDD strongly induces AhR-dependent CYP1
gene expression, whereas DIM is a relatively weak CYP1 inducer. DIM strongly
inhibits estrogen receptor-alpha expression and estrogen signaling, whereas TCDD
has a notably weaker effect on these processes. Small interfering RNA knockdown
of AhR confirms that the effects of DIM and TCDD are indeed AhR dependent. Our
findings reveal that DIM and TCDD each elicit a unique pattern of change in
pathways that control estrogen action; such patterns may determine if an AhR
ligand has beneficial or adverse health effects.

PMID: 19223575

Clin Cancer Res. 2009 Jan 15;15(2):543-52.

Enhancement of docetaxel anticancer activity by a novel diindolylmethane compound
in human non-small cell lung cancer.

Ichite N, Chougule MB, Jackson T, Fulzele SV, Safe S, Singh M.

College of Pharmacy and Pharmaceutical Sciences. Florida A&M University,
Tallahassee, Florida 32307, USA.

PURPOSE: This study was conducted to examine the cytotoxic effects of a
peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, 1,1-bis
(3'-indolyl)-1-(p-biphenyl) methane (DIM-C-pPhC(6)H(5)), alone and in combination
with docetaxel in vitro in A549 lung cancer cells and in vivo in nude mice
bearing A549 orthotopic lung tumors. EXPERIMENTAL DESIGN: Isobolographic method
was used to calculate combination index values from cell viability data.
Apoptosis was evaluated in A549 cells by terminal deoxynucleotidyl
transferase-mediated nick end labeling assay and measurement of cleaved
poly(ADP-ribose) polymerase level. Expression of proteins was studied by Western
blotting. A549 cells were implanted to induce orthotopic lung tumors in nude mice
and the efficacy of docetaxel, DIM-C-pPhC(6)H(5), or combination was determined.
Apoptosis and cleaved caspase-3 expression in the harvested tissues were studied
by terminal deoxynucleotidyl transferase-mediated nick end labeling and
immunohistochemistry, respectively. RESULTS: The combination index values
(0.36-0.9) suggested synergistic to additive effects of docetaxel +
DIM-C-pPhC(6)H(5) and resulted in the highest increase in percentage of apoptotic
cells and expression of cleaved poly(ADP-ribose) polymerase, Bax, and N-cadherin
compared with treatment with either agent. The combination also enhanced
procaspase-3 and -9 cleavage. In vivo, docetaxel + DIM-C-pPhC(6)H(5) reduced lung
weights by 57% compared with 39% by docetaxel or 22% by DIM-C-pPhC(6)H(5) alone,
induced apoptosis in 43% of the tumor cells compared with 29% and 22% in tumors
treated with docetaxel and DIM-C-pPhC(6)H(5), respectively, and increased
procaspase-3 cleavage compared with either agent alone. CONCLUSIONS: These
findings suggest potential benefit for use of docetaxel and DIM-C-pPhC(6)H(5)
combination in lung cancer treatment.

PMID: 19147759

J Nutr. 2009 Jan;139(1):26-32. Epub 2008 Dec 3.

Targeting of aryl hydrocarbon receptor-mediated activation of cyclooxygenase-2
expression by the indole-3-carbinol metabolite 3,3'-diindolylmethane in breast
cancer cells.

Degner SC, Papoutsis AJ, Selmin O, Romagnolo DF.

Department of Nutritional Sciences, University of Arizona, Tucson, AZ 85721, USA.

Ligands of the aryl hydrocarbon receptor (AhR) include the environmental
xenobiotic 2,3,7,8 tetrachlorodibenzo(p)dioxin (TCDD), polycyclic aryl
hydrocarbons, and the dietary compounds 3, 3'-diindolylmethane (DIM), a
condensation product of indol-3-carbinol found in Brassica vegetables, and the
phytoalexin resveratrol (RES). The AhR and its cofactors regulate the expression
of target genes at pentameric (GCGTG) xenobiotic responsive elements (XRE).
Because the activation of cyclooxygenase-2 (COX-2) expression by AhR ligands may
contribute to inflammation and tumorigenesis, we investigated the epigenetic
regulation of the COX-2 gene by TCDD and the reversal effects of DIM in MCF-7
breast cancer cells. Results of DNA binding and chromatin immunoprecipitation
(ChIP) studies documented that the treatment with TCDD induced the association of
the AhR to XRE harbored in the COX-2 promoter and control CYP1A1 promoter
oligonucleotides. The TCDD-induced binding of the AhR was reduced by
small-interfering RNA for the AhR or the cotreatment with synthetic
(3-methoxy-4-naphthoflavone) or dietary AhR antagonists (DIM, RES). In time
course ChIP studies, TCDD induced the rapid (15 min) occupancy by the AhR, the
histone acetyl transferase p300, and acetylated histone H4 (AcH4) at the COX-2
promoter. Conversely, the cotreatment of MCF-7 cells with DIM (10 micromol/L)
abrogated the TCDD-induced recruitment of the AhR and AcH4 to the COX-2 promoter
and the induction of COX-2 mRNA and protein levels. Taken together, these data
suggest that naturally occurring modulators of the AhR such as DIM may be
effective agents for dietary strategies against epigenetic activation of COX-2
expression by AhR agonists.

PMID: 19056653

J Oncol. 2009;2009:491985. Epub 2009 Mar 24.

Modulation of CXCR4, CXCL12, and Tumor Cell Invasion Potential In Vitro by
Phytochemicals.

Hsu EL, Chen N, Westbrook A, Wang F, Zhang R, Taylor RT, Hankinson O.

Molecular Toxicology Interdepartmental Doctoral Program, University of California
Los Angeles, Los Angeles, CA 90095, USA.

CXCR4 is a chemokine receptor frequently overexpressed on primary tumor cells.
Organs to which these cancers metastasize secrete CXCL12, the unique ligand for
CXCR4, which stimulates invasion and metastasis to these sites. Similar to our
previous work with the chemoprotective phytochemical, 3,3'-diindolylmethane
(DIM), we show here that genistein also downregulates CXCR4 and CXCL12 and
subsequently lowers the migratory and invasive potentials of breast and ovarian
cancer cells. Moreover, genistein and DIM elicit a significantly greater
cumulative effect in lowering CXCR4 and CXCL12 levels than either compound alone.
Our data suggest a novel mechanism for the protective effects of phytochemicals
against cancer progression and indicate that in combination, these compounds may
prove even more efficacious.

PMID: 19325924

Mol Pharmacol. 2009 Jan;75(1):35-43. Epub 2008 Oct 7.

Suppression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nitric-oxide
synthase 2 expression in astrocytes by a novel diindolylmethane analog protects
striatal neurons against apoptosis.

Carbone DL, Popichak KA, Moreno JA, Safe S, Tjalkens RB.

Department of Environmental and Radiological Health Sciences, Colorado State
University, Collins, CO 80523, USA.

The progressive debilitation of motor functions in Parkinson's disease (PD)
results from degeneration of dopaminergic neurons within the substantia nigra
pars compacta of the midbrain. Long-term inflammatory activation of microglia and
astrocytes plays a central role in the progression of PD and is characterized by
activation of the nuclear factor-kappaB (NF-kappaB) signaling cascade and
subsequent overproduction of inflammatory cytokines and nitric oxide (NO).
Suppression of this neuroinflammatory phenotype has received considerable
attention as a potential target for chemotherapy, but there are no currently
approved drugs that sufficiently address this problem. The data presented here
demonstrate the efficacy of a novel anti-inflammatory diindolylmethane class
compound, 1,1-bis(3'-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu), in
suppressing NF-kappaB-dependent expression of inducible nitric-oxide synthase
(NOS2) and NO production in astrocytes exposed to the parkinsonian neurotoxicant
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through a mechanism distinct
from that described for the thiazolidinedione-class compound, rosiglitazone.
Chromatin immunoprecipitations revealed that micromolar concentrations of
DIM-C-pPhtBu prevented association of the p65 subunit of NF-kappaB with enhancer
elements in the Nos2 promoter but had little effect on DNA binding of either
peroxisome proliferator-activated receptor-gamma (PPAR-gamma) or the nuclear
corepressor NCoR2. Treatment with DIM-C-pPhtBu concomitantly suppressed NO
production and protein nitration in MPTP-activated astrocytes and completely
protected cocultured primary striatal neurons from astrocyte-dependent apoptosis.
These data demonstrate the efficacy of DIM-C-pPhtBu in preventing the activation
of NF-kappaB-dependent inflammatory genes in primary astrocytes and suggest that
this class of compounds may be effective neuroprotective anti-inflammatory agents
in vivo.

PMID: 18840677


J Nutr. 2008 Dec;138(12):2379-85.

3,3'-Diindolylmethane and genistein decrease the adverse effects of estrogen in
LNCaP and PC-3 prostate cancer cells.

Smith S, Sepkovic D, Bradlow HL, Auborn KJ.

Department of Microbiology and Immunology, Albert Einstein College of Medicine,
Bronx, NY 10461, USA.

Evidence suggests that 17beta-estradiol (E2) contributes to the risk of prostate
cancer (PCa), whereas the phytochemicals genistein from soy and
3,3'-diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous
vegetables, decrease the risk of PCa. This study examined the potential of these
phytochemicals to reduce the adverse effects of E2 on PCa. In LNCaP PCa cells (E2
sensitive), DIM decreased E2-induced proliferation. Genistein increased
proliferation at low concentrations and decreased proliferation at higher
concentrations; DIM abolished the increased proliferation by genistein. The E2
stimulation in LNCaP cells was consistent with dependence on the androgen
receptor, as evidenced by the inhibition of E2-induced proliferation with the
antiandrogen casodex, E2 stimulation of an androgen response element luciferase
reporter, and E2 stimulation of prostate-specific antigen (PSA) protein
expression. Both genistein and DIM abrogated the E2 stimulation of PSA. Genistein
and DIM altered major E2 metabolism pathways in LNCaP and PC-3 (E2 insensitive)
PCa cells by increasing the expression of the 2-hydoxylation enzyme cytochrome
P450 1A1 (CYP1A1) and the O-methylating enzyme catechol-o-methyltransferase
(COMT) as determined by real-time RT-PCR. The increase in COMT mRNA occurred only
when the combination of DIM and genistein (15 micromol/L) was used. Quantitation
by MS indicated increased 2-hydroxyestrogen and decreased 16alpha-hydroxyestrone,
a result that should result in less estrogenicity and increased amounts of the
anticancer metabolite 2-methoxyestrone. We conclude that DIM and genistein
decrease the effects of E2 that have the potential to promote PCa.

PMID: 19022961

Pharm Res. 2008 Sep;25(9):2117-24. Epub 2008 Apr 22.

Chemoprevention of pancreatic cancer: characterization of Par-4 and its
modulation by 3,3' diindolylmethane (DIM).

Azmi AS, Ahmad A, Banerjee S, Rangnekar VM, Mohammad RM, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, 9374 Scott Hall, 540 E Canfield, Detroit, Michigan 48201, USA.

PURPOSE: Cancer chemoprevention is defined as the use of natural, synthetic, or
biological agents to suppress, reverse or prevent the carcinogenic process from
turning into aggressive cancer. Prostate apoptosis response-4 (Par-4) is a unique
pro-apoptotic protein that selectively induces apoptosis in prostate cancer
cells. However, its role in other malignancies has not been fully explored. This
study tries to identify the functional significance of Par-4 in pancreatic
cancer. METHODS: Multiple molecular techniques such as Western blot analysis,
trypan blue assay for cell viability, MTT assay for cell growth inhibition and
Histone/DNA ELISA for apoptosis were used. RESULTS: Western blot analysis
revealed that 3,3'-diindolylmethane (DIM) a chemopreventive agent, specifically
its more bioavailable formulation, DIM, at low doses (20 micromol/L) induces
Par-4, in L3.6pl and Colo-357 pancreatic cancer cells. At similar doses, DIM
reduced cell viability and caused cell growth inhibition and apoptosis. Moreover,
DIM pre-treatment sensitized the cells to cytotoxic action of chemotherapeutic
drug gemcitabine through up-regulation of Par-4. CONCLUSION: The induction of
Par-4 is indirectly related to increased sensitivity and cell death through
apoptosis. To our knowledge the results reported here showed, for the first time,
the induction of Par-4 by chemopreventive agents, in general, and DIM, in
particular, in pancreatic cancer cells in vitro.

PMID: 18427961

 

Food Chem Toxicol. 2008 Jul;46(7):2451-8. Epub 2008 Apr 6.

Inhibitory effects of a dietary phytochemical 3,3'-diindolylmethane (DIM) on the
phenobarbital-induced hepatic CYP mRNA expression and CYP-catalyzed reactions in
female rats.

Parkin DR, Lu Y, Bliss RL, Malejka-Giganti D.

Veterans Affairs Medical Center, Minneapolis, MN 55417, USA.

3,3'-diindolylmethane (DIM), derived from indole-3-carbinol (I3C), is used as a
dietary supplement for its putative anticancer effects that include suppression
of mammary tumor growth in female rats. The mechanism of action DIM may involve
its interaction(s) with hepatic cytochromes P450 (CYPs) catalyzing oxidations of
17beta-estradiol (E2). Our study showed that DIM added to hepatic microsomes of
female Sprague-Dawley rats was primarily a competitive inhibitor of
beta-naphthoflavone (beta-NF)- or I3C-induced CYP1A1 probe activity, and a potent
mixed or uncompetitive inhibitor of phenobarbital (PB)-induced CYP2B1 or CYP2B2
probe activity, respectively. Microsomal metabolites of DIM were tentatively
identified as two mono-hydroxy isomers of DIM, each formed preferentially by
CYP1A1- or CYP2B1/2-catalyzed reaction. Evaluation of the effects of co-treatment
of rats with PB and DIM by a full factorial ANOVA showed that DIM decreased the
PB-induced CYP2B1 and CYP2B2 mRNA expression levels, and the rates of 2- and
4-hydroxylation of E2, and total E2 metabolite formation. The results suggest
that interactions of DIM, and/or its mono-hydroxy metabolites, with CYP2B1 and
CYP2B2 found to occur in hepatic microsomes upon addition of DIM or co-treatment
of rats with DIM affect the rates of relevant oxidations of E2, and potentially
protect against estrogen-dependent tumorigenesis.

PMID: 18486294

 

Carcinogenesis. 2008 Jun;29(6):1139-47. Epub 2008 May 5.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell
and tumor growth through activation of c-jun N-terminal kinase.

Lei P, Abdelrahim M, Cho SD, Liu S, Chintharlapalli S, Safe S.

Institute of Biosciences and Technology, Texas A&M University Health Science
Center, Houston, TX 77030-3303, USA.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) activate the orphan
receptors peroxisome proliferator-activated receptor gamma (PPARgamma) and Nur77
and induce receptor-dependent and -independent apoptotic pathways in colon and
other cancer cells. Structure-activity studies show that the p-bromo
(DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation
of Nur77 and PPARgamma, induce expression of CCAAT/enhancer-binding protein
homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series
of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the
position of the phenyl substituents (para >/= meta >/= ortho) and required a free
indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated
death receptor 5 (CHOP dependent), cleavage of caspase 8 and poly (ADP ribose)
polymerase (PARP) that is consistent with activation of the extrinsic pathway of
apoptosis. These responses were associated with the activation of c-jun
N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of
the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to
classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and
thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 that
is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also
observed in athymic nude mice bearing RKO cell xenografts and treated with 30
mg/kg/day DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation
in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer
cells and tumors is related to a novel ER stress-independent activation of JNK.

PMID: 18460448

 

Cancer Let. 2008 Jun 28;265(1):113-23. Epub 2008 Apr 2.

CXCR4 and CXCL12 down-regulation: a novel mechanism for the chemoprotection of
3,3'-diindolylmethane (DIM) for breast and ovarian cancers.

Hsu EL, Chen N, Westbrook A, Wang F, Zhang R, Taylor RT, Hankinson O.

Molecular Toxicology Interdepartmental Doctoral Program, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA.

Cruciferous vegetables are thought to protect against numerous types of cancer.
3,3'-Diindolylmethane (DIM) is an acid-catalyzed product generated during the
consumption of cruciferous vegetables and appears to be chemoprotective for
breast cancer. The interaction between the chemokine receptor, CXCR4, and its
unique ligand, CXCL12, is known to mediate the progression and metastasis of
breast and other cancers. Organs to which these cancers metastasize secrete
CXCL12, which binds to CXCR4 expressed on the surface of primary cancer cells.
This process subsequently stimulates the invasive properties of the cancer cells
and attracts them to the preferred organ sites of metastases. We have found that
DIM down-regulates both CXCR4 and CXCL12 in MCF-7 and MDA-MB-231 breast cancer
cells as well as in BG-1 ovarian cancer cells at the transcriptional level and in
an estrogen-independent manner. We demonstrate that the potential of MDA-MB-231
and BG-1 cells for chemotaxis and invasion towards CXCL12, but not towards IL-6
or fetal bovine serum, respectively, is inhibited by DIM. Furthermore, we show
that DIM down-regulates CXCR4 under hypoxia and CXCL12 under estradiol-inducing
conditions. Our data suggest that one mechanism whereby DIM protects against
breast, ovarian, and possibly other cancers is through the repression of CXCR4
and/or CXCL12, thereby lowering the invasive and metastatic potential of these
cells.

PMID: 18378071

 

Mol Cancer Ther. 2008 Jun;7(6):1708-19.

Apoptosis-inducing effect of erlotinib is potentiated by 3,3'-diindolylmethane (DIM) in
vitro and in vivo using an orthotopic model of pancreatic cancer.

Ali S, Banerjee S, Ahmad A, El-Rayes BF, Philip PA, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI, USA.

Blockade of epidermal growth factor receptor (EGFR) by EGFR tyrosine kinase
inhibitors is insufficient for effective antitumor activity because of
independently activated survival pathways. A multitargeted approach may therefore
improve the outcome of anti-EGFR therapies. In the present study, we determined
the effects of 3,3'-diindolylmethane on cell viability and apoptosis with
erlotinib in vitro and in vivo using an orthotopic animal tumor model. BxPC-3 and
MIAPaCa cells with varying levels of EGFR and nuclear factor-kappaB (NF-kappaB)
DNA-binding activity were treated with DIM (20 micromol/L), erlotinib (2
micromol/L), and the combination. Cell survival and apoptosis was assessed by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and histone-DNA
ELISA. Electrophoretic mobility shift assay was used to evaluate NF-kappaB
DNA-binding activity. We found significant reduction in cell viability by both
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic
assays, induction of apoptosis, down-regulation of EGFR phosphorylation,
NF-kappaB DNA-binding activity, and expression of antiapoptotic genes in BxPC-3
cells when treated with the combination of erlotinib and DIM compared with
either agent alone. In contrast, no such effect was observed in MIAPaCa cells by
similar treatment. Most importantly, these in vitro results were recapitulated in
animal model showing that DIM in combination with erlotinib was much more
effective as an antitumor agent compared with either agent alone. These results
suggest that the utilization of DIM could be a useful strategy for achieving
better treatment outcome in patients with activated status of EGFR and NF-kappaB
in their tumors.

PMID: 18566242

Biochem Pharmacol. 2008 May 1;75(9):1858-67.

3,3'-diindolylmethane (DIM) reduces levels of HIF-1alpha and HIF-1 activity in hypoxic
cultured human cancer cells.

Riby JE, Firestone GL, Bjeldanes LF.

Department of Nutritional Sciences and Toxicology, 217 Morgan Hall, University of
California, Berkeley, CA 94720, USA.

3,3'-diindolylmethane (DIM) is a chemopreventive and chemotherapeutic
phytochemical derived from the metabolism of indoles found at high concentrations
in cruciferous vegetables. We have previously shown that DIM exhibits
anti-angiogenic properties in cultured vascular endothelial cells and in Matrigel
plug assays in rodents. In the present study, we demonstrate that DIM reduces the
level of hypoxia-inducible factor (HIF)-1alpha in hypoxic tumor cell lines, as
well as HIF-1 transcriptional activity as measured by a reporter assay. Moreover,
DIM inhibited the expression of HIF-1-responsive endogenous genes, resulting in
the reduced expression of key hypoxia responsive factors, VEGF, furin, enolase-1,
glucose transporter-1 and phosphofructokinase. DIM reduced the level of
HIF-1alpha in hypoxic cells by increasing the rate of the prolylhydroxylase- and
proteasome-mediated degradation of HIF-1alpha, and by decreasing the rate of
HIF-1alpha transcription. Using enzyme kinetics studies, we established that DIM
interacts with the oligomycin-binding site on the F0 transmembrane component of
mitochondrial F1F0-ATPase. The contributions of the resulting increases in levels
of ROS and O2 in hypoxic cells to the inhibitory effects of DIM on HIF-1alpha
expression are discussed. These studies are the first to show that DIM can
decrease the accumulation and activity of the key angiogenesis regulatory factor,
HIF-1alpha, in hypoxic tumor cells.

PMID: 18329003

Pharm Res. 2008 Apr 22

Chemoprevention of Pancreatic Cancer: Characterization of Par-4 and its
Modulation by 3,3' Diindolylmethane (DIM).

Azmi AS, Ahmad A, Banerjee S, Rangnekar VM, Mohammad RM, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, 9374 Scott Hall, 540 E Canfield, Detroit, Michigan, 48201, USA.

PURPOSE: Cancer chemoprevention is defined as the use of natural, synthetic, or
biological agents to suppress, reverse or prevent the carcinogenic process from
turning into aggressive cancer. Prostate apoptosis response-4 (Par-4) is a unique
pro-apoptotic protein that selectively induces apoptosis in prostate cancer
cells. However, its role in other malignancies has not been fully explored. This
study tries to identify the functional significance of Par-4 in pancreatic
cancer. METHODS: Multiple molecular techniques such as Western blot analysis,
trypan blue assay for cell viability, MTT assay for cell growth inhibition and
Histone/DNA ELISA for apoptosis were used. RESULTS: Western blot analysis
revealed that 3,3'-diindolylmethane (DIM) a chemopreventive agent, specifically
its more bioavailable formulation, DIM, at low doses (20 mumol/L) induces
Par-4, in L3.6pl and Colo-357 pancreatic cancer cells. At similar doses, DIM
reduced cell viability and caused cell growth inhibition and apoptosis. Moreover,
DIM pre-treatment sensitized the cells to cytotoxic action of chemotherapeutic
drug gemcitabine through up-regulation of Par-4. CONCLUSION: The induction of
Par-4 is indirectly related to increased sensitivity and cell death through
apoptosis. To our knowledge the results reported here showed, for the first time,
the induction of Par-4 by chemopreventive agents, in general, and DIM, in
particular, in pancreatic cancer cells in vitro.

PMID: 18427961

Cancer Let. 2008 Mar 28

CXCR4 and CXCL12 down-regulation: A novel mechanism for the chemoprotection of
3,3'-diindolylmethane (DIM) for breast and ovarian cancers.

Hsu EL, Chen N, Westbrook A, Wang F, Zhang R, Taylor RT, Hankinson O.

Molecular Toxicology Interdepartmental Doctoral Program, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA; Department of Pathology
and Laboratory Medicine, University of California-Los Angeles, Los Angeles, CA
90095-1732, USA; Jonsson Comprehensive Cancer Center, University of
California-Los Angeles, Los Angeles, CA 90095-1732, USA.

Cruciferous vegetables are thought to protect against numerous types of cancer.
3,3'-Diindolylmethane (DIM) is an acid-catalyzed product generated during the
consumption of cruciferous vegetables and appears to be chemoprotective for
breast cancer. The interaction between the chemokine receptor, CXCR4, and its
unique ligand, CXCL12, is known to mediate the progression and metastasis of
breast and other cancers. Organs to which these cancers metastasize secrete
CXCL12, which binds to CXCR4 expressed on the surface of primary cancer cells.
This process subsequently stimulates the invasive properties of the cancer cells
and attracts them to the preferred organ sites of metastases. We have found that
DIM down-regulates both CXCR4 and CXCL12 in MCF-7 and MDA-MB-231 breast cancer
cells as well as in BG-1 ovarian cancer cells at the transcriptional level and in
an estrogen-independent manner. We demonstrate that the potential of MDA-MB-231
and BG-1 cells for chemotaxis and invasion towards CXCL12, but not towards IL-6
or fetal bovine serum, respectively, is inhibited by DIM. Furthermore, we show
that DIM down-regulates CXCR4 under hypoxia and CXCL12 under estradiol-inducing
conditions. Our data suggest that one mechanism whereby DIM protects against
breast, ovarian, and possibly other cancers is through the repression of CXCR4
and/or CXCL12, thereby lowering the invasive and metastatic potential of these
cells.

PMID: 18378071

Cancer Res. 2008 Mar 15;68(6):1927-34.

Mammalian target of rapamycin repression by 3,3'-diindolylmethane (DIM) inhibits
invasion and angiogenesis in platelet-derived growth factor-D-overexpressing PC3
cells.

Kong D, Banerjee S, Huang W, Li Y, Wang Z, Kim HR, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA.

Platelet-derived growth factor-D (PDGF-D) is a newly recognized growth factor
known to regulate many cellular processes, including cell proliferation,
transformation, invasion, and angiogenesis. Recent studies have shown that PDGF-D
and its cognate receptor PDGFR-beta are expressed in prostate tumor tissues,
suggesting that PDGF-D might play an important role in the development and
progression of prostate cancer. However, the biological role of PDGF-D in
tumorigenesis remains elusive. In this study, we found that PDGF-D-overexpressing
PC3 cells (PC3 cells stably transfected with PDGF-D cDNA and referred to as PC3
PDGF-D) exhibited a rapid growth rate and enhanced cell invasion that was
associated with the activation of mammalian target of rapamycin (mTOR) and
reduced Akt activity. Rapamycin repressed mTOR activity and concomitantly
resulted in the activation of Akt, which could attenuate the therapeutic effects
of mTOR inhibitors. In contrast, DIM significantly inhibited both mTOR and Akt in

PC3 PDGF-D cells, which were correlated with decreased cell proliferation and invasion.
Moreover, conditioned medium from PC3 PDGF-D cells significantly increased the
tube formation of human umbilical vein endothelial cells, which was inhibited by
DIM treatment concomitant with reduced full-length and active form of PDGF-D.
Our results suggest that DIM could serve as a novel and efficient
chemopreventive and/or therapeutic agent by inactivation of both mTOR and Akt
activity in PDGF-D-overexpressing prostate cancer.

PMID: 18339874

Zhonghua Yi Xue Za Zhi. 2008 Mar 11;88(10):661-4.

3,3-diindolylmethane (DIM) enhances the inhibitory effect of idarubicin on the growth
of human prostate cancer cells (Article in Chinese)

Zhao YY, Zhou L, Pan YZ, Zhao LJ, Liu YN, Yu H, Li Y, Zhao XJ.

Prostate Diseases Prevention and Treatment Research Center, Jilin University,
Changchun 130021, China.

OBJECTIVE: To study the effects of idarubicin (IDA) combined with 3,
3-diindolylmethane (DIM) on the growth inhibition of human prostate cancer cells.
METHODS: Human prostate cancer cells of the line PC-3M were cultured and then
divided into the following groups: control group with solvent added into the
culture fluid; IDA groups, with IDA of the terminal concentrations of 0.5, 1 or 5
mg/L added into the culture fluid; DIM groups, with DIM of the terminal
concentrations of 30, 60 or 100 micromol/L added into the culture fluid; and DIM
+ IDA groups, with 0. 5 mg/L IDA and DIM 30, 60 or 100 micromol/L added into the
culture fluid. 48 h later the cell growth inhibition rate was detected by MTT
assay. Flow cytometry and acridine orange staining were used to detect the cell
cycle and apoptosis. RT-PCR and Western blotting were used to detect the mRNA and
protein expression of caspase 9, an apoptosis gene. RESULTS: Both IDA and DIM
dose-dependently inhibited the growth of the PC-3M cells. The growth inhibition
rate of the 60 micromol/L DIM + 0.5 mg/L IDA group was 69.9%, almost 10 times as
that of the 0.5 mg/L IDA group. The apoptosis rate of the 60 micromol/L DIM + 0.
5 mg/L IDA group was 47.0%, significantly higher than that of the 0.5 mg/L IDA
group (3.2%, P < 0.05). RT-PCR and Western blotting showed that the combination
of DIM and IDA significantly enhanced the mRNA and protein expression of caspase
9. CONCLUSION: DIM enhances the growth inhibition effect of IDA on human prostate
cancer cells by the mechanism of induction of apoptosis.

PMID: 18642764

Mol Cancer Ther. 2008 Feb;7(2):341-9.

Induction of growth arrest and apoptosis in human breast cancer cells by
3,3-diindolylmethane (DIM) is associated with induction and nuclear localization of
p27kip.

Wang Z, Yu BW, Rahman KM, Ahmad F, Sarkar FH.

Department of Pathology, Karmanos Cancer Institute, Wayne State University School
of Medicine, Detroit, MI 48201, USA.

3,3'-Diindolylmethane (DIM) is a stable condensation product of
indole-3-carbanol, a potential breast cancer chemoprevention agent. Human breast
cancer cell lines were studied to better understand its mechanisms. In vitro
experiments were done in MCF-7, T47D, BT-20 and BT-474 cells using MTT, ELISA,
immunoblotting assays, reverse transcription-PCR, protein half-life, confocal
microscopy, cell fractionation, and immunoprecipitation assays. We found that DIM
inhibited the growth of all four breast cancer cell lines (IC(50)s, 25-56
micromol/L). Because BT-20 and BT-474 overexpressed Her-2 and activated Akt, and
BT-20 lacks estrogen receptor, these were studied further. In both cell lines,
DIM appeared to induce expression of p27(kip) protein before the loss of cell
viability and apoptosis. In BT-20 cells, DIM also inhibited expression of
activated Akt, but this appeared after p27(kip) induction. In both cell lines,
DIM induced p27(kip) transcript expression within 6 h. DIM prolonged the p27(kip)
protein half-life in BT-20 but not BT-474 cells. We also showed, for the first
time, that DIM induced nuclear localization of p27(kip) in both cell lines.
Moreover, in BT-20 cells, DIM induced a decrease in p27(kip) phosphorylation at
Thr(187), and its association with the 14-3-3 protein, which helped to explain
the protein half-life increase and nuclear localization, respectively. DIM
modulates p27(kip) through transcription, prolongation of protein half-life, and
nuclear localization. These effects appear to be independent of Her-2, Akt, or
estrogen receptor status and should support further study for its chemoprevention
potential in breast cancer.

PMID: 18281517

J Nutr. 2008 Jan;138(1):17-23.

 

3,3'-Diindolylmethane (DIM) suppresses the inflammatory response to lipopolysaccharide

in murine macrophages.

 

Cho HJ, Seon MR, Lee YM, Kim J, Kim JK, Kim SG, Park JH.

 

Center for Efficacy Assessment and Development of Functional Foods and Drugs,

Hallym University, Chuncheon 200-702, South Korea.

 

3,3'-Diindolylmethane (DIM), a major acid-condensation product of

indole-3-carbinol, has been shown to have multiple anticancer effects in

experimental models. Because recurrent or chronic inflammation has been

implicated in the development of a variety of human cancers, this study examined

the antiinflammatory effects of DIM and the underlying mechanisms using

lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. DIM

significantly decreased the release of nitric oxide (NO), prostaglandin (PG)E2,

tumor necrosis factor alpha, interleukin (IL)-6, and IL-1beta by RAW264.7 cells

treated with LPS. DIM inhibited LPS-induced increases in protein levels of

inducible NO synthase (iNOS), which were accompanied by decreased iNOS mRNA

levels and transcriptional activity. The mRNA levels of phospholipase A2

decreased, whereas neither cyclooxygenases-2 protein nor transcript was altered

by DIM. In addition, DIM suppressed LPS-induced nuclear factor-kappaB (NF-kappaB)

transcriptional activity, NF-kappaB DNA-binding activity, translocation of p65

(RelA) to the nucleus, and degradation of inhibitor of kappaB alpha. Furthermore,

DIM decreased LPS-induced transcriptional activity of activator protein (AP)-1,

AP-1 DNA-binding activity, and phosphorylation of stress-activated protein

kinase/Jun-N-terminal kinase and c-Jun. We demonstrate that DIM inhibits

LPS-induced release of proinflammatory mediators in murine macrophages.

Downregulation of NF-kappaB and AP-1 signaling may be one of the mechanisms by

which DIM inhibits inflammatory responses.

 

PMID: 18156398

 

Mol Cancer Ther. 2007 Nov;6(11):3071-9.

 

Extended treatment with physiologic concentrations of dietary phytochemicals

results in altered gene expression, reduced growth, and apoptosis of cancer

cells.

 

Moiseeva EP, Almeida GM, Jones GD, Manson MM.

 

Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester,

University Road, Leicester LE1 7RH, United Kingdom.

 

Dietary phytochemicals exhibit chemopreventive potential in vivo through

persistent low-dose exposures, whereas mechanistic in vitro studies with these

agents generally use a high-dose single treatment. Because the latter approach is

not representative of an in vivo steady state, we investigated antitumor activity

of curcumin, 3,3'-diindolylmethane (DIM), epigallocatechin gallate (EGCG),

genistein, or indole-3-carbinol (I3C) in breast cancer MDA-MB-231 cells, exposed

in long-term culture to low concentrations, achievable in vivo. Curcumin and EGCG

increased cell doubling time. Curcumin, EGCG, and I3C inhibited clonogenic growth

by 55% to 60% and induced 1.5- to 2-fold higher levels of the basal caspase-3/7

activity. No changes in expression of cell cycle-related proteins or survivin

were found; however, I3C reduced epidermal growth factor receptor expression,

contributing to apoptosis. Because some phytochemicals are shown to inhibit DNA

and histone modification, modulation of expression by the agents in a set of

genes (cadherin-11, p21Cip1, urokinase-type plasminogen activator, and

interleukin-6) was compared with changes induced by inhibitors of DNA methylation

or histone deacetylation. The phytochemicals modified protein and/or RNA

expression of these genes, with EGCG eliciting the least and DIM the most changes

in gene expression. DIM and curcumin decreased cadherin-11 and increased

urokinase-type plasminogen activator levels correlated with increased cell

motility. Curcumin, DIM, EGCG, and genistein reduced cell sensitivity to

radiation-induced DNA damage without affecting DNA repair. This model has

revealed that apoptosis and not arrest is likely to be responsible for growth

inhibition. It also implicated new molecular targets and activities of the agents

under conditions relevant to human exposure.

 

PMID: 18025290 [PubMed - in process]

  

Mol Cancer Ther. 2007 Oct;6(10):2757-65. Epub 2007 Oct 3.

 

Inactivation of NF-kappaB by 3,3'-diindolylmethane (DIM) contributes to increased

apoptosis induced by chemotherapeutic agent in breast cancer cells.

 

Rahman KM, Ali S, Aboukameel A, Sarkar SH, Wang Z, Philip PA, Sakr WA, Raz A.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, 715 HWCRC, 4100 John R, Detroit, MI 48201, USA.

 

Constitutive activation of Akt or nuclear factor-kappaB (NF-kappaB) has been

reported to play a role in de novo resistance of cancer cells to chemotherapeutic

agents, which is a major cause of treatment failure in cancer chemotherapy.

Previous studies have shown that 3,3'-diindolylmethane (DIM), a major in vivo

acid-catalyzed condensation product of indole-3-carbinol, is a potent inducer of

apoptosis, inhibitor of tumor angiogenesis, and inactivator of Akt/NF-kappaB

signaling in breast cancer cells. However, little is known regarding the

inactivation of Akt/NF-kappaB that leads to chemosensitization of breast cancer

cells to chemotherapeutic agents, such as Taxotere. Therefore, we examined

whether the inactivation Akt/NF-kappaB signaling caused by DIM could sensitize

breast cancer cells to chemotherapeutic agents both in vitro and in vivo.

MDA-MB-231 cells were simultaneously treated with 15 to 45 micromol/L DIM and

0.5 to 1.0 nmol/L Taxotere for 24 to 72 h. Cell growth inhibition assay,

apoptosis assay, electrophoretic mobility shift assay, and Western blotting were

done. The combination treatment of 30 micromol/L DIM with 1.0 nmol/L Taxotere

elicited significantly greater inhibition of cell growth compared with either

agent alone. The combination treatment induced greater apoptosis in MDA-MB-231

cells compared with single agents. Moreover, we found that NF-kappaB activity was

significantly decreased in cells treated with DIM and Taxotere. We also have

tested our hypothesis using transfection studies, followed by combination

treatment with DIM/Taxotere, and found that combination treatment significantly

inhibited cell growth and induced apoptosis in MDA-MB-231 breast cancer cells

mediated by the inactivation of NF-kappaB, a specific target in vitro and in

vivo. These results were also supported by animal experiments, which clearly

showed that DIM sensitized the breast tumors to Taxotere, which resulted in

greater antitumor activity mediated by the inhibition of Akt and NF-kappaB.

Collectively, our results clearly suggest that inhibition of Akt/NF-kappaB

signaling by DIM leads to chemosensitization of breast cancer cells to

Taxotere, which may contribute to increased growth inhibition and apoptosis in

breast cancer cells. The data obtained from our studies could be a novel

breakthrough in cancer therapeutics by using nontoxic agents, such as DIM, in

combination with other conventional therapeutic agents, such as Taxotere.

  

PMID: 17913854

 

J Nutr Biochem. 2007 Aug 16 [Epub ahead of print]

 

3,3'-Diindolylmethane (DIM) stimulates murine immune function in vitro and in vivo.

 

Xue L, Pestka JJ, Li M, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM), a major condensation product of indole-3-carbinol,

exhibits chemopreventive properties in animal models of cancer. Recent studies

have shown that DIM stimulates interferon-gamma (IFN-gamma) production and

potentiates the IFN-gamma signaling pathway in human breast cancer cells via a

mechanism that includes increased expression of the IFN-gamma receptor. The goal

of this study was to test the hypothesis that DIM modulates the murine immune

function. Specifically, the effects of DIM were evaluated in a panel of murine

immune function tests that included splenocyte proliferation, reactive oxygen

species (ROS) generation, cytokine production and resistance to viral infection.

DIM was found to induce proliferation of splenocytes as well as augment mitogen-

and interleukin (IL)-2-induced splenocyte proliferation. DIM also stimulated the

production of ROS by murine peritoneal macrophage cultures. Oral administration

of DIM, but not intraperitoneal injection, induced elevation of serum cytokines

in mice, including IL-6, granulocyte colony-stimulating factor (G-CSF), IL-12 and

IFN-gamma. Finally, in a model of enteric virus infection, oral DIM

administration to mice enhanced both clearance of reovirus from the GI tract and

the subsequent mucosal IgA response. Thus, DIM is a potent stimulator of immune

function. This property might contribute to the cancer inhibitory effects of this

indole.

 

PMID: 17707631 [PubMed - as supplied by publisher]

  

J Biol Chem. 2007 Jul 20;282(29):21542-50. Epub 2007 May 23.

 

Regulation of FOXO3a/beta-catenin/GSK-3beta signaling by 3,3'-diindolylmethane (DIM)

contributes to inhibition of cell proliferation and induction of apoptosis in

prostate cancer cells.

 

Li Y, Wang Z, Kong D, Murthy S, Dou QP, Sheng S, Reddy GP, Sarkar FH.

 

Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State

University School of Medicine, Detroit, Michigan 48201, USA.

 

Previous studies from our laboratory have shown anti-proliferative and

pro-apoptotic effects of 3,3'-diindolylmethane (DIM) through regulation of Akt

and androgen receptor (AR) in prostate cancer cells. However, the mechanism by

which DIM regulates Akt and AR signaling pathways has not been fully

investigated. It has been known that FOXO3a and glycogen synthase kinase-3beta

(GSK-3beta), two targets of activated Akt, interact with beta-catenin, regulating

cell proliferation and apoptotic cell death. More importantly, FOXO3a, GSK-3beta,

and beta-catenin are all AR coregulators and regulate the activity of AR,

mediating the development and progression of prostate cancers. Here, we

investigated the molecular effects of DIM, a formulated DIM with higher

bioavailability, on Akt/FOXO3a/GSK-3beta/beta-catenin/AR signaling in

hormone-sensitive LNCaP and hormone-insensitive C4-2B prostate cancer cells. We

found that DIM significantly inhibited the phosphorylation of Akt and FOXO3a

and increased the phosphorylation of beta-catenin, leading to the inhibition of

cell growth and induction of apoptosis. We also found that DIM significantly

inhibited beta-catenin nuclear translocation. By electrophoretic mobility shift

and chromatin immunoprecipitation assays, we found that DIM inhibited FOXO3a

binding to the promoter of AR and promoted FOXO3a binding to the p27(KIP1)

promoter, resulting in the alteration of AR and p27(KIP1) expression, the

inhibition of cell proliferation, and the induction of apoptosis in both

androgen-sensitive and -insensitive prostate cancer cells. These results suggest

that DIM-induced cell growth inhibition and apoptosis induction are partly

mediated through the regulation of Akt/FOXO3a/GSK-3beta/beta-catenin/AR

signaling. Therefore, DIM could be a promising non-toxic agent for possible

treatment of hormone-sensitive but most importantly hormone-refractory prostate

cancers.

 

PMID: 17522055

 

Mol Med. 2007 Jan-Feb;13(1-2):69-78.

 

Interplay of genes regulated by estrogen and diindolylmethane (DIM) in breast cancer

cell lines.

 

Mulvey L, Chandrasekaran A, Liu K, Lombardi S, Wang XP, Auborn KJ, Goodwin L.

 

Feinstein Institute for Medical Research, Manhasset, NY 11030, USA.

 

Diindolylmethane (DIM), a biologically active congener of indole-3-carbinol (I3C)

derived from cruciferous vegetables, is a promising agent for the prevention of

estrogen-sensitive cancers. Both DIM and estrogen affect transcription of genes

by binding receptors, such as aryl hydrocarbon receptor (AhR) or estrogen

receptors (ER). Gene regulation by DIM and estradiol (E2) can be very complex.

While DIM typically binds the AhR, this complex can directly associate with the

ER, recruit co-activators that bind to estrogen-responsive promoters, and

activate transcription. Alternately, DIM can bind the ER directly. In this study,

we have analyzed gene expression using microarray profiling and quantitative real

time-polymerase chain reaction in MCF7 breast cancer cells treated with E2 (1 nM)

or DIM (25 microM) alone or in combination for 16 h. The interplay of E2 and DIM

was reflected in the expression of a subset of genes (<90) in which the

combination of E2 and DIM acted either additively or antagonistically to alter

gene expression.

 

PMID: 17515958

 

Cancer Res. 2007 Apr 1;67(7):3310-9.

 

Inhibition of angiogenesis and invasion by 3,3'-diindolylmethane (DIM) is mediated by

the nuclear factor-kappaB downstream target genes MMP-9 and uPA that regulated

bioavailability of vascular endothelial growth factor in prostate cancer.

 

Kong D, Li Y, Wang Z, Banerjee S, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, Michigan, USA.

 

Progression of prostate cancer is believed to be dependent on angiogenesis

induced by tumor cells. 3,3'-Diindolylmethane (DIM) has been shown to repress

neovascularization in a Matrigel plug assay and inhibit cell proliferation,

migration, invasion, and capillary tube formation of cultured human umbilical

vein endothelial cells. However, the molecular mechanism, by which DIM inhibits

angiogenesis and invasion, has not been fully elucidated. Therefore, we sought to

explore the molecular mechanism by which DIM inhibits angiogenesis and invasion,

specifically by investigating the role of angiogenic factors secreted by prostate

cancer cells which control all steps of angiogenesis. We found that DIM inhibited

angiogenesis and invasion by reducing the bioavailability of vascular endothelial growth

factor (VEGF) via repressing extracellular matrix-degrading proteases, such as

matrix metalloproteinase (MMP)-9 and urokinase-type plasminogen activator (uPA),

in human prostate cancer cells and reduced vascularity (angiogenesis) in vivo

using Matrigel plug assay. We also found that DIM treatment inhibited DNA

binding activity of nuclear factor-kappaB (NF-kappaB), which is known to mediate

the expression of many NF-kappaB downstream target genes, including VEGF, IL-8,

uPA, and MMP-9, all of which are involved in angiogenesis, invasion, and

metastasis. Our data suggest that inhibition of NF-kappaB DNA binding activity by

DIM contributes to the regulated bioavailability of VEGF by MMP-9 and uPA and,

in turn, inhibits invasion and angiogenesis, which could be mechanistically

linked with the antitumor activity of DIM as observed previously by our

laboratory in a prostate cancer animal model.

 

PMID: 17409440

 

Carcinogenesis. 2007 Jul;28(7):1471-7. Epub 2007 Feb 28.

 

Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane (DIM)

on proliferation of human colon cancer cells in vitro.

 

Pappa G, Strathmann J, Löwinger M, Bartsch H, Gerhäuser C.

 

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center

(DKFZ), C010-2 Chemoprevention, Im Neuenheimer Feld 280, 69120 Heidelberg,

Germany.

 

Isothiocyanates (ITCs) and indoles derived from cruciferous vegetables possess

growth-inhibiting and apoptosis-inducing activities in cancer cell lines in

vitro. ITCs like sulforaphane (SFN) are cytotoxic, whereas indoles including

indole-3-carbinol or its condensation product 3,3'-diindolylmethane (DIM) are

acting by cytostatic mechanisms in human colon cancer cell lines. In the present

study, we have investigated the impact of defined combinations of SFN and DIM

(ratio 1:4, 1:2, 1:1, 2:1 and 4:1) on cell proliferation, cell-cycle progression

and apoptosis induction in cultured 40-16 colon carcinoma cells. Calculations of

combination effects were based on the method of Chou et al. (1984) Adv. Enzyme

Regul., 22, 27-55, and were expressed as a combination index (CI) with CI < 1, CI

= 1 or CI > 1 representing synergism, additivity or antagonism, respectively.

Interestingly, at a total drug concentration of 2.5 microM, all combinations of

SFN and DIM were antagonistic. With increasing concentrations, the antagonistic

effect gradually turned into a synergistic interaction at the highest combined

cytotoxic concentration of 40 microM. Cell-cycle analyses with SFN:DIM ratios of

1:1, 1:2 and 1:4 and total concentrations between 10 and 25 microM confirmed

antagonism at low and additive effects at higher doses. SFN (10 microM) in

combination with DIM (10 microM) resulted in strong G(2)/M cell-cycle arrest,

which was not observed with either compound alone. Our results indicate that

cytotoxic concentrations of SFN:DIM combinations affect cell proliferation

synergistically. At low total concentrations (below 20 microM), which are

physiologically more relevant, the combined broccoli compounds showed

antagonistic interactions in terms of cell growth inhibition. These data stress

the need for elucidating mechanistic interactions for better predicting

beneficial health effects of bioactive food components.

 

PMID: 17331956

   

J Nutr. 2007 Jan;137(1):31-6.

 

Activation of caspase-8 contributes to 3,3'-Diindolylmethane-induced apoptosis in

colon cancer cells.

 

Kim EJ, Park SY, Shin HK, Kwon DY, Surh YJ, Park JH.

 

Center for Efficacy Assessment and Development of Functional Foods and Drugs,

Hallym University, Chuncheon, 200-702, Korea.

 

3,3'-Diindolylmethane (DIM) is the major in vivo product of acid-catalyzed

oligomerization of indole-3-carbinol, which is a promising anticancer agent

present in cruciferous vegetables and has itself been reported to have

anticarcinogenic properties. This study examined DIM-mediated regulation of

apoptosis in the HCT116 (wild-type p53) and HT-29 (mutant p53) human colon cancer

cell lines. DIM (0-30 micromol/L) substantially decreased the number of viable

cells and induced apoptosis of HCT116 and HT-29 cells in a

concentration-dependent manner. Western-blot analyses of total cell lysates

revealed that DIM increased the activation of caspase-3, -7, -8, and -9 and

enhanced poly(ADP-ribose) polymerase cleavage in both HCT116 and HT-29 cells. In

addition, DIM increased the translocation of cytochrome c and Smac/Diablo from

the mitochondria to the cytoplasm. In concert with the caspase-8 activation by

DIM, increased levels of Fas and truncated Bid were observed. DIM did not affect

the protein levels of p53, Bcl-2, Bax, or Fas ligand (FasL) in HCT116 cells. In

HT-29 cells, however, DIM decreased Bcl-2 levels, although the protein levels of

Bax or FasL were not affected. The caspase-8 inhibitor Z-IETD-FMK attenuated the

DIM-induced apoptosis, indicating that increased activation of this enzyme

contributed to the increase in p53-independent apoptosis that was observed in

colon cancer cells. We have demonstrated that DIM induces apoptosis in colon

cancer cells, providing insights into the mechanisms underlying its

antitumorigenic activities.

 

PMID: 17182797

  

Mol Pharmacol. 2007 Feb;71(2):558-69. Epub 2006 Nov 8.

 

1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes inhibit growth, induce

apoptosis, and decrease the androgen receptor in LNCaP prostate cancer cells

through peroxisome proliferator-activated receptor gamma-independent pathways.

 

Chintharlapalli S, Papineni S, Safe S.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University, 4466

TAMU, Vet. Res. Bldg. 409, College Station, TX 77843-4466, USA.

 

1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) containing

para-trifluoromethyl, t-butyl, and phenyl groups are a novel class of peroxisome

proliferator-activated receptor (PPAR)gamma agonists. In LNCaP prostate cancer

cells, these compounds induce PPARgamma-dependent transactivation, inhibit cell

proliferation, and induce apoptosis. In addition, these PPARgamma agonists

modulate a number of antiproliferative and proapoptotic responses, including

induction of p27, activating transcription factor 3, and nonsteroidal

anti-inflammatory drug-activated gene-1 and down-regulation of cyclin D1 and

caveolin-1. Moreover, the PPARgamma antagonist 2-chloro-5-nitrobenzanilide

(GW9662) does not inhibit these effects. The C-DIM compounds also abrogate

androgen receptor (AR)-mediated signaling and decrease prostate-specific antigen

(PSA) and AR protein expression, and these responses were PPARgamma-independent.

The effects of C-DIMs on AR and PSA were due to decreased AR and PSA mRNA

expression in LNCaP cells. Thus, this series of methylene-substituted

diindolylmethane derivatives simultaneously activate multiple pathways in LNCaP

cells, including ablation of androgen-responsiveness and down-regulation of

caveolin-1. Both of these responses are associated with activation of

proapoptotic pathways in this cell line.

 

PMID: 17093136

 

Am J Pathol. 2006 Nov;169(5):1833-42.

 

Fas-mediated apoptosis in cholangiocarcinoma cells is enhanced by

3,3'-diindolylmethane (DIM) through inhibition of AKT signaling and FLICE-like

inhibitory protein.

 

Chen Y, Xu J, Jhala N, Pawar P, Zhu ZB, Ma L, Byon CH, McDonald JM.

 

Department of Pathology, University of Alabama at Birmingham, LHRB 511, 1530 3rd

Ave. South, Birmingham, AL 35294, USA.

 

Stimulation of Fas-mediated apoptosis has been promoted as a potential therapy

for many cancers, including cholangiocarcinoma. We have previously reported that

Fas-resistant, but not Fas-sensitive, cholangiocarcinoma cells are tumorigenic in

nude mice. The present studies sought to identify molecular targets that promote

Fas-mediated apoptosis in cholangiocarcinoma. We found that Fas-resistant

cholangiocarcinoma cells exhibited increased constitutive phosphorylation of AKT

compared with Fas-sensitive cells. Increased phosphorylation of AKT was also

demonstrated in human cholangiocarcinoma tumors and was evident in a mouse

xenograft cholangiocarcinoma model. Furthermore, we found that

3,3'-diindolylmethane (DIM), a vegetable autolysis product, promoted Fas-mediated

apoptosis of cholangiocarcinoma cells. DIM inhibited phosphorylation of AKT and

activation of FLICE-like-inhibitory-protein (FLIP). Inhibition of

phosphatidylinositol 3-kinase/AKT decreased FLIP activation and promoted

Fas-mediated apoptosis. By contrast, adenovirus-mediated constitutively activated

AKT protected cholangiocarcinoma cells from Fas-mediated apoptosis. Decreased

activation of extracellular signal-regulated kinase and nuclear factor-kappaB and

increased activation of caspase-3, -8, and -9 were associated with inhibition of

AKT and FLIP. These results support AKT and FLIP as potential molecular targets

and DIM as a potent compound for cholangiocarcinoma intervention.

 

PMID: 17071604

 

Cancer Res. 2006 Oct 15;66(20):10064-72.

 

Down-regulation of androgen receptor by 3,3'-diindolylmethane (DIM) contributes to

inhibition of cell proliferation and induction of apoptosis in both

hormone-sensitive LNCaP and insensitive C4-2B prostate cancer cells.

 

Bhuiyan MM, Li Y, Banerjee S, Ahmed F, Wang Z, Ali S, Sarkar FH.

 

Departments of Pathology and Internal Medicine, Karmanos Cancer Institute, Wayne

State University School of Medicine, Detroit, Michigan 48201, USA.

 

Despite the initial efficacy of androgen deprivation therapy, most patients with

advanced prostate cancer eventually progress to hormone-refractory prostate

cancer, for which there is no curative therapy. Previous studies from our

laboratory and others have shown the antiproliferative and proapoptotic effects

of 3,3'-diindolylmethane (DIM) in prostate cancer cells. However, the molecular

mechanism of action of DIM has not been investigated in androgen receptor

(AR)-positive hormone-responsive and -nonresponsive prostate cancer cells.

Therefore, we investigated the effects of DIM, a formulated DIM with greater

bioavailability, on AR, Akt, and nuclear factor kappaB (NF-kappaB) signaling in

hormone-sensitive LNCaP (AR+) and hormone-insensitive C4-2B (AR+) prostate cancer

cells. We found that DIM significantly inhibited cell proliferation and induced

apoptosis in both cell lines. By Akt gene transfection, reverse

transcription-PCR, Western blot analysis, and electrophoretic mobility shift

assay, we found a potential crosstalk between Akt, NF-kappaB, and AR.

Importantly, DIM significantly inhibited Akt activation, NF-kappaB DNA binding

activity, AR phosphorylation, and the expressions of AR and prostate-specific

antigen, suggesting that DIM could interrupt the crosstalk. Confocal studies

revealed that DIM inhibited AR nuclear translocation, leading to the

down-regulation of AR target genes. Moreover, DIM significantly inhibited C4-2B

cell growth in a severe combined immunodeficiency-human model of experimental

prostate cancer bone metastasis. These results suggest that DIM-induced cell

proliferation inhibition and apoptosis induction are partly mediated through the

down-regulation of AR, Akt, and NF-kappaB signaling. These observations provide a

rationale for devising novel therapeutic approaches for the treatment of

hormone-sensitive, but more importantly, hormone-refractory prostate cancer by

using DIM alone or in combination with other therapeutics.

 

PMID: 17047070

   

Cancer Res. 2006 May 1;66(9):4952-60.

 

Gene expression profiling revealed survivin as a target of

3,3'-diindolylmethane-induced cell growth inhibition and apoptosis in breast

cancer cells.

 

Rahman KW, Li Y, Wang Z, Sarkar SH, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, Michigan 48201, USA.

 

The phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, and

its major acid-catalyzed reaction product 3,3'-diindolylmethane (DIM) showed

anticancer activity mediated by its pleiotropic effects on cell cycle

progression, apoptosis, carcinogen bioactivation, and DNA repair. To further

elucidate the molecular mechanism(s) by which 3,3'-diindolylmethane exerts its

effects on breast cancer cells, we have used microarray gene expression profiling

analysis. We found a total of 1,238 genes altered in

3,3'-diindolylmethane-treated cells, among which 550 genes were down-regulated

and 688 genes were up-regulated. Clustering analysis showed significant

alterations in some genes that are critically involved in the regulation of cell

growth, cell cycle, apoptosis, and signal transduction, including down-regulation

of survivin. Previous studies have shown that antiapoptotic protein survivin is

overexpressed in many human cancers, including breast cancer. However, very

little or no information is available regarding the consequence of

down-regulation of survivin for cancer therapy. We, therefore, hypothesized that

down-regulation of survivin as observed by 3,3'-diindolylmethane could be an

important approach for the treatment of breast cancer. We have tested our

hypothesis using multiple molecular approaches and found that

3,3'-diindolylmethane inhibited cell growth and induced apoptosis in MDA-MB-231

breast cancer cells by down-regulating survivin, Bcl-2, and cdc25A expression and

also caused up-regulation of p21(WAF1) expression, which could be responsible for

cell cycle arrest. Down-regulation of survivin by small interfering RNA before

3,3'-diindolylmethane treatment resulted in enhanced cell growth inhibition and

apoptosis, whereas overexpression of survivin by cDNA transfection abrogated

3,3'-diindolylmethane-induced cell growth inhibition and apoptosis. These results

suggest that targeting survivin by 3,3'-diindolylmethane could be a new and novel

approach for the prevention and/or treatment of breast cancer.

 

PMID: 16651453

 

Cancer Res. 2006 May 1;66(9):4880-7.

 

3,3'-Diindolylmethane (DIM) is a novel mitochondrial H(+)-ATP synthase inhibitor that

can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human

breast cancer cells.

 

Gong Y, Sohn H, Xue L, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

Epidemiologic evidence suggests that high dietary intake of Brassica vegetables,

such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis

in multiple organs. 3,3'-Diindolylmethane, one of the active products derived

from Brassica vegetables, is a promising antitumor agent. Previous studies in our

laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in

human breast cancer MCF-7 cells by a mechanism that included increased expression

of p21. In the present study, the upstream events leading to p21 overexpression

were further investigated. We show for the first time that 3,3'-diindolylmethane

is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20

micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of

mitochondrial inner membrane, decreased cellular ATP level, and significantly

stimulated mitochondrial reactive oxygen species (ROS) production. ROS

production, in turn, led to the activation of stress-activated pathways involving

p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580

and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase

(JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In

addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced

activation of p38 and JNK and induction of p21, indicating that oxidative stress

is the major trigger of these events. To further support the role of ROS in

3,3'-diindolylmethane-induced p21 overexpression, we showed that

3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial

respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane

did not stimulate ROS production. Thus, we have established the critical role of

enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21

up-regulation in human breast cancer cells.

 

PMID: 16651444

 

J Surg Res. 2006 May 15;132(2):208-13. Epub 2006 Mar 31.

 

3,3'-diindolylmethane (DIM) and paclitaxel act synergistically to promote apoptosis in

HER2/Neu human breast cancer cells.

 

McGuire KP, Ngoubilly N, Neavyn M, Lanza-Jacoby S.

 

Department of Surgery, Jefferson Medical College, Thomas Jefferson University,

Philadelphia, Pennsylvania 19107, USA.

 

BACKGROUND: HER2/neu positive breast tumors are difficult to treat. About 25 to

30% of invasive breast tumors overexpress the HER2/neu oncogene. These tumors are

aggressive and become resistant to chemotherapeutic drugs. 3'3'-diindolylmethane

(DIM), the active metabolite of indole-3-carbinol, a naturally occurring compound

found in cruciferous vegetables, has been found to have anti-cancer properties in

both humans and animals. DIM has been shown to induce cell cycle arrest and

apoptosis in animal breast cancer models. Because HER2/neu overexpression confers

resistance to paclitaxel, and DIM has anti-tumor effects, we hypothesized that

DIM will enhance the cytotoxic effects of paclitaxel, a common taxane drug, on

human Her2/neu breast cancer cells by potentiating its effect on cell cycle and

stimulating apoptosis. METHODS: The MDA-MB-435eB1 human Her2/neu breast

cancer cells were treated with varying concentrations of DIM and paclitaxel. The cells

were analyzed at different time points (24, 48, and 72 h). Proliferation was

measured by a commercial cell proliferation assay (Promega Procheck Assay).

Cell-cycle analysis and apoptosis were determined by flow cytometry. Western blot

analysis was performed on to determine the effect of DIM and/or paclitaxel on the

proteins involved in apoptosis, and epidermal growth factor-induced activation of

HER2/neu and ERK1/2 signaling proteins. RESULTS: Both DIM and paclitaxel

exhibited time and concentration dependent inhibition of cell proliferation.

TUNEL assay indicated that the combination also increased the number of apoptotic

cells more than either agent alone. The presence of cleaved poly (ADP-Ribose)

polymerase (PARP) significantly increased in the combination treatment, whereas

Bcl-2 is decreased. DIM alone decreased the activation of the Her2/neu receptor;

the combination decreased the activation of ERK1/ERK2. CONCLUSIONS: DIM in

combination with paclitaxel synergistically inhibits growth of Her2/neu human

breast cancer cells through G2M phase cell-cycle arrest and induction of

apoptosis/necrosis. The Her2/neu receptor and its downstream signaling protein

ERK1/2 appear to be involved in DIM's affect on cell growth and differentiation,

whereas apoptosis appears to be mediated through the mitochondrial pathway

(Bcl-2/PARP). It appears DIM, a naturally occurring, nontoxic compound, may be a

beneficial addition to a traditional (taxane-based) chemotherapy regimen.

 

PMID: 16580691

 

Mol Cancer Ther. 2006 Mar;5(3):556-63.

 

Multiple, disparate roles for calcium signaling in apoptosis of human prostate

and cervical cancer cells exposed to diindolylmethane (DIM).

 

Savino JA 3rd, Evans JF, Rabinowitz D, Auborn KJ, Carter TH.

 

Department of Biological Sciences, St. John's University, Jamaica, New York, USA.

 

Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables,

causes growth arrest and apoptosis of cancer cells in vitro. DIM also induces

endoplasmic reticulum (ER) stress, and thapsigargin, a specific inhibitor of the

sarcoplasmic reticulum/ER calcium-dependent ATPase, enhances this effect. We

asked whether elevated cytosolic free calcium [Ca2+]i is required for

cytotoxicity of DIM and thapsigargin in two cancer cells lines (C33A, from

cervix, and DU145, from prostate). [Ca2+]i was measured in real-time by FURA-2

fluorescence. We tested whether DIM, thapsigargin, and DIM + thapsigargin cause

apoptosis, measured by nucleosome release, under conditions that prevented

elevation of [Ca2+]i, using both cell-permeable and cell-impermeable forms of the

specific calcium chelator BAPTA. DIM, like thapsigargin, rapidly mobilized ER

calcium. C33A and DU145 responded differently to perturbations in Ca2+

homeostasis, suggesting that DIM induces apoptosis by different mechanisms in

these two cell lines and/or that calcium mobilization also activates different

survival pathways in C33A and DU145. Apoptosis in C33A was independent of

increased [Ca2+]i, suggesting that depletion of ER Ca2+ stores may be sufficient

for cell killing, whereas apoptosis in DU145 required elevated [Ca2+]i for full

response. Inhibitor studies using cyclosporin A and KN93 showed that Ca2+

signaling is important for cell survival but the characteristics of this response

also differed in the two cell lines. Our results underscore the complex and

variable nature of cellular responses to disrupted Ca2+ homeostasis and suggest

that alteration Ca2+ homeostasis in the ER can induce cellular apoptosis by both

calcium-dependent and calcium-independent mechanisms.

 

PMID: 16546969

 

Chem Res Toxicol. 2006 Mar;19(3):436-42.

 

Fate of 3,3'-diindolylmethane (DIM) in cultured MCF-7 human breast cancer cells.

 

Staub RE, Onisko B, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

3,3'-Diindolylmethane (DIM) is a major in vivo product of the cancer preventative

agent indole-3-carbinol that is found in vegetables of the genus Brassica. Here,

we report on the metabolic fate of radiolabeled DIM in MCF-7 cells. DIM was

slowly metabolized to several sulfate conjugates of oxidized DIM products that

were primarily detected in the medium. The radioactivity detected in cells was

predominantly unmodified DIM (81-93%) at all time intervals up to 72 h treatment.

Co-treatment of MCF-7 cells with quercetin slowed the rate that oxidized DIM

products accumulated in the medium, while indole[3,2-b]carbazole (ICZ)

co-treatment accelerated their production. ICZ is an inducer of P450 1A2, while

quercetin is a specific inhibitor of this isoform, suggesting that P450 1A2 is

primarily responsible for the oxidation of DIM, probably through 2,3-epoxidation

similar to 3-methylindole. Sulfate conjugates of oxidized DIM metabolites were

cleaved by sulfatase digestion and identified by LC/MS as

3-(1H-indole-3-ylmethyl)-2-oxindole (2-ox-DIM), bis(1H-indol-3-yl)methanol

(3-methylenehydroxy-DIM), 3-[hydroxy-(1H-indol-3-yl)-methyl]-1,3-dihydro-2-oxindole

(3-methylenehydroxy-2-ox-DIM), and 3-hydroxy-3-(1H-indole-3-ylmethyl)-2-oxindole

(3-hydroxy-2-ox-DIM). Derivatives of 2-ox-DIM represented greater than 30% of the

radioactivity in the sulfatase-digested medium. Although oxindole formation was

the primary metabolic pathway in MCF-7 cells, synthetic 2-ox-DIM was inactive in

a 4-ERE-luciferase reporter assay and, therefore, probably not responsible for

the estrogenic activity previously observed for DIM. Unmodified DIM rapidly

accumulated in the nuclear membranes representing approximately 35-40% of the

radioactivity after 0.5-2 h treatment. Uptake of radiolabeled DIM appeared to be

a passive partitioning into the nuclear membranes and was not dependent upon the

cell cytosol. The nuclear uptake of DIM was not saturable and could not be

blocked by pretreatment with unlabeled DIM (100 microM). Further, treatments in

serum-free medium increased the uptake of radiolabeled DIM by the MCF-7 cells.

These findings show that the uptake of DIM by membranes significantly increases

its localized concentration, which may contribute to its biological activities.

 

PMID: 16544949

  

Mutat Res. 2006 Jul 25;599(1-2):76-87. Epub 2006 Feb 24.

 

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in

human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae.

 

Pappa G, Lichtenberg M, Iori R, Barillari J, Bartsch H, Gerhäuser C.

 

Division of Toxicology and Cancer Risk Factors, German Cancer Research Center,

DKFZ, 69120 Heidelberg, Germany.

 

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as

well as the indoles indole-3-carbinol and its condensation product

3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce

apoptosis. In this study, we compared the cell growth inhibitory potential of the

four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+))

and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116).

Using sulforhodamin B staining to assess cell proliferation, we found that the

isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell

growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four

compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of

treatment. Apoptosis induction was analyzed by immunoblotting of

poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC

(10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM)

induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines,

suggestive of a p53-independent mechanism of apoptosis induction. In cultured

40-16 cells, activation of caspase-9 and -7 detected by Western blotting

indicated involvement of the mitochondrial pathway. We detected time- and

concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L)

as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane

only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly

correlated with apoptosis induction measured by PARP cleavage. Taken together, we

demonstrated that the glucosinolate breakdown products investigated in this study

have distinct profiles of cell growth inhibition, potential to induce

p53-independent apoptosis and to modulate Bcl-2 family protein expression in

human colon cancer cell lines.

 

PMID: 16500682

   

Mol Pharmacol. 2006 Apr;69(4):1320-7. Epub 2005 Dec 29.

 

3,3'-diindolylmethane (DIM) is a novel topoisomerase II alpha catalytic inhibitor that

induces S-phase retardation and mitotic delay in human hepatoma HepG2 cells.

 

Gong Y, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, 119 Morgan Hall, University of

California, Berkeley, CA 94720-3104, USA.

 

Epidemiological evidence suggests that high consumption of Brassica genus

vegetables, such as broccoli, cabbage, and Brussels sprouts, is very effective in

reducing the risks of several types of cancers. 3,3'-Diindolylmethane (DIM), one

of the most abundant and biologically active dietary compounds derived from

Brassica genus vegetables, displays remarkable antitumor activity against several

experimental tumors. In the present study, we demonstrate for the first time that

DIM is a novel catalytic topoisomerase IIalpha inhibitor. In supercoiled DNA

relaxation assay and kinetoplast DNA decatenation assay, DIM strongly inhibited

DNA topoisomerase IIalpha and also partially inhibited DNA topoisomerases I and

IIbeta. DIM did not stabilize DNA cleavage complex and did not prevent

etoposide-induced DNA cleavage complex formation. Further experiments showed that

DIM inhibited topoisomerase IIalpha-catalyzed ATP hydrolysis, which is a

necessary step for the enzyme turnover. In cultured human hepatoma HepG2 cells,

DIM blocked DNA synthesis and mitosis in a concentration-dependent manner, which

was consistent with the outcome of topoisomerase inhibition in these cell-cycle

phases. Our results identified a new mode of action for this intriguing dietary

component that might be exploited for therapeutic development.

 

PMID: 16385077

 

Biochem Biophys Res Commun. 2006 Feb 10;340(2):718-25. Epub 2005 Dec 20.

 

3,3'-Diindolylmethane (DIM) downregulates pro-survival pathway in hormone independent

prostate cancer.

 

Garikapaty VP, Ashok BT, Tadi K, Mittelman A, Tiwari RK.

 

Department of Microbiology and Immunology, New York Medical College,

Valhalla, NY, 10595, USA.

 

Epidemiological evidences suggest that the progression and promotion of prostate

cancer (CaP) can be modulated by diet. Since all men die with prostate cancer

rather than of the disease, it is of particular interest to prevent or delay the

progression of the disease by chemopreventive strategies. We have been studying

the anticancer properties of compounds present in cruciferous vegetables such as

indole-3-carbinol (I3C). Diindolylmethane (DIM) is a dimer of I3C that is formed

under acidic conditions and unlike I3C is more stable with higher anti-cancer

effects. In the present report, we demonstrate that DIM is a potent

anti-proliferative agent compared to I3C in the hormone independent DU 145 CaP

cells. The anti-prostate cancer effect is mediated by the inhibition of the Akt

signal transduction pathway as DIM, in sharp contrast to I3C, induces the

downregulation of Akt, p-Akt, and PI3 kinase. DIM also induced a G1 arrest in DU

145 cells by flow cytometry and downstream concurrent inhibition of cell cycle

parameters such as cyclin D1, cdk4, and cdk6. Our data suggest a need for further

development of DIM, as a chemopreventive agent for CaP, which justifies

epidemiological evidences and molecular targets that are determinants for CaP

dissemination/progression. The ingestion of DIM may benefit CaP patients and

reduce disease recurrence by eliminating micro-metastases that may be present in

patients who undergo radical prostatectomy.

 

PMID: 16380095

 

Mol Cancer Ther. 2005 Dec;4(12):1972-81.

 

Down-regulation of c-FLIP contributes to the sensitization effect of

3,3'-diindolylmethane (DIM) on TRAIL-induced apoptosis in cancer cells.

 

Zhang S, Shen HM, Ong CN.

 

Department of Community, Occupational and Family Medicine, Faculty of Medicine,

National University of Singapore, 16 Medical Drive, Singapore 117597, Singapore.

 

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of

the tumor necrosis factor superfamily, which has been shown to preferentially

induce apoptosis in cancer cells without adverse effects on normal cells.

However, there are still some cancer cells, especially those with high

malignancy, resistant to TRAIL-induced apoptosis, impeding the clinical

anticancer efficiency of TRAIL. In this report, we showed that

3,3'-diindolylmethane, an indole compound derived from cruciferous vegetables, is

capable of overcoming TRAIL resistance by sensitizing TRAIL-induced apoptosis in

human cancer cells. Noncytotoxic concentrations of 3,3'-diindolylmethane

significantly enhanced TRAIL-resistant cancer cells to TRAIL-induced apoptosis

via promoting the caspase cascade, a process independent of nuclear factor-kappaB

activation and cell surface TRAIL receptor expression. In the search of the

molecular mechanisms involved in the sensitization activity of

3,3'-diindolylmethane, we found that combined treatment of 3,3'-diindolylmethane

and TRAIL led to significant down-regulation of the cellular FLICE inhibitory

protein expression (c-FLIP). Furthermore, we provided evidence showing that the

reduced c-FLIP level is predominately mediated by the ubiquitin-proteasome

degradation system. These findings reveal a novel anticancer property of

3,3'-diindolylmethane and suggest that this compound could have potential use in

cancer therapy to overcome TRAIL resistance.

 

PMID: 16373712

 

Prostate. 2006 Apr 1;66(5):453-62.

 

Synthetic dimer of indole-3-carbinol: second generation diet derived anti-cancer

agent in hormone sensitive prostate cancer.

 

Garikapaty VP, Ashok BT, Tadi K, Mittelman A, Tiwari RK.

 

Department of Microbiology & Immunology, New York Medical College, Valhalla, New

York 10595, USA.

 

BACKGROUND: Cruciferous vegetables have been found to have anti-prostate cancer

effects. The active compounds mediating these effects include indoles such as

indole-3-carbinol (I3C) and isothiocyanates. I3C is unstable having tissue tropic

effects and clinical utility has been partly addressed by the synthesis of a more

stable dimer diindolylmethane (DIM). METHODS: Anti-proliferative activity was

measured by XTT assay and cytosolic proteins quantitated by Western blot

analysis. RESULTS: DIM (IC(50) 50 microM) is a better anti-proliferative agent

than I3C (IC(50) 150 microM) in androgen dependent LNCaP cells, inhibits DNA

synthesis, and growth of R1881 stimulated LNCaP cells. Androgen receptor (AR),

cyclin D1, and cdk4, induced by R1881, are downregulated by DIM. DIM

downregulates phosphorylated Akt and phosphatidyl inositol 3-kinase and

downstream inhibition of cyclin D1 and cdk4. CONCLUSION: These studies provide

evidence that DIM is a second-generation chemopreventive agent with a viable

cellular target and has clinical potential as an anti-prostate cancer

chemopreventive. (c) 2005 Wiley-Liss, Inc.

 

PMID: 16353249

 

Carcinogenesis. 2006 Apr;27(4):717-28. Epub 2005 Dec 6.

 

3,3'-diindolylmethane (DIM) and its derivatives induce apoptosis in pancreatic

cancer cells through endoplasmic reticulum stress-dependent upregulation of DR5.

 

Abdelrahim M, Newman K, Vanderlaag K, Samudio I, Safe S.

 

Institute of Biosciences and Technology, The Texas A&M University System Health

Science Center, 2121 W. Holcombe Boulevard, Houston, TX 77030, USA.

 

3,3'-Diindolylmethane (DIM), ring-substituted DIMs and

1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methanes (C-DIMs) inhibit growth of

Panc-1 and Panc-28 pancreatic cancer cells. Although DIMs (diarylmethanes) and

selected C-DIMs (triarylmethanes), such as the p-t-butyl derivative

(DIM-C-pPhtBu), activate the aryl hydrocarbon receptor and peroxisome

proliferator-activated receptor gamma, respectively, this study shows that both

DIM and DIM-C-pPhtBu induce common receptor-independent pathways. Both DIM and

DIM-C-pPhtBu increased endoplasmic reticulum (ER) staining and ER calcium release

in Panc-1 cells, and this was accompanied by increased expression of glucose

related protein 78 and C/EBP homologous transcription factor (CHOP/GADD153)

proteins. Similar results were observed after treatment with thapsigargin (Tg), a

prototypical inducer of ER stress. The subsequent downstream effects of

DIM/DIM-C-pPhtBu- and Tg-induced ER stress included CHOP-dependent induction of

death receptor DR5 and subsequent cleavage of caspase 8, caspase 3, Bid and PARP.

Activation of both receptor-dependent and receptor-independent (ER stress)

pathways by DIM and DIM-C-pPhtBu in pancreatic cancer cells enhances the efficacy

and potential clinical importance of these compounds for cancer chemotherapeutic

applications.

 

PMID: 16332727

  

Mol Pharmacol. 2006 Feb;69(2):430-9. Epub 2005 Nov 2.

 

Activation and potentiation of interferon-gamma signaling by

3,3'-diindolylmethane in MCF-7 breast cancer cells.

 

Riby JE, Xue L, Chatterji U, Bjeldanes EL, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM), a natural autolytic product in plants of the

Brassica genus, including broccoli, cauliflower, and Brussels sprouts, exhibits

promising cancer protective activities, especially against mammary neoplasia in

animal models. We observed previously that DIM induced a G(1) cell-cycle arrest

and strong induction of cell-cycle inhibitor p21 expression and promoter activity

in both estrogen-responsive and -independent breast cancer cell lines. We showed

recently that DIM up-regulates the expression of interferon gamma (IFNgamma) in

human MCF-7 breast cancer cells. This novel effect may contribute to the

anticancer effects of DIM because IFNgamma plays an important role in preventing

the development of primary and transplanted tumors. In this study, we observed

that DIM activated the IFNgamma signaling pathway in human breast cancer cells.

DIM activated the expression of the IFNgamma receptor (IFNGR1) and

IFNgamma-responsive genes p56- and p69-oligoadenylate synthase (OAS). In

cotreatments with IFNgamma, DIM produced an additive activation of endogenous

p69-OAS and of an OAS-Luc reporter and a synergistic activation of a GAS-Luc

reporter. DIM synergistically augmented the IFNgamma induced phosphorylation of

signal transducer and activator of transcription factor 1, further evidence of

DIM activation of the IFNgamma pathway. DIM and IFNgamma produced an additive

inhibition of cell proliferation and a synergistic increase in levels of major

histocompatibility complex class-1 (MHC-1) expression, accompanied by increased

levels of mRNAs of MHC-1-associated proteins and transporters. These results

reveal novel immune activating and potentiating activities of DIM in human tumor

cells that may contribute to the established effectiveness of this dietary indole

against various tumors types.

 

PMID: 16267208

 

Biochem Biophys Res Commun. 2005 Nov 25;337(3):1019-25. Epub 2005 Oct 3.

 

3,3'-Diindolylmethane (DIM), a cruciferous vegetable derived synthetic

anti-proliferative compound in thyroid disease.

 

Tadi K, Chang Y, Ashok BT, Chen Y, Moscatello A, Schaefer SD, Schantz SP,

Policastro AJ, Geliebter J, Tiwari RK.

 

Department of Microbiology and Immunology, New York Medical College, Valhalla,

NY, USA.

 

Considerable epidemiological evidence exists to link thyroid disease with

differing patterns of dietary consumption, in particular, cruciferous vegetables.

We have been studying the anti-thyroid cancer (TCa) activity of indole-3-carbinol

(I3C) found in cruciferous vegetables and its acid catalyzed dimer,

3,3'-diindolylmethane (DIM). There are no studies as yet to elucidate the effect

of these compounds on the altered proliferative patterns in goiter or thyroid

neoplasia. In this study, we tested the anti-proliferative effects of I3C and DIM

on four different thyroid cancer cell lines representative of papillary (B-CPAP

and 8505-C) and follicular carcinoma of the thyroid (CGTH-W-1 and ML-1), and

primary human goiter cells. Cell survival and IC50 values for I3C and DIM were

calculated by the XTT assay and cell cycle distribution analysis was done by flow

cytometry. DIM was found to be a better anti-proliferative agent than I3C in both

papillary and follicular TCa resulting in a greater cytotoxic effect at a

concentration over three fold lower than predicted by the molar ratio of DIM and

I3C. The anti-proliferative activity of DIM in follicular TCa was mediated by a

G1 arrest followed by induction of apoptosis. DIM also inhibited the growth of

primary goiter cells by 70% compared to untreated controls. Contrary to

traditional belief that cruciferous vegetables are "goitrogenic", DIM has

anti-proliferative effects in glandular thyroid proliferative disease. Our

preclinical studies provide a strong rationale for the clinical exploration of

DIM as an adjuvant to surgery in thyroid proliferative disease.

 

PMID: 16219298

 

Carcinogenesis. 2006 Mar;27(3):541-50. Epub 2005 Sep 30.

 

Inhibition of growth factor-induced Ras signaling in vascular endothelial cells

and angiogenesis by 3,3'-diindolylmethane (DIM).

 

Chang X, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM), an indole derivative produced on consumption of

broccoli and other cruciferous vegetables, has been shown to have multiple

anticancer effects in both in vivo and in vitro models. The present study was

carried out to clarify the mechanism of DIM's antiangiogenic activity. We found

that DIM can inhibit vascular endothelial growth factor (VEGF)-induced cell

proliferation and DNA synthesis in human umbilical vascular endothelial cells

(HUVECs). Consistent with this inhibition, VEGF-induced extracellular

signal-regulated kinase (ERK1/2) phosphorylation was greatly reduced. However,

VEGF receptor phosphorylation induced by VEGF was not affected by DIM, indicating

that DIM does not exert a direct and specific effect on the tyrosine kinase

activity of this receptor. Further studies showed that DIM had a similar

inhibitory effect on ERK1/2 phosphorylation induced by a variety of growth

factors. Furthermore, Ras-GTP content, which dramatically increased after HUVECs

were challenged by either individual growth factors or serum, was reduced by

approximately 80% with 25 muM DIM treatment, which in turn resulted in the

reduced activities of Raf and MEK, culminating in the drop of ERK1/2 activation.

Overexpression of constitutively active GTPase mutant, Ras G12V, in HUVECs

reversed the inhibitory effect of DIM on ERK1/2 activation. In a rodent Matrigel

plug model, the presence of DIM strongly reduced VEGF-induced neovascularization,

indicating that DIM is active in vivo. These data provide evidence that DIM

inhibits Ras signaling induced by VEGF and other growth factors, which interferes

with its downstream biological effects necessary for angiogenesis.

 

PMID: 16199440  

  

Toxicol Appl Pharmacol. 2006 Mar 1;211(2):115-23. Epub 2005 Jul 25.

 

Indole-3-carbinol, but not its major digestive product 3,3'-diindolylmethane (DIM),

induces reversible hepatocyte hypertrophy and cytochromes P450.

 

Crowell JA, Page JG, Levine BS, Tomlinson MJ, Hebert CD.

 

Division of Cancer Prevention, National Cancer Institute, Bethesda, MD

20892-7322, USA. [email protected]

 

Indole-3-carbinol (I-3-C) and 3,3'-diindolylmethane (DIM) have been shown to

reduce the incidence and multiplicity of cancers in laboratory animal models.

Based on the observation that I-3-C induced hepatocyte hypertrophy when

administered orally for 13 weeks to rats, a treatment and recovery study was

undertaken to test the hypothesis that the induction of hepatocyte hypertrophy

and cytochrome P450 (CYP) activity by I-3-C are adaptive, reversible responses.

Additionally, we directly compared the effects of I-3-C to those of its principle

metabolite DIM. Rats were treated orally for 28 days with 2 doses of I-3-C (5 and

50 mg I-3-C/kg body weight/day) and DIM (7.5 and 75 mg DIM/kg body weight/day)

and then one-half of the animals were not treated for an additional 28 days.

Organ weights, histopathology, and the CYP enzyme activities of 1A1/2, 2B1/2,

2C9, 2D6, 2E1, 3A4, and 19 A were measured both after treatment and after

recovery. Oral administration of 50 mg I-3-C/kg body weight/day to rats for 28

days significantly increased liver weights and CYP enzyme activities. The effects

in males were more pronounced and persistent after recovery than the effects in

females. The increased organ weights returned to control values after treatment.

Conversely, DIM did not alter liver weights and had no effect on CYP activities

after the 28-day treatment. Some changes in CYP activities were measured after

the DIM recovery period but the magnitudes of the changes were considered

biologically insignificant. The results show that I-3-C, but not DIM, induces

reversible adaptive responses in the liver.

 

PMID: 16043203

  

J Agric Food Chem. 2005 May 18;53(10):3895-901.

 

Effects of Brussels sprout juice on the cell cycle and adhesion of human

colorectal carcinoma cells (HT29) in vitro.

 

Smith TK, Lund EK, Clarke RG, Bennett RN, Johnson IT.

 

Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA,

United Kingdom.

 

Consumption of Brassica vegetables is associated with a reduced risk of cancer of

the alimentary tract in animal models and human populations. We used raw juice

extracted from Brussels sprouts rich in the glucosinolate sinigrin to explore the

effect of naturally occurring glucosinolate breakdown products on cell cycle

progression and apoptosis in human colorectal carcinoma cells (HT29). Juice was

prepared from sprout tissue immediately before use, and the glucosinolate

breakdown products were determined by gas chromatography mass spectrometry and

liquid chromatography mass spectrometry. The cell cycle was analyzed by flow

cytometry on detached and adherent cells, and apoptosis was measured in the

detached population by annexin V staining. Twenty-four hours after challenge with

juice (10 microL/mL), 7-13% of adherent cells had detached from the substratum

but the majority (82%) of these cells had not entered apoptosis, whereas only 33%

of detached control cells were not apoptotic (p < 0.05). The main glucosinolate

breakdown products were as follows: the sinigrin breakdown product,

1-cyano-2,3-epithiopropane (ca. 38 mM); the gluconapin hydrolysis product,

3-butenyl isothiocyanate (ca. 2.2.mM); the glucobrassicin metabolite, ascorbigen

(ca. 8 mM); and low concentrations of other indole glucosinolate-derived

hydrolysis products such as neoascorbigen and 3,3'-diindolylmethane. A variety of

biologically active glucosinolate breakdown products are released by mechanical

disruption of raw Brussels sprout tissue, but contrary to previous assumptions,

allyl isothiocyanate is not the main compound responsible for the inhibition of

cell proliferation.

 

PMID: 15884814

   

Oncogene. 2005 Mar 31;24(14):2343-53.

 

DIM stimulates IFNgamma gene expression in human breast cancer cells via the

specific activation of JNK and p38 pathways.

 

Xue L, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California, 119

Morgan Hall, Berkeley, CA 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM) is a promising anticancer agent derived from Brassica

vegetables, but the mechanisms of DIM action are largely unknown. We have shown

that DIM can upregulate the expression and stimulate the secretion of

interferon-gamma (IFNgamma) in the human MCF-7 breast cancer cell line. This

novel effect may provide important clues to explain the anticancer effects of DIM

because it is well known that IFNgamma plays an important role in preventing the

development of primary and transplanted tumors. Utilizing promoter deletions, we

show here that the region between -108 and -36 bp in the IFNgamma promoter, which

contains two conserved and essential regulatory elements, is required for

DIM-induced IFNgamma expression. DIM activates both JNK and p38 pathways, induces

the phosphorylation of c-Jun and ATF-2, and increases the binding of the

homodimer or heterodimer of c-Jun/ATF-2 to the proximal AP-1.CREB-ATF-binding

element. Moreover, studies with specific enzyme inhibitors showed that up-stream

Ca2+-dependent kinase(s) is required for the inducing effects of DIM in MCF-7

cells. These results establish that DIM-induced IFNgamma expression in human

breast tumor cells is mediated by activation of both JNK and p38 pathways, which

is ultimately dependent on intracellular calcium signaling.

 

PMID: 15735741

  

Xenobiotica. 2004 Jul;34(7):619-32.

 

Phytochemical-induced changes in gene expression of carcinogen-metabolizing

enzymes in cultured human primary hepatocytes.

 

Gross-Steinmeyer K, Stapleton PL, Liu F, Tracy JH, Bammler TK, Quigley SD, Farin

FM, Buhler DR, Safe SH, Strom SC, Eaton DL.

 

Department of Environmental and Occupational Health Sciences, University of

Washington, Seattle, WA 98105, USA.

 

The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM),

isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC)

and sulforaphane (SFN) protect animals against chemically induced tumours.

Putative chemoprotective mechanisms include modulated expression of hepatic

biotransformation enzymes. However, few, if any, studies have used human primary

cells as test models. 2. The present study investigated the effects of these

phytochemicals on the expression of four carcinogenesis-relevant

enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1)

and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated

human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was

up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription

was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM

exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and

87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold

at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in

protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5

x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on

GSTA1 transcription were seen. 4. The findings show novel and unexpected effects

of these phytochemicals on the expression of human hepatic biotransformation

enzymes that play key roles in chemical-induced carcinogenesis.

 

PMID: 15672752

 

Biochem Biophys Res Commun. 2005 Mar 4;328(1):63-9.

 

Indole-3-carbinol and 3,3'-diindolylmethane induce expression of NAG-1 in a

p53-independent manner.

 

Lee SH, Kim JS, Yamaguchi K, Eling TE, Baek SJ.

 

Laboratory of Environmental Carcinogenesis, Department of Pathobiology, College

of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA.

 

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo

product 3,3'-diindolylmethane (DIM), have been reported to suppress cancer

development. However, the responsible molecular mechanisms are not fully

understood. Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is a

TGF-beta superfamily gene associated with pro-apoptotic and anti-tumorigenic

activities. The present study was performed to investigate whether I3C and DIM

influence NAG-1 expression and to provide the potential molecular mechanism of

their effects on anti-tumorigenesis. The I3C repressed cell proliferation and

induced NAG-1 expression in a concentration-dependent manner. In addition, DIM

increased the expression of NAG-1 as well as activating transcription factor 3

(ATF3), and the induction of ATF3 was earlier than that of NAG-1. The DIM

treatment increased luciferase activity of NAG-1 in HCT-116 cells transfected

with NAG-1 promoter construct. The results suggest that I3C represses cell

proliferation through up-regulation of NAG-1 and that ATF3 may play a pivotal

role in DIM-induced NAG-1 expression in human colorectal cancer cells.

Furthermore, the mixture of I3C with resveratrol enhances NAG-1 expression,

suggesting the synergistic effect of these two unrelated compounds on NAG-1

expression.

 

PMID: 15670751

 

Cancer Res. 2005 Jan 1;65(1):364-71.

 

Inhibition of nuclear translocation of nuclear factor-{kappa}B contributes to

3,3'-diindolylmethane-induced apoptosis in breast cancer cells.

 

Rahman KW, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, MI 48201, USA.

 

Dietary indole-3-carbinol (I3C), a natural compound present in vegetables of the

genus Brassica, showed clinical benefits and caused apoptosis in breast cancer

cells. Our laboratory and others have shown that I3C induces apoptosis in breast

cancer cells mediated by inactivation of Akt and nuclear factor-kappaB

(NF-kappaB) pathway. 3,3'-Diindolylmethane (DIM), a major in vivo acid-catalyzed

condensation product of I3C, also showed some benefit in breast cancer. However,

the precise molecular mechanism(s) by which DIM induces apoptosis in breast

cancer cells has not been fully elucidated. Hence, we investigated whether

DIM-induced apoptosis of breast cancer cells could also be mediated by

inactivation of Akt and NF-kappaB. We found that DIM induces apoptotic processes

in MCF10A derived malignant (MCF10CA1a) cell lines but not in nontumorigenic

parental MCF10A cells. DIM specifically inhibits Akt kinase activity and

abrogates the epidermal growth factor-induced activation of Akt in breast cancer

cells, similar to those observed for I3C. We also found that DIM reduces

phosphorylation of IkappaBalpha, an inhibitor of NF-kappaB. Our confocal

microscopy study clearly showed that DIM blocks the translocation of p65, a

subunit of NF-kappaB to the nucleus. DNA binding analysis and transfection

studies with IkappaB kinase cDNA revealed that overexpression of IkappaB kinase

mediates IkappaBalpha phosphorylation, which activates NF-kappaB, and this

activation was completely abrogated by DIM treatment. Taken together, these

results showed for the first time that the inactivation of Akt and NF-kappaB

activity also plays important roles in DIM-induced apoptosis in breast cancer

cells, which seems to be more relevant to in vivo situations.

 

PMID: 15665315

 

Carcinogenesis. 2005 Apr;26(4):771-8. Epub 2005 Jan 20.

 

3,3'-Diindolylmethane inhibits angiogenesis and the growth of transplantable

human breast carcinoma in athymic mice.

 

Chang X, Tou JC, Hong C, Kim HA, Riby JE, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

Studies have linked the consumption of broccoli and other cruciferous vegetables

to a reduced risk of breast cancer. The phytochemical indole-3-carbinol (I3C),

present in cruciferous vegetables, and its major acid-catalyzed reaction product

3,3'-diindolylmethane (DIM) have bioactivities relevant to the inhibition of

carcinogenesis. In this study, the effect of DIM on angiogenesis and

tumorigenesis in a rodent model was investigated. We found that DIM produced a

concentration-dependent decrease in proliferation, migration, invasion and

capillary tube formation of cultured human umbilical vein endothelial cells

(HUVECs). Consistent with its antiproliferative effect, which was significant at

only 5 microM DIM, this indole caused a G1 cell cycle arrest in actively

proliferating HUVECs. Furthermore, DIM downregulated the expression of

cyclin-dependent kinases 2 and 6 (CDK2, CDK6), and upregulated the expression of

CDK inhibitor, p27(Kip1), in HUVECs. We observed further in a complementary in

vivo Matrigel plug angiogenesis assay that, compared with vehicle control,

neovascularization was inhibited up to 76% following the administration of 5

mg/kg DIM to female C57BL/6 mice. Finally, this dose of DIM also inhibited the

growth of human MCF-7 cell tumor xenografts by up to 64% in female athymic

(nu/nu) mice, compared with the vehicle control. This is the first study to show

that DIM can strongly inhibit the development of human breast tumor in a

xenograft model and to provide evidence for the antiangiogenic properties of this

dietary indole.

 

PMID: 15661811

 

Nutr Cancer. 2004;50(2):161-7.

 

Pilot study: effect of 3,3'-diindolylmethane supplements on urinary hormone

metabolites in postmenopausal women with a history of early-stage breast cancer.

 

Dalessandri KM, Firestone GL, Fitch MD, Bradlow HL, Bjeldanes LF.

 

Department of Molecular and Cell Biology, University of California, Berkeley,

94720-3200, USA.

 

Dietary indoles, present in Brassica plants such as cabbage, broccoli, and

Brussels sprouts, have been shown to provide potential protection against

hormone-dependent cancers. 3,3'-Diindolylmethane (DIM) is under study as one of

the main protective indole metabolites. Postmenopausal women aged 50-70 yr from

Marin County, California, with a history of early-stage breast cancer, were

screened for interest and eligibility in this pilot study on the effect of DIM supplements

on urinary hormone metabolites. The treatment group received daily DIM (108 mg

DIM/day) supplements for 30 days, and the control group received a placebo capsule

daily for 30 days. Urinary metabolite analysis included 2-hydroxyestrone (2-OHE1),

16-alpha hydroxyestrone (16alpha-OHE1), DIM, estrone (El), estradiol(E2), estriol (E3),

6beta-hydroxycortisol (6beta-OHC), and cortisol in the first morning urine sample

before intervention and 31 days after intervention. Nineteen women completed the

study,for a total of 10 in the treatment group and 9 in the placebo group.

DIM-treated subjects, relative to placebo, showed a significant increase in

levels of2-OHE1 (P=0. 020), DIM (P =0. 045), and cortisol (P = 0.039), and a

nonsignificant increase of 47% in the 2-OHE1/16alpha-OHE1 ratio from 1.46 to 2.14

(P=0.059). In this pilot study, DIM increased the 2-hydroxylation of estrogen

urinary metabolites.

 

PMID: 15623462

 

J Nutr. 2004 Dec;134(12 Suppl):3493S-3498S.

 

Indole-3-carbinol and prostate cancer.

 

Sarkar FH, Li Y.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, MI, 48201, USA.

 

Epidemiological and dietary studies have revealed an association between high

dietary intake of cruciferous vegetables and decreased prostate cancer risk. Our

studies have shown that indole-3-carbinol (I3C), a common phytochemical in

cruciferous vegetables, and its in vivo dimeric product 3,3'-diindolylmethane

(DIM) upregulate the expression of phase I and phase II enzymes, suggesting

increased capacity for detoxification and inhibition of carcinogens. Studies from

our laboratory and others have found that I3C can induce G1 cell-cycle arrest and

apoptosis in prostate cancer cells. In addition, we found, by microarray gene

expression profiling, that I3C and DIM regulate many genes that are important for

the control of cell cycle, cell proliferation, signal transduction, and other

cellular processes, suggesting the pleiotropic effects of I3C and DIM on prostate

cancer cells. We recently found that I3C functions as an inhibitor of Akt and

nuclear factor kappaB (NF-kappaB), which play important roles in cell survival

and which are believed to be potential targets in cancer therapy. Studies have

already shown that the inactivation of Akt and NF-kappaB is responsible for

chemosensitization of chemoresistant cancer cells. Because there is no effective

treatment strategy for hormone-dependent and, most importantly,

hormone-independent and metastatic prostate cancer, our strategies to sensitize

prostate cancer cells to a chemotherapeutic agent by I3C and DIM is a novel

breakthrough that could be used for devising novel therapies for prostate cancer.

In conclusion, the results from our laboratory and from others provide ample

evidence for the benefit of I3C and DIM for the prevention and the treatment of

prostate cancer.

 

PMID: 15570059

 

Chem Biol Interact. 2004 Nov 20;150(2):161-70.

 

Lack of antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin's (TCDDs) induction of

cytochrome P4501A1 (CYP1A1) by the putative selective aryl hydrocarbon receptor

modulator 6-alkyl-1,3,8-trichlorodibenzofuran (6-MCDF) in the mouse hepatoma cell

line Hepa-1c1c7.

 

Fretland AJ, Safe S, Hankinson O.

 

Department of Pathology and Laboratory Medicine, Jonsson Comprehensive Cancer

Center, Center for Health Sciences, University of California, Los Angeles, CA

90095-1732, USA.

 

Regulation of gene expression by the aryl hydrocarbon (AHR) receptor is a

much-studied pathway of molecular toxicology. Activation of AHR by the xenobiotic

ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is hypothesized as the

mechanism by which TCDD exerts its toxic and carcinogenic effects. Paradoxically,

some studies have shown that TCDD acts as an antiestrogen. This has led to the

hypothesis that so-called selective aryl hydrocarbon receptor modulators

(SAhRMs), AHR ligands that retain the antiestrogenic effects but lack the

transcriptional effects of TCDD associated with toxicity, may be utilized as

cancer chemotherapeutics in conjunction with other antiestrogenic compounds such

as tamoxifen. The present study attempts to further define the molecular

mechanism of action of the putative SAhRMs, 6-alkyl-1,3,8-trichlorodibenzofuran

(6-MCDF), and diindolylmethane (DIM), focusing particularly on the former. We

tested 6-MCDF and DIM for the recruitment of AHR and RNA polymerase II (pol II)

to the regulatory region of the AHR responsive gene, cytochrome P4501A1 (CYP1A1),

using the chromatin immunoprecipitation (ChIP) assay in the mouse hepatoma cell

line Hepa-1c1c7 (Hepa-1). We also tested the level of CYP1A1 induction in Hepa-1

cells using quantitative real-time PCR. We show no difference in the recruitment

of AHR or pol II to the regulatory region of CYP1A1 in response to TCDD, 6-MCDF,

or co-treatment with both TCDD and 6-MCDF. Our results also show no antagonism of

CYP1A1 induction with co-treatment of Hepa-1 cells with TCDD and 6-MCDF. These

data suggest that 6-MCDF exhibits agonist activity with respect to induction of

CYP1A1 in the Hepa-1 cell line.

 

PMID: 15535986

 

Mutat Res. 2004 Nov 2;555(1-2):53-64.

 

Cell signaling pathways altered by natural chemopreventive agents.

 

Sarkar FH, Li Y.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, 715 Hudson Webber Cancer Research Center, 110 E Warren, Detroit, MI

48201, USA

 

Epidemiological studies have indicated a significant difference in the incidence

of cancers among ethnic groups, who have different lifestyles and have been

exposed to different environmental factors. It has been estimated that more than

two-thirds of human cancers, which are contributed by mutations in multiple

genes, could be prevented by modification of lifestyle including dietary

modification. The consumption of fruits, soybean and vegetables has been

associated with reduced risk of several types of cancers. The in vitro and in

vivo studies have demonstrated that some dietary components such as isoflavones,

indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM), curcumin,

(-)-epigallocatechin-3-gallate (EGCG), apigenin, etc., have shown inhibitory

effects on human and animal cancers, suggesting that they may serve as

chemopreventive agents. Experimental studies have also revealed that these

components regulate the molecules in the cell signal transduction pathways

including NF-kappaB, Akt, MAPK, p53, AR, and ER pathways. By modulating cell

signaling pathways, these components, among other mechanisms, activate cell death

signals and induce apoptosis in precancerous or cancer cells, resulting in the

inhibition of cancer development and/or progression. This article reviews current

studies regarding the effects of natural chemopreventive agents on cancer-related

cell signaling pathways and provides comprehensive knowledge of the biological

and molecular roles of chemopreventive agents in cancer cells.

 

PMID: 15476851

  

Br J Cancer. 2004 Oct 4;91(7):1358-63.

 

Induction of apoptosis in human prostate cancer cell line, PC3, by

3,3'-diindolylmethane through the mitochondrial pathway.

 

Nachshon-Kedmi M, Yannai S, Fares FA.

 

Faculty of Food Engineering and Biotechnology, Technion-Israel Institute of

Technology, Haifa 32000, Israel.

 

Prostate cancer is the most common malignancy and the second leading cause of

male death in Western countries. Prostate cancer mortality results from

metastases to the bones and lymph nodes and progression from androgen-dependent

to androgen-independent disease. Although androgen ablation was found to be

effective in treating androgen-dependent prostate cancer, no effective

life-prolonging therapy is available for androgen-independent cancer.

Epidemiological studies have shown a strong correlation between consumption of

cruciferous vegetables and a lower risk of prostate cancer. These vegetables

contain glucosinolates, which during metabolism give rise to several breakdown

products, mainly indole-3-carbinol (I3C), which may be condensed to polymeric

products, especially 3,3'-diindolylmethane (DIM). It was previously shown that

these indole derivatives have significant inhibitory effects in several human

cancer cell lines, which are exerted through induction of apoptosis. We have

previously reported that I3C and DIM induce apoptosis in prostate cancer cell

lines through p53-, bax-, bcl-2- and fasL-independent pathways. The objective of

this study was examination of the apoptotic pathways that may be involved in the

effect of DIM in the androgen-independent prostate cancer cell line, PC3, in

vitro. Our results suggest that DIM induces apoptosis in PC3 cells, through the

mitochondrial pathway, which involves the translocation of cytochrome c from the

mitochondria to the cytosol and the activation of initiator caspase, 9, and

effector caspases, 3 and 6, leading to poly ADP-ribose polymerase (PARP) cleavage

and induction of apoptosis. Our findings may lead to the development of new

therapeutic strategies for the treatment of androgen-independent prostate cancer.

 

PMID: 15328526

 

Prostate. 2004 Oct 1;61(2):153-60.

 

Therapeutic activity of 3,3'-diindolylmethane on prostate cancer in an in vivo

model.

 

Nachshon-Kedmi M, Fares FA, Yannai S.

 

Faculty of Food Engineering and Biotechnology, Technion-Israel Institute of

Technology, Haifa, Israel.

 

BACKGROUND: Prostate cancer (PC) is the second leading cancer-related death in

men in Western countries. Hence, efficient anti-carcinogenic and therapeutic

compounds against PC are badly needed. We have previously shown that

3,3'-diindolylmethane (DIM) has a suppressive effect on the growth of human

breast and PC cell lines. The objective of this study was examination of the

potential therapeutic effects of DIM in an in vivo model. METHODS: TRAMP-C2, a

mouse PC cell line, was injected into the flank of male C57BL/6 mice. When tumors

appeared, mice were injected intraperitoneally with either corn oil (vehicle) or

DIM (2.5, 5, or 10 mg per kg body weight) 3-times a week, for 3 weeks, and tumor

volumes were measured bi-weekly with calibermeters. Later, the tumors were

removed, their final weights and volumes were measured, and tumor sections were

tested for histological studies. RESULTS: DIM had a significant inhibitory

effect, caused by diminished tumor growth. Histological examination of tumors

from treated groups revealed apoptosis and decreased cell proliferation, compared

with the controls. DIM didn't affect body weights or kidney and liver

functioning. CONCLUSIONS: The inhibitory action of DIM on tumor growth was

demonstrated in vivo. Hence, this compound at the concentrations tested may offer

an effective and non toxic therapeutic means against tumor growth in rodents, and

may serve as a potential natural anti-carconigenic compound in humans. Copyright

2004 Wiley-Liss, Inc

 

PMID: 15305338

 

Cell Stress Chaperones. 2004 Mar;9(1):76-87.

 

Endoplasmic reticulum stress as a correlate of cytotoxicity in human tumor cells

exposed to diindolylmethane in vitro.

 

Sun S, Han J, Ralph WM Jr, Chandrasekaran A, Liu K, Auborn KJ, Carter TH.

 

North Shore-Long Island Jewish Research Institute, Manhasset, NY 11030, USA.

 

The dietary phytochemical indole-3-carbinol (I3C) protects against cervical

cancer in animal model studies and in human clinical trials. I3C and its

physiologic condensation product diindolylmethane (DIM) also induce apoptosis of

tumor cells in vitro and in vivo, suggesting that these phytochemicals might be

useful as therapeutic agents as well as for cancer prevention. Deoxyribonucleic

acid microarray studies on transformed keratinocytes and tumor cell lines exposed

to pharmacologic concentrations of DIM in vitro are consistent with a cellular

response to nutritional deprivation or disruptions in protein homeostasis such as

endoplasmic reticulum (ER) stress. In this report we investigate whether specific

stress response pathways are activated in tumor cells exposed to DIM and whether

the ER stress response might contribute to DIM's cytotoxicity. Induction of the

stress response genes GADD153, GADD34 and GADD45A, XBP-1, GRP78,

GRP94, and asparagine synthase was documented by Western blot and real-time reverse

transcriptase-polymerase chain reaction in C33A cervical cancer cells, and

induction of a subset of these was also observed in cancer cell lines from breast

(MCF-7) and prostate (DU145). The results are consistent with activation of more

than 1 stress response pathway in C33A cells exposed to 75 microM DIM.

Phosphorylation elF2alpha was rapidly and transiently increased, followed by

elevated levels of ATF4 protein. Activation of IRE1alpha was indicated by a rapid

increase in the stress-specific spliced form of XBP-1 messenger ribonucleic acid

and a rapid and persistent phosphorylation of JNK1 and JNK2. Transcriptional

activation dependent on an ATF6-XBP-1 binding site was detected by transient

expression in MCF-7, C33A, and a transformed epithelial cell line (HaCaT);

induction of the GADD153 (CHOP) promoter was also confirmed by transient

expression. Cleavage of caspase 12 was observed in both DIM-treated and untreated

C33A cells but did not correlate with cytotoxicity, whereas caspase 7 was cleaved

at later times, coinciding with the onset of apoptosis. The results support the

hypothesis that cytotoxic concentrations of DIM can activate cellular stress

response pathways in vitro, including the ER stress response. Conversely, DIM was

especially cytotoxic to stressed cells. Thapsigargin and tunicamycin, agents that

induce ER stress, sensitized cells to the cytotoxic effects of DIM to differing

degrees; nutrient limitation had a similar, but even more pronounced, effect.

Because DIM toxicity in vitro is enhanced in cells undergoing nutritional

deprivation and ER stress, it is possible that stressed cells in vivo, such as

those within developing solid tumors, also have increased sensitivity to killing

by DIM.

 

PMID: 15270080

 

Cancer Detect Prev. 2004;28(1):72-9.

 

Differences in the hepatic P450-dependent metabolism of estrogen and tamoxifen in

response to treatment of rats with 3,3'-diindolylmethane and its parent compound

indole-3-carbinol.

 

Parkin DR, Malejka-Giganti D.

 

Veterans Affairs Medical Center, Minneapolis, MN 55417, USA.

 

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo

product 3,3'-diindolylmethane (DIM), have been reported to suppress

estrogen-responsive cancers. This effect may be mediated through the modification

of cytochrome P450 (CYP) complement and activities leading to estrogen

detoxification. We examined the effects of a 4-day treatment of female

Sprague-Dawley rats with DIM at 8.4 and 42 mg/kg body weight (bwt), on the

hepatic CYP protein level, CYP1A1, 1A2, 2B1/2 and 3A1/2 probe activities and

CYP-dependent metabolism of 17beta-estradiol (E2) and estrone (E1). At 42 mg/kg

bwt, DIM effected a small increase (2.8-fold) in CYP1A1 activity, and at both

dose levels it reduced CYP3A1/2 activity by approximately 40%. At the higher dose

level, DIM decreased the rates of oxidation of E2 to 4-OH-E2, 4-OH-E1,

6alpha-OH-E2 and 6(alpha+beta)-OH-E1 by 39, 44, 71 and 60%, respectively, and E1

to 6(alpha+beta)-OH-E1 by 39%. These effects were considerably different from

those of I3C reported by us previously. We also examined the effects of DIM and

I3C on the hepatic microsomal metabolism of tamoxifen (TAM). Whereas metabolism

of TAM was unaffected by DIM, formation of N-desmethyl-TAM (and its presumed

derivative) was increased approximately 3-fold by I3C at 250 mg/kg bwt. Since

N-desmethyl-TAM is transformed to a genotoxic metabolite, dietary exposure to I3C

may enhance hepatic carcinogenicity of TAM in the rat. The differences between

I3C and DIM in CYP-mediated activities and metabolism indicate that DIM is not a

proximate intermediate in the mechanism of action of I3C.

 

PMID: 15041081

  

Mol Endocrinol. 2004 Feb;18(2):291-302. Epub 2003 Nov 26.

 

Potent ligand-independent estrogen receptor activation by 3,3'-diindolylmethane

is mediated by cross talk between the protein kinase A and mitogen-activated

protein kinase signaling pathways.

 

Leong H, Riby JE, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

We investigated the mechanism of ligand-independent activation of the estrogen

receptor (ER) by 3,3'-diindolylmethane (DIM), a promising anticancer agent

derived from vegetables of the Brassica genus, in Ishikawa and HEC-1B human

endometrial cancer cells. DIM stimulated the activity of an ER-responsive

reporter by over 40-fold, equivalent to the maximum induction produced by

estradiol (E2), whereas cotreatment of cells with the ER antagonist, ICI-182,780

(ICI), abolished the stimulatory effect of DIM. DIM also induced the expressions

of the endogenous genes, TGF-alpha, alkaline phosphatase, and progesterone

receptor similar to levels induced by E2. Induction of gene expression by DIM was

inhibited by the protein synthesis inhibitor, cycloheximide. In addition,

cotreatment of cells with the protein kinase A (PKA) inhibitor, H89, or the MAPK

inhibitor, PD98059, reduced DIM activation of the ER by 75% and 50%,

respectively. Simultaneous treatment of cells with both inhibitors completely

abolished the effect of DIM. DIM stimulated MAPK activity and induced

phosphorylation of the endogenous PKA target, cAMP response element binding

protein (CREB), in a PKA-dependent manner. Expression of MCREB, a

nonphosphorylatable CREB mutant, partially abolished activation of the ER by DIM.

These results demonstrate that DIM is a mechanistically novel activator of the ER

that requires PKA-dependent phosphorylation of CREB.

 

PMID: 14645498

 

Curr Opin Otolaryngol Head Neck Surg. 2003 Dec;11(6):433-41.

 

Overview of recurrent respiratory papillomatosis.

 

Wiatrak BJ.

 

Department of Pediatrics and Surgery, University of Alabama at Birmingham, USA.

 

PURPOSE OF REVIEW: The purpose of this article is to review recent literature

regarding pediatric recurrent respiratory papillomatosis (RRP) published within

the last year. By reviewing and assessing these articles, a more clear

understanding regarding the etiology and management of pediatric RRP can be

obtained, allowing physicians to better care for their pediatric RRP patients.

RECENT FINDINGS: Pediatric RRP continues to be an extremely difficult management

problem for otolaryngologists. This disease process continues to be a significant

burden on the health care system and is a significant cause of morbidity in

affected patients and their families. The incidence of RRP continues to be

approximately 3.96 per 100,000 in the pediatric population. It has been noted

recently that approximately 7 of every 1000 children born to mothers with vaginal

condyloma develop pediatric RRP. Although the mainstay of surgical management has

traditionally been the CO2 laser, newer surgical techniques have demonstrated

efficacy in the management of pediatric RRP patients, including powered

instrumentation and the pulse-dye laser. The traditional adjuvant medical

therapies used for pediatric RRP continue to be commonly used, including

interferon-alpha2a, retinoic acid, and indol-3-carbinol/diindolylmethane

(I3C/DIM). Recently cidofovir has demonstrated efficacy in selected patients. In

addition, current research regarding vaccine therapy for pediatric RRP has shown

promise. Basic science research in the field of immunology has demonstrated

multiple defects in cell-mediated immunity, which has shed further light on the

etiology of pediatric RRP. SUMMARY: Pediatric RRP continues to be a highly morbid

disease process. New surgical and medical therapies offer hope for better control

of this disease in affected patients. Recent advances in immunologic research

offer the hope of immune system modulation and augmentation as potential future

treatment modalities to better control this disease process.

 

PMID: 14631175

 

Mol Cell Biol. 2003 Nov;23(21):7920-5.

 

Agonist and chemopreventative ligands induce differential transcriptional

cofactor recruitment by aryl hydrocarbon receptor.

 

Hestermann EV, Brown M.

 

Department of Molecular Oncology, Dana-Farber Cancer Institute, Brigham and

Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

 

Aryl hydrocarbon receptor (AHR) is a transcription factor whose activity is

regulated by environmental agents, including several carcinogenic agonists. We

measured recruitment of AHR and associated proteins to the human cytochrome

P4501A1 gene promoter in vivo. Upon treatment with the agonist

beta-naphthoflavone, AHR is rapidly associated with the promoter and recruits the

three members of the p160 family of coactivators as well as the p300 histone

acetyltransferase, leading to recruitment of RNA polymerase II (Pol II) and

induction of gene transcription. AHR, coactivators, and Pol II cycle on and off

the promoter, with a period of approximately 60 min. In contrast, the

chemopreventative AHR ligand 3,3'-diindolylmethane promotes AHR nuclear

translocation and p160 coactivator recruitment but, remarkably, fails to recruit

Pol II or cause histone acetylation. This novel mechanism of receptor antagonism

may account for the antitumor properties of chemopreventative compounds targeting

the AHR.

 

PMID: 14560034

  

J Nutr. 2003 Jul;133(7 Suppl):2470S-2475S.

 

Indole-3-carbinol and 3-3'-diindolylmethane antiproliferative signaling pathways

control cell-cycle gene transcription in human breast cancer cells by regulating

promoter-Sp1 transcription factor interactions.

 

Firestone GL, Bjeldanes LF.

 

Department of Molecular and Cell Biology and Cancer Research Laboratory,

University of California at Berkeley, Berkeley, CA 94720-3200, USA.

 

Indole-3-carbinol (I3C), a compound that occurs naturally in Brassica vegetables

such as cabbage and broccoli, can induce a G1 cell-cycle arrest of human MCF-7

breast cancer cells that is accompanied by the selective inhibition of

cyclin-dependent kinase 6 (Cdk6) expression and stimulation of p21(Waf1/Cip1)

gene expression. Construction and transfection of a series of promoter-reporter

plasmids demonstrate that the indole-regulated changes in Cdk6 and p21(Waf1/Cip1)

levels are due to specific effects on their corresponding promoters. Mutagenic

analysis reveals that I3C signaling targets a composite transcriptional element

in the Cdk6 promoter that requires both Sp1 and Ets transcription factors for

transactivation function. Analysis of protein-DNA complexes formed with nuclear

proteins isolated from I3C-treated and -untreated cells demonstrates that the Sp1

DNA element in the Cdk6 promoter interacts with an I3C-inhibited protein-protein

complex that contains the Sp1 transcription factor. In indole-treated cells, a

fraction of [(3)H]I3C was converted into its natural diindole product

(3)H-labeled 3-3'-diindolylmethane ([(3)H]DIM), which accumulates in the nucleus;

this suggests that DIM may have a role in the transcriptional activities of I3C.

Mutagenic analysis of the p21(Waf1/Cip1) promoter reveals that in transfected

breast cancer cells, DIM (as well as I3C) stimulates p21(Waf1/Cip1) transcription

through an indole-responsive region of the promoter that contains multiple Sp1

consensus sequences. Furthermore, DIM treatment regulates the presence of a

nuclear Sp1 DNA-binding activity. Our results demonstrate that both the Cdk6 and

p21(Waf1/Cip1) promoters are newly defined downstream targets of the

indole-signaling pathway, and that the observed transcriptional effects are due

to a combination of the cellular activities of I3C and DIM.

 

PMID: 12840223

 

Food Chem Toxicol. 2003 Jun;41(6):745-52.

 

Indole-3-carbinol and 3,3'-diindolylmethane induce apoptosis in human prostate

cancer cells.

 

Nachshon-Kedmi M, Yannai S, Haj A, Fares FA.

 

Faculty of Food Engineering and Biotechnology, Technion- Israel Institute of

Technology, 32000, Haifa, Israel.

 

Cruciferous vegetables contain glucobrassicin which, during metabolism, yields

indole-3-carbinol (I3C). In a low pH environment I3C is converted into polymeric

products, among which 3,3'-diindolylmethane (DIM) is the main one. The apoptotic

effects of I3C and DIM were exhibited in human breast cancer cells. The

objectives of this study were: (a) examination of the potential effects of I3C

and DIM on the proliferation and induction of apoptosis in human prostate cancer

cell lines with different p53 status; (b) to try to characterise the mechanism(s)

involved in these effects. Our results indicate that both indole derivatives

suppress the growth of these cells in a dose- and time-dependent manner, by

inducing apoptosis. It appears that these indolic compounds may offer effective

means against prostate cancer. Induction of apoptosis was p53-independent.

Moreover, the indole derivatives employed did not affect the levels of bcl-2, bax

and fasL.

 

PMID: 12738179

  

J Nutr. 2003 Apr;133(4):1011-9.

 

Gene expression profiles of I3C- and DIM-treated PC3 human prostate cancer cells

determined by cDNA microarray analysis.

 

Li Y, Li X, Sarkar FH.

 

Department of Pathology, Karmanos Cancer Institute, Wayne State University School

of Medicine, Detroit, MI 48201, USA.

 

Studies from our laboratory and others have shown that indole-3-carbinol (I3C)

and its in vivo dimeric product, 3,3'-diindolylmethane (DIM), inhibit the growth

of PC3 prostate cancer cells and induce apoptosis by inhibiting nuclear factor

(NF)-kappaB and Akt pathways. To obtain comprehensive gene expression profiles

altered by I3C- and DIM-treated PC3 cells, we utilized cDNA microarray to

interrogate the expression of 22,215 known genes using the Affymetrix Human

Genome U133A Array. We found a total of 738 genes that showed a greater than

twofold change after 24 h of DIM treatment. Among these genes, 677 genes were

down-regulated and 61 were up-regulated. Similarly, 727 genes showed a greater

than twofold change in expression, with down-regulation of 685 genes and

up-regulation of 42 genes in I3C-treated cells. The altered expressions of genes

were observed as early as 6 h and were more evident with longer treatment. Upon

cluster analysis, we found that both I3C and DIM up-regulated the expression of

genes that are related to the Phase I and Phase II enzymes, suggesting their

increased capacity for detoxification of carcinogens or chemicals. We also found

that I3C and DIM down-regulated the expression of genes that are critically

involved in the regulation of cell growth, cell cycle, apoptosis, signal

transduction, Pol II transcription factor and oncogenesis. Real-time reverse

transcription-polymerase chain reaction analysis was conducted to confirm the

cDNA microarray data, and the results were consistent. We conclude that I3C and

DIM affected the expression of a large number of genes that are related to the

control of carcinogenesis, cell survival and physiologic behaviors. This may help

determine the molecular mechanism(s) by which I3C and DIM exert their pleiotropic

effects on PC3 prostate cancer cells; in addition, this information could be

further exploited for devising chemopreventive and/or therapeutic strategies for

prostate cancer.

 

PMID: 12672912

 

J Biol Chem. 2003 Jun 6;278(23):21136-45. Epub 2003 Mar 27.

 

Plant-derived 3,3'-Diindolylmethane is a strong androgen antagonist in human

prostate cancer cells.

 

Le HT, Schaldach CM, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, The University of California,

Berkeley, California 94720-3104, USA.

 

3,3'-Diindolylmethane (DIM) is a major digestive product of indole-3-carbinol, a

potential anticancer component of cruciferous vegetables. Our results indicate

that DIM exhibits potent antiproliferative and antiandrogenic properties in

androgen-dependent human prostate cancer cells. DIM suppresses cell proliferation

of LNCaP cells and inhibits dihydrotestosterone (DHT) stimulation of DNA

synthesis. These activities were not produced in androgen-independent PC-3 cells.

Moreover, DIM inhibited endogenous PSA transcription and reduced intracellular

and secreted PSA protein levels induced by DHT in LNCaP cells. Also, DIM

inhibited, in a concentration-dependent manner, the DHT-induced expression of a

prostate-specific antigen promoter-regulated reporter gene construct in

transiently transfected LNCaP cells. Similar effects of DIM were observed in PC-3

cells only when these cells were co-transfected with a wild-type androgen

receptor expression plasmid. Using fluorescence imaging with green fluorescent

protein androgen receptor and Western blot analysis, we demonstrated that DIM

inhibited androgen-induced androgen receptor (AR) translocation into the nucleus.

Results of receptor binding assays indicated further that DIM is a strong

competitive inhibitor of DHT binding to the AR. Results of structural modeling

studies showed that DIM is remarkably similar in conformational geometry and

surface charge distribution to an established synthetic AR antagonist, although

the atomic compositions of the two substances are quite different. Taken together

with our published reports of the estrogen agonist activities of DIM, the present

results establish DIM as a unique bifunctional hormone disrupter. To our

knowledge, DIM is the first example of a pure androgen receptor antagonist from

plants.

 

PMID: 12665522

   

Carcinogenesis. 2002 Aug;23(8):1297-305.

 

3,3'-Diindolylmethane (DIM) induces a G(1) cell cycle arrest in human breast

cancer cells that is accompanied by Sp1-mediated activation of p21(WAF1/CIP1)

expression.

 

Hong C, Kim HA, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM) is a promising cancer chemopreventive agent derived

from Brassica food plants. To determine whether this natural indole has a direct

growth inhibitory effect on human breast cancer cells, we examined the cell cycle

regulatory effects of DIM in estrogen-dependent (MCF-7) and estrogen-independent

(MDA-MB-231) human breast cancer cell lines. Results of flow cytometry studies

showed that DIM treatment produced a marked increase (from 51 to 79%) in the

proportion of cells in the G(1) phase of the cell cycle, regardless of

estrogen-receptor status. Analyses of G(1)-acting cell cycle components indicated

that the enzymatic activity of cyclin-dependent kinase (CDK) 2 was also strongly

reduced. Western blot analyses showed that, concurrent with the DIM-induced cell

cycle arrest, DIM stimulated a rapid and pronounced increase in levels of the CDK

inhibitor, p21(WAF1/CIP1) (p21). Northern blot analysis demonstrated that DIM

increased p21 mRNA expression with a maximal 6-7-fold induction, and exposure to

cycloheximide did not block the response. Similar increases in expression of p21

protein and mRNA were observed in both MCF-7 and MDA-MB-231 human

breast cancer cells, suggesting that DIM induction of p21 expression is independent of

estrogen-receptor signaling and p53. Transient transfection of 5'-deletion

constructs of the p21 promoter demonstrated that the first 291 bp segment of the

proximal promoter, which contains six promoter specific transcription factor 1

(Sp1) elements, maintained DIM responsiveness. Consistent with a role for Sp1 in

this response, a reporter construct driven by three consensus Sp1 binding sites

was responsive to DIM. In addition, electrophoretic mobility shift assays showed

that DIM induced the binding of Sp1 and Sp3 to the consensus Sp1 responsive

element. Thus, our observations have uncovered an antiproliferative pathway for

DIM that implicates Sp1/Sp3-induced expression of p21 as a target for cell cycle

control in human breast cancer cells.

 

PMID: 12151347

 

Nutr Cancer. 2001;41(1-2):57-63.

 

Quantitative determination of 3,3'-diindolylmethane in urine of individuals

receiving indole-3-carbinol.

 

Sepkovic DW, Bradlow HL, Bell M.

 

Institute for Biomedical Research, Hackensack University Medical Center,

Hackensack, NJ 07601, USA.

 

Indole-3-carbinol (I3C) or, more correctly, its acid condensation products act as

chemoprotective agents via several mechanisms. It induces the expression of

cytochrome P-450 1A1, which shifts the estrogen metabolic pathway in favor of C-2

hydroxylation and away from the formation of 16 alpha-hydroxyestrone, a suspected

endogenous carcinogen. Increased 16 alpha-hydroxylation of estrogen is associated

with greater risk of cancer of the cervix, breast, endometrium, and larynx. The

production of 4-hydroxyestrone is also inhibited by I3C. I3C can induce a G1 cell

cycle arrest in human MCF-7 breast cancer cells. It can suppress aberrant crypt

foci. I3C significantly inhibits the cell adhesion, spreading, and invasion

associated with an upregulation of PTEN (a tumor suppressor gene) and E-cadherin

(a regulator of cell-cell adhesion) expression in T47-D human breast cancer

cells. Thus I3C exhibits anticancer activities by suppressing breast tumor cell

growth and metastatic spread. A gas chromatography-mass spectrometry method for

the quantitation of diindolylmethane, the principal acid condensation product of

I3C, has been developed for use in determining compliance in subjects who have

been treated with I3C. The method utilizes a 1-ml urine sample. We have used this

method to correlate I3C ingestion with regression of cervical intraepithelial

neoplasia in a population of women at risk for cervical cancer. The assay

provides an objective marker of consumption using a noninvasive biological fluid

and illustrates that diindolylmethane may be used as a marker of compliance in

I3C dietary intervention studies.

 

PMID: 12094629

 

Antivir Ther. 2002 Mar;7(1):1-9.

 

Therapy for recurrent respiratory papillomatosis.

 

Auborn KJ.

 

Department of Otolaryngology, Long Island Jewish Medical Center, Albert Einstein

College of Medicine, New Hyde Park, NY, USA.

 

Human papillomaviruses types 6 or 11 are aetiological agents of recurrent

respiratory papillomatosis, a disease characterized by benign exophytic tumours

usually on the vocal cords. Surgery debulks the tumours, but these growths

generally recur at regular intervals. Adjunct medical treatments, aimed at

containing the virus and growth of tumours, include indole-3-carbinol or its

dimer diindolylmethane, interferon, photodynamic therapy and others. Preventive

and therapeutic vaccines hold promise for eliminating the virus.

 

PMID: 12008782

 

Altern Med Rev. 2002 Apr;7(2):112-29.

 

Estrogen metabolism and the diet-cancer connection: rationale for assessing the

ratio of urinary hydroxylated estrogen metabolites.

 

Lord RS, Bongiovanni B, Bralley JA.

 

MetaMetrix Clinical Laboratory, 4855 Peachtree Industrial Boulevard, Suite 201,

Norcross, GA, 30092, USA.

 

Estrogens are known for their proliferative effects on estrogen-sensitive tissues

resulting in tumorigenesis. Results of experiments in multiple laboratories over

the last 20 years have shown that a large part of the cancer-inducing effect of

estrogen involves the formation of agonistic metabolites of estrogen, especially

16-alpha-hydroxyestrone. Other metabolites, such as 2-hydroxyestrone and

2-hydroxyestradiol, offer protection against the estrogen-agonist effects of

16-alpha-hydroxyestrone. An ELISA method for measuring 2- and

16-alpha-hydroxylated estrogen (OHE) metabolites in urine is available and the

ratio of urinary 2-OHE/16-alpha-OHE (2/16-alpha ratio) is a useful biomarker for

estrogen-related cancer risk. The CYP1A1 enzyme that catalyzes 2-hydroxyestrone

(2-OHE1) formation is inducible by dietary modification and supplementation with

the active components of cruciferous vegetables, indole-3-carbinol (I-3-C), or

diindolylmethane (DIM). Other dietary components, especially omega-3

polyunsaturated fatty acids and lignans in foods like flax seed, also exert

favorable effects on estrogen metabolism. Thus, there appear to be effective

dietary means for reducing cancer risk by improving estrogen metabolism. This

review presents the accumulated evidence to help clinicians evaluate the merit of

using tests that measure estrogen metabolites and using interventions to modify

estrogen metabolism.

 

PMID: 11991791

 

Biochem Pharmacol. 2002 Mar 15;63(6):1085-97.

 

Bcl-2 family-mediated apoptotic effects of 3,3'-diindolylmethane (DIM) in human

breast cancer cells.

 

Hong C, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley 94720-3200, USA.

 

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of the putative

anticancer agent indole-3-carbinol (I3C), which is present in vegetables of the

Brassica genus. At concentrations above 10 microM, DIM inhibited DNA synthesis

and cell proliferation in both estrogen receptor replete (MCF-7) and deficient

(MDA-MB-231) human breast cancer cells in a concentration- and time-dependent

manner. These antiproliferative effects were accompanied by characteristic

indications of programmed cell death in both cell lines, including

externalization of phosphatidylserine, chromatin condensation, and DNA

fragmentation. Furthermore, Western and Northern blot analyses, as well as

coimmunoprecipitation assays, revealed that in both MCF-7 and MDA-MB-231 cells,

DIM treatment decreased total transcript and protein levels of the apoptosis

inhibitory protein Bcl-2, and the amount of Bcl-2 bound to the pro-apoptotic

protein Bax. DIM treatment also caused an increase in Bax protein levels, but did

not affect the level of Bax that was bound to Bcl-2. As a functional test of the

role of Bcl-2 down-regulation in the DIM-induced apoptotic response, ectopic

expression of Bcl-2 in MCF-7 cells was shown to attenuate the apoptotic effect of

DIM. These results demonstrate that DIM can induce apoptosis in breast cancer

cells independent of estrogen receptor status by a process that is mediated by

the modulated expression of the Bax/Bcl-2 family of apoptotic regulatory factors.

 

PMID: 11931841

 

Chem Res Toxicol. 2002 Feb;15(2):101-9.

 

Fate of indole-3-carbinol in cultured human breast tumor cells.

 

Staub RE, Feng C, Onisko B, Bailey GS, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of California,

Berkeley, California 94720, USA.

 

Indole-3-carbinol (I3C), a natural component of Brassica vegetables, is a

promising cancer preventive agent that can reduce the incidence of tumors in

reproductive organs when administered in the diet. Here we report on the

metabolic fate of radiolabeled I3C in MCF-7 cells. I3C was surprisingly inert to

metabolism by these cells with a half-life in medium of approximately 40 h.

[(3)H]I3C levels in media declined at a similar rate whether incubation was with

cultured cells or in cell-free medium. Neither [(3)H]I3C nor its modified

products accumulated in MCF-7 cells and only low levels of intact I3C were

detected in cellular fractions. In contrast, I3C represented over 30% of the

radioactivity in media even after 72 h. In cytosolic fractions, the

3-(cystein-S-ylmethyl) and 3-(glutathion-S-ylmethyl) conjugates of [(3)H]I3C were

the primary conversion products identified after 16 h, representing approximately

50% and approximately 15% of the radioactivity in these fractions, respectively.

The reaction of I3C with thiols appears to be nonenzymatic since the cysteine

conjugate is produced when I3C is incubated in cell-free medium containing

additional cysteine. Both cellular and extracellular proteins were

nonspecifically modified with [(3)H]I3C. In medium, proteins are radiolabeled

even in the absence of cells, indicating again that enzymatic activation was not

required. I3C was also oxidized to indole-3-carboxaldehyde and

indole-3-carboxylic acid in culture medium independent of cells. Unexpectedly,

3,3'-diindolylmethane (DIM), an I3C product with in vitro and in vivo biological

activity, was detected in cellular fractions and appeared to accumulate in the

nucleus, representing approximately 40% of this fraction after 72 h treatment.

These findings suggest that MCF-7 cells do not vigorously metabolize I3C and that

the major route of reaction is with cellular thiols such as glutathione and

proteins. The accumulation of DIM in the nucleus suggests that this product may

have a role in the cellular biological activities of I3C.3

 

PMID: 11849035

   

Carcinogenesis. 2001 Nov;22(11):1809-17.

 

Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells

result from an estrogen receptor-mediated increase in transforming growth

factor-alpha expression.

 

Leong H, Firestone GL, Bjeldanes LF.

 

Department of Nutritional Sciences and Toxicology, University of

California-Berkeley, Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM), a major in vivo product of indole-3-carbinol (I3C),

is a promising anticancer agent derived from vegetables of the Brassica genus

including broccoli, Brussels sprouts and cabbage. We report here that DIM has a

potent cytostatic effect in cultured human Ishikawa endometrial cancer cells. A

combination of northern blot and quantitative PCR analyses revealed that DIM

induced the level of TGF-alpha transcripts by approximately 4-fold within 24 h of

indole treatment. DIM also induced a 4-fold increase in the activity of the

estrogen response marker, alkaline phosphatase (AP). Co-treatment of cells with

the estrogen receptor (ER) antagonist ICI, or with the inhibitor of PKA-mediated

activation of the ER, H89, ablated the DIM induction of both TGF-alpha expression

and AP activity. Furthermore, DIM increased the maximum stimulatory effect of

estrogen on TGF-alpha expression. Co-treatment with the protein synthesis

inhibitor, cycloheximide, abolished the inductive effects of DIM, indicating

differences in the mechanistic requirements of DIM and estrogen. DIM treatment

also stimulated levels of secreted TGF-alpha protein by >10-fold. The ectopic

addition of TGF-alpha inhibited the growth of Ishikawa cells, whereas incubation

with a TGF-alpha antibody partially reversed the growth inhibitory effects of

DIM. Taken together, these results extend our previous findings of the ligand

independent estrogen receptor agonist activity of DIM, and uncover an essential

role for the stimulation in TGF-alpha expression and the TGF-alpha activated

signal transduction pathway in the potent cytostatic effects of DIM in

endometrial cancer cells.

 

PMID: 11698343

 

Cancer Res. 2001 Aug 15;61(16):6120-30.

 

Dietary indoles and isothiocyanates that are generated from cruciferous

vegetables can both stimulate apoptosis and confer protection against DNA damage

in human colon cell lines.

 

Bonnesen C, Eggleston IM, Hayes JD.

 

Biomedical Research Centre, Ninewells Hospital and Medical School, University of

Dundee, Dundee DD1 9SY, Scotland, United Kingdom.

 

The natural indoles 3,3'-diindolylmethane (DIM), ascorbigen (ASG),

indole-3-carbinol (I3C), and indolo[3,2-b]carbazole (ICZ), as well as the natural

isothiocyanates sulforaphane (SUL), benzyl isothiocyanate (BITC) and phenethyl

isothiocyanate (PEITC), all possess cancer chemopreventive properties. It is now

shown that DIM, ICZ, SUL, and BITC can each stimulate apoptosis in human colon

adenocarcinoma LS-174 and Caco-2 cells. Treatment of LS-174 cells with nontoxic

doses of DIM, ASG, I3C, or ICZ affected an increase of up to 21-fold in

cytochrome P450 1A1 (CYP1A1). None of these indoles caused an elevation in either

aldo-keto reductase 1C1 (AKR1C1) or the gamma-glutamylcysteine synthetase heavy

subunit (GCS(h)), but DIM, I3C, and ICZ produced a very modest increase in

NAD(P)H:quinone oxidoreductase 1 (NQO1). By contrast, nontoxic doses of SUL,

BITC, or PEITC failed to induce expression of CYP1A1 in LS-174 cells, but caused

an increase of between 11- and 17-fold in the protein levels of AKR1C1, NQO1, and

GCS(h). Treatment of the colon cell line with ICZ or SUL caused increases in the

levels of mRNA for CYP1A1, AKR1C1, and NQO1 that were consistent with the enzyme

data. Exposure of Caco-2 cells to media containing indoles or isothiocyanates

gave similar results to those obtained using LS-174 cells. Evidence is presented

that the ability of indoles and isothiocyanates to stimulate either xenobiotic

response element- or antioxidant response element-driven gene expression accounts

for the two groups of phytochemicals inducing different gene batteries.

Pretreatment of LS-174 cells for 24 h with ICZ and SUL before exposure for 24 h

to benzo(a)pyrene (BaP) reduced to <20% the number of single-strand DNA breaks

produced by the carcinogen. Neither ICZ alone nor SUL alone were able to confer

the same degree of protection against DNA damage produced by BaP as they achieved

in combination. Similar results were obtained with H(2)O(2) as the genotoxic

agent. Together, these phytochemicals may prevent colon tumorigenesis by both

stimulating apoptosis and enhancing intracellular defenses against genotoxic

agents.

 

PMID: 11507062

 

Breast Cancer Res Treat. 2001 Mar;66(2):147-57.

 

Methyl-substituted diindolylmethanes as inhibitors of estrogen-induced growth of

T47D cells and mammary tumors in rats.

 

McDougal A, Gupta MS, Morrow D, Ramamoorthy K, Lee JE, Safe SH.

 

Department of Veterinary Physiology & Pharmacology, Texas A &M University,

College Station 77843-4466, USA.

 

Diindolylmethane (DIM) is formed by acid catalyzed dimerization of the

phytochemical indole-3-carbinol, and both compounds inhibit formation and/or

growth of mammary tumors in rodents. In this study, we have investigated the aryl

hydrocarbon receptor (AhR) agonist activity and inhibitory AhR-estrogen receptor

crosstalk induced by the following methyl-substituted DIMs: 1,1'-dimethyl-,

2,2'-dimethyl-, 5,5'-dimethyl-, 6,6'-dimethyl-, and 7,7'-dimethylDIM and

1,1',2,2'-tetramethylDIM. The six compounds bound to the rat cytosolic AhR in a

transformation assay but, at concentrations < or = 10 microM, exhibited minimal

to non-detectable AhR agonist or antagonist activities associated with CYP1A1

induction. In contrast, the methyl-substituted DIMs inhibited estrogen-induced

T47D human breast cancer cell growth and the four most active compounds (1,1'-,

2,2'-, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM) inhibited one or more

estrogen-induced responses in the 21-day-old female B6C3F1 mice at a dose of 100

mg/kg/day (X3). Induction of hepatic CYP1A1-dependent activity was not observed

at this high dose. The antitumorigenic activity of these compounds was examined

in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumor model in which the

DIM analogs were orally administered (by gavage in corn oil) at a dose of 1

mg/kg/day (X10). 1,1'-DimethylDIM, 5,5'-dimethylDIM and 1,1',2,2'-tetramethylDIM

significantly inhibited mammary tumor growth, and this was not accompanied by

changes in organ/body weights or histopathology. These studies demonstrate that

methyl-substituted DIMs are selective AhR modulators (SAhRMs) with potential for

clinical treatment of breast cancer.

 

PMID: 11437101

 

Toxicol Sci. 2001 May;61(1):40-8.

 

2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce

cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells.

 

Sanderson JT, Slobbe L, Lansbergen GW, Safe S, van den Berg M.

 

Research Institute for Toxicology, Utrecht University, P.O. Box 80176, 3508 TD

Utrecht, The Netherlands.

 

Diindolylmethane (DIM) is an acid-catalyzed condensation product of

indole-3-carbinol, a constituent of cruciferous vegetables, and is formed in the

stomach. DIM alters estrogen metabolism and inhibits carcinogen-induced mammary

tumor growth in rodents. DIM is a weak agonist for the aryl hydrocarbon (Ah)

receptor and blocks the effects of estrogens via inhibitory Ah receptor-estrogen

receptor cross-talk. DIM and various structural analogs were examined in H295R

cells for effects on 3 cytochrome P450 (CYP) enzymes involved in estrogen

synthesis and/or metabolism: CYP1A1, CYP1B1, and CYP19 (aromatase). Aromatase

activity was measured by conversion of 1 beta-(3)H-androstenedione to estrone and

(3)H(2)O. H295R cells were exposed to the test chemicals dissolved in dimethyl

sulfoxide for 24 h prior to analyses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

(0--30 nM) and DIM (0--10 microM) induced ethoxyresorufin-O-deethylase (EROD)

activity, as a measure of CYP1A1 and possibly 1B1 activity, with EC(50) values of

about 0.3 nM and 3 microM, respectively. DIM, but not TCDD, induced aromatase

activity with an apparently maximal 2-fold increase at 10 microM; higher

concentrations of DIM and many of its analogs were cytotoxic. TCDD (30 nM)

significantly increased CYP1A1 and 1B1 mRNA levels, but had no effect on mRNA for

CYP19. DIM (3 microM) significantly increased mRNA levels for all three CYPS: DIM

analogs with substitutions on the 5 and 5' position (3 microM) induced aromatase

and EROD activity, together with mRNA levels of CYP1A1, 1B1, and 19; analogs that

were substituted on the central carbon of the methane group showed little or no

inductive activity toward the CYPS: In conclusion, DIM and several of its analogs

appear to induce CYPs via multiple yet distinct pathways in H295R human

adrenocortical carcinoma cells.

 

PMID: 11294972

 

Gene. 2001 Jan 10;262(1-2):207-14.

 

Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7

breast cancer cells using suppression subtractive hybridization.

 

Chen I, Hsieh T, Thomas T, Safe S.

 

Department of Veterinary Physiology and Pharmacology, A&M University, College

Station, TX 77843, USA.

 

Aryl hydrocarbon receptor (AhR) agonists inhibit 17beta-estradiol (E2) induced

growth of MCF-7 human breast cancer cells in vitro and rodent mammary tumor

growth in vivo. Genes associated with inhibitory AhR-estrogen receptor (ER)

crosstalk were investigated in MCF-7 human breast cancer cells using

poly(A)(+)RNA from cells treated with either 1 nM E2 (target) or E2 plus 1 nM

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 microM

diindolylmethane (DIM) as AhR agonists in MCF-7 cells. Suppression subtractive

hybridization (SSH) was subsequently used to identify 33 genes with sequence

homology to known human genes that are induced by E2 and inhibited by AhR

agonists in MCF-7 cells; two unknown genes were also identified. Many of these

genes are involved in cell proliferation and these include cell cycle regulators

(cdc28/cdc2-associated protein), nucleotide synthases (thymidylate synthase),

early intermediate genes (early growth response alpha, EGRalpha) and other

proteins involved in signaling pathways (calmodulin, ATP synthase alpha subunit).

Thus SSH has identified a diverse spectrum of new genes that are affected by

inhibitory AhR-ER crosstalk and among this group are a subset of genes that may

be critical for the in vivo antitumorigenic effects of AhR agonists.

 

PMID: 11179685

 

Drug Metab Dispos. 2000 Aug;28(8):930-6.

 

Concurrent flavin-containing monooxygenase down-regulation and cytochrome P-450

induction by dietary indoles in rat: implications for drug-drug interaction.

 

Katchamart S, Stresser DM, Dehal SS, Kupfer D, Williams DE.

 

Department of Environmental and Molecular Toxicology, Oregon State University,

Corvallis, Oregon, USA.

 

Our laboratory has previously shown that dietary administration of

indole-3-carbinol (I3C) to male Fischer 344 rats has the very unusual property of

inducing hepatic levels of a number of cytochrome P450s (CYPs), especially

CYP1A1, while markedly inhibiting the levels of flavin-containing monooxygenase

(FMO) 1 protein and its catalytic activity. We hypothesized that rats fed I3C or

3,3'-diindolylmethane (DIM), one of its major acid condensation products formed

in vivo, should exhibit a marked shift in the metabolic profiles of drugs or

xenobiotics that are substrates for both monooxygenase systems. Male rats were

fed AIN-76A powdered diets containing 0, 1000, or 2500 ppm I3C or DIM for 4

weeks. Dietary I3C and DIM reduced FMO1 protein levels (8% reduction with I3C and

84% with DIM at 1000 ppm, and 90% reduction with I3C and 97% with DIM at 2500

ppm) in hepatic microsomes. The ratio of FMO (N-oxygenation)- to CYP

(N-demethylation)-mediated metabolism of N,N-dimethylaniline decreased in liver

microsomes from I3C- or DIM-fed rats from near unity to 0.02 at the highest

dietary doses. FMO-mediated N-oxygenation (nicotine N-1'-oxide) was decreased,

whereas CYP-mediated (nornicotine and nicotine delta (1,5)-iminium ion)

metabolism of nicotine was unchanged in liver microsomes from rats fed I3C or

DIM. Similarly, the ratio of FMO to CYP metabolites of tamoxifen decreased due to

a reduction in N-oxygenation. This study demonstrates alteration of FMO- and

CYP-mediated drug metabolism in vitro by dietary I3C or DIM and suggests the

potential for altered toxicity of tamoxifen and nicotine in vivo.

 

PMID: 10901703

 

Biochem Pharmacol. 2000 Jul 15;60(2):167-77.

 

Ligand-independent activation of estrogen receptor function by 3,

3'-diindolylmethane in human breast cancer cells.

 

Riby JE, Chang GH, Firestone GL, Bjeldanes LF.

 

Division of Nutritional Sciences and Toxicology, University of California,

Berkeley, CA 94720, USA.

 

3,3'-Diindolylmethane (DIM), a major in vivo product of acid-catalyzed

oligomerization of indole-3-carbinol (I3C), is a promising anticancer agent

present in vegetables of the Brassica genus. We investigated the effects of DIM

on estrogen-regulated events in human breast cancer cells and found that DIM was

a promoter-specific activator of estrogen receptor (ER) function in the absence

of 17beta-estradiol (E(2)). DIM weakly inhibited the E(2)-induced proliferation

of ER-containing MCF-7 cells and induced proliferation of these cells in the

absence of steroid, by approximately 60% of the E(2) response. DIM had little

effect on proliferation of ER-deficient MDA-MB-231 cells, suggesting that it is

not generally toxic at these concentrations. Although DIM did not bind to the ER

in this concentration range, as shown by a competitive ER binding assay, it

activated the ER to a DNA-binding species. DIM increased the level of transcripts

for the endogenous pS2 gene and activated the estrogen-responsive pERE-vit-CAT

and pS2-tk-CAT reporter plasmids in transiently transfected MCF-7 cells. In

contrast, DIM failed to activate transcription of the simple E(2)- and

diethylstilbesterol-responsive reporter construct pATC2. The estrogen antagonist

ICI 182780 (7alpha-[9-[(4,4,5,5,

5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3, 17beta-diol) was

effective against DIM-induced transcriptional activity of the pERE-vit-CAT

reporter, which further supports the hypothesis that DIM is acting through the

ER. We demonstrated that ligand-independent activation of the ER in MCF-7 cells

could be produced following treatment with the D1 dopamine receptor agonist

SKF-82958 [(+/-)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,

5-tetrahydro-1H-3-benzazepinehydrobromide]. We also demonstrated that the agonist

effects of SKF-82958 and DIM, but not of E(2), could be blocked by co-treatment

with the protein kinase A (PKA) inhibitor H-89

(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide). These results have

uncovered a promoter-specific, ligand-independent activation of ER signaling for

DIM that may require activation by PKA, and suggest that this major I3C product

may be a selective activator of ER function.

 

PMID: 10825461

 

Cancer Lett. 2000 Apr 14;151(2):169-79.

 

Inhibition of carcinogen-induced rat mammary tumor growth and other

estrogen-dependent responses by symmetrical dihalo-substituted analogs of

diindolylmethane.

 

McDougal A, Sethi Gupta M, Ramamoorthy K, Sun G, Safe SH.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station, TX 77843-4466, USA.

 

90%) by the haloDIMs at concentrations of 5 or 10 microM, and only 4,

4'-dichloroDIM alone increased cell proliferation. With the exception of

5,5'-difluoroDIM, the remaining compounds also inhibited E2-induced growth of

MCF-7 human breast cancer cells. DihaloDIMs (100 mg/kg/dayx3) were not estrogenic

in the immature female B6C3F1 mouse uterus; however, in animals co-treated with

E2 (0.02 microg/mouse), 5,5'-dichloro- and 6,6'-dichloroDIM inhibited uterine

progesterone receptor (PR) binding and uterine peroxidase activity, whereas

5,5'-dichloro- and 5,5'-dichloro-2,2'-dimethylDIM inhibited only the latter

response. The antitumorigenic activities of the dihaloDIMs were determined by

their inhibition of carcinogen-induced mammary tumor growth in female

Sprague-Dawley rats. 4,4'-Dichloro-, 5,5'-dibromo- and 6,6'-dichloroDIM,

significantly inhibited mammary tumor growth at doses of 1 mg/kg every second

day, and no significant changes in organ weights or liver and kidney

histopathology were observed. These three compounds were more active than DIM in

the same in vivo assay.

 

PMID: 10738111

   

Toxicol Sci. 1999 Dec;52(2):178-88.

 

The anti-estrogenicity of Ah receptor agonists in carp (Cyprinus carpio)

hepatocytes.

 

Smeets JM, van Holsteijn I, Giesy JP, van den Berg M.

 

Research Institute of Toxicology, Utrecht University, The Netherlands.

 

Cultured hepatocytes of female carp (Cyprinus carpio) were coexposed for 4 days

to 200 nM 17beta-estradiol (E2), and concentration ranges of nine known Ah

receptor (AhR) agonists: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),

3,3'4,4'5-pentachlorobiphenyl (PCB 126), 2,3'4,4'5-pentachlorobiphenyl (PCB 118),

beta-naphthoflavone (BNF), benzo(a)pyrene (BaP), benzo(a)anthracene (BaA),

diindolylmethane (DIM), 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) and

hexachlorobenzene (HCB). TCDD caused a greater than 100-fold induction of

cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase

(EROD), with an EC50 of 6 pM. Based on EC50 values, the order of potency as CYP1A

inducers was TCDD > PCB 126 > BNF > BaP > BaA > PCB 118. DIM and MCDF

caused a lower maximum CYP1A induction (< 9-fold), whereas HCB caused no EROD

induction at concentrations up to 6 microM. TCDD, PCB 126, BNF, BaP, and DIM also

caused a concentration-dependent suppression of the secretion of the yolk protein

vitellogenin (Vtg), relative to E2-treated hepatocytes. Suppression of Vtg

secretion was not directly correlated with EROD activity, and the antiestrogenic

effects occurred at higher concentrations than the induction of CYP1A. This

indicates that the anti-estrogenicity was not caused by increased metabolism of

E2 due to induction of CYP1A. Nevertheless, the order of potency of the tested

compounds for suppression of Vtg secretion was comparable to the order of potency

for CYP1A induction. This concurrence suggests that the anti-estrogenicity of

these compounds is AhR-mediated, but does not involve CYP1A. This could be

relevant for feral fish populations, as they are frequently exposed to AhR

agonists, to an extent that AhR-mediated effects are observed.

 

PMID: 10630570

 

Toxicol Sci. 1999 Nov;52(1):1-8.

 

Symposium on mechanisms of action of naturally occurring anticarcinogens.

 

Safe S, Wargovich MJ, Lamartiniere CA, Mukhtar H.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station 77843-4466, USA.

 

PMID: 10568692

 

Food Chem Toxicol. 1999 Jun;37(6):609-18.

 

Effect of some indole derivatives on xenobiotic metabolism and xenobiotic-induced

toxicity in cultured rat liver slices.

 

Renwick AB, Mistry H, Barton PT, Mallet F, Price RJ, Beamand JA, Lake BG.

 

BIBRA International, Carshalton, Surrey, UK.

 

In this study the effect of some indole derivatives on xenobiotic metabolizing

enzymes and xenobiotic-induced toxicity has been examined in cultured

precision-cut liver slices from male Sprague-Dawley rats. While treatment of rat

liver slices for 72 hours with 2-200 microM of either indole-3-carbinol (I3C) or

indole-3-acetonitrile (3-ICN) had little effect on cytochrome P-450

(CYP)-dependent enzyme activities, enzyme induction was observed after in vivo

administration of I3C. The treatment of rat liver slices with 50 microM

3,3'-diindolylmethane (DIM; a dimer derived from I3C under acidic conditions) for

72 hours resulted in a marked induction of CYP-dependent enzyme activities. DIM

appears to be a mixed inducer of CYP in rat liver slices having effects on CYP1A,

CYP2B and CYP3A subfamily isoforms. Small increases in liver slice reduced

glutathione levels and glutathione S-transferase activity were also observed

after DIM treatment. While aflatoxin B1 and monocrotaline produced a

concentration-dependent inhibition of protein synthesis in 72-hour-cultured rat

liver slices, cytotoxicity was markedly reduced in liver slices cultured with 50

microM DIM. These results demonstrate that cultured rat liver slices may be

employed to evaluate the effects of chemicals derived from cruciferous and other

vegetables on CYP isoforms. In addition, liver slices can also be utilized to

examine the ability of such chemicals to modulate xenobiotic-induced toxicity.

 

PMID: 10478829

 

Biochem Pharmacol. 1999 Sep 1;58(5):825-34.

 

Cytostatic and antiestrogenic effects of

2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary

indole-3-carbinol.

 

Chang YC, Riby J, Chang GH, Peng BC, Firestone G, Bjeldanes LF.

 

Division of Nutritional Sciences and Toxicology, University of California,

Berkeley 94720, USA.

 

Under acidic conditions, indole-3-carbinol (13C) is converted to a series of

oligomeric products thought to be responsible for the biological effects of

dietary 13C. Chromatographic separation of the crude acid mixture of 13C, guided

by cell proliferation assay in human MCF-7 cells, resulted in the isolation of

2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr-1) as a major antiproliferative

component. LTr-1 inhibited the growth of both estrogen-dependent (MCF-7) and

-independent (MDA-MB-231) breast cancer cells by approximately 60% at a

non-lethal concentration of 25 microM. LTr-1 had no apparent effect on the

proliferation of MCF-7 cells in the absence of estrogen. LTr-1 was a weak ligand

for the estrogen receptor (ER) (IC50 70 microM) and efficiently inhibited the

estradiol (E2)-induced binding of the ER to its cognate DNA responsive element.

The antagonist effects of LTr-1 also were exhibited in assays of endogenous pS2

gene expression and in cells transiently transfected with an estrogen-responsive

reporter construct (pERE-vit-CAT). LTr-1 activated both binding of the aryl

hydrocarbon (Ah) receptor to its cognate DNA responsive element and expression of

the Ah receptor-responsive gene CYP1A1. LTr-1 was a competitive inhibitor of

CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity. In summary, these

results demonstrated that LTr-1, a major in vivo product of I3C, could inhibit

the proliferation of both estrogen-dependent and -independent breast tumor cells

and that LTr-1 is an antagonist of estrogen receptor function and a weak agonist

of Ah receptor function.

 

PMID: 10449193

 

Carcinogenesis. 1998 Sep;19(9):1631-9.

 

Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of

diindolylmethane.

 

Chen I, McDougal A, Wang F, Safe S.

 

Department of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station 77843-4466, USA.

 

Phytochemicals such as indole-3-carbinol (I3C) and sulforaphane are components of

cruciferous vegetables which exhibit antitumorigenic activity associated with

altered carcinogen metabolism and detoxification. Diindolylmethane (DIM) is a

major acid-catalyzed metabolite of I3C formed in the gut that binds to the aryl

hydrocarbon receptor (AhR) and treatment of MCF-7 human breast cancer cells with

10-50 microM DIM resulted in rapid formation of the nuclear AhR complex and

induction of CYP1A1 gene expression was observed at concentrations >50 microM.

Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin

(TCDD), a high affinity AhR ligand, inhibits 17beta-estradiol (E2)-induced

responses in MCF-7 cells and growth of E2-dependent 7,12-dimethylbenzanthracene

(DMBA)-induced mammary tumors in female Sprague-Dawley rats. Results of this

study show that like TCDD, DIM inhibits E2-induced proliferation of MCF-7 cells,

reporter gene activity in cells transiently transfected with an E2-responsive

plasmid (containing a frog vitellogenin A2 gene promoter insert) and

down-regulates the nuclear estrogen receptor. Moreover, DIM (5 mg/kg every other

day) also inhibits DMBA-induced mammary tumor growth in Sprague-Dawley rats and

this was not accompanied by induction of hepatic CYP1A1-dependent activity. Thus,

DIM represents a new class of relatively non-toxic AhR-based antiestrogens that

inhibit E2-dependent tumor growth in rodents and current studies are focused on

development of analogs for clinical treatment of breast cancer.

 

PMID: 9771935

 

Xenobiotica. 1998 Aug;28(8):803-11.

 

3,3'-Diindolylmethane induces CYP1A2 in cultured precision-cut human liver

slices.

 

Lake BG, Tredger JM, Renwick AB, Barton PT, Price RJ.

 

BIBRA International, Carshalton, UK.

 

1. The effect of 3,3'-diindolylmethane (DIM), an indole derivative derived from

cruciferous vegetables, on cytochrome P450 (CYP) isoforms in the CYP1A and CYP3A

subfamilies has been studied in 72-h cultured human liver slices. 2. In cultured

human liver slices 50 microM DIM induced 7-ethoxyresorufin O-deethylase and to a

lesser extent 7-methoxyresorufin O-demethylase activities. 3. Western

immunoblotting of liver slice microsomes was performed with antibodies to rat

CYP1A2 and human CYP3A4. Compared with control liver slice microsomes (dimethyl

sulphoxide-only treated), DIM induced levels of CYP1A2 but had little effect on

levels of CYP3A4. The treatment of human liver slices with 2 microg/ml of the

polycholorinated biphenyl mixture Aroclor 1254 also resulted in an induction of

levels of CYP1A2, but had no effect on CYP3A4. 4. These results demonstrate that

DIM induces CYP1A isoforms in cultured human liver slices. Some variability in

the magnitude of induction of enzyme activities by DIM was observed in four human

liver samples examined. For 7-ethoxyresorufin O-deethylase, the magnitude of

induction by 50 microM DIM ranged from 2.3- to 19.3-fold. 5. These results

demonstrate that cultured human liver slices can be used to evaluate the effect

of chemicals derived from cruciferous and other vegetables on CYP isoforms.

 

PMID: 9741959

 

Anticancer Drugs. 1998 Feb;9(2):141-8.

 

Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products

on human colon cancer cells in vitro.

 

Gamet-Payrastre L, Lumeau S, Gasc N, Cassar G, Rollin P, Tulliez J.

 

INRA, Laboratoire des Xénobiotiques, Toulouse, France.

 

Glucosinolates hydrolysis products are attracting increasing attention since many

studies have suggested that they may be involved in the anticarcinogenic property

of cruciferous vegetables. In this study, we show that diindolylmethane (DIM) and

sulforaphane, produced during the hydrolysis of glucobrassicin and glucoraphanin,

respectively, exert a dose-dependent cytotoxicity on human colon adenocarcinoma

HT29 cells. Moreover, these products are able to inhibit quiescent cells to

re-enter the cell cycle. Interestingly, our results clearly show that low doses

of DIM and sulforaphane, although very effective on undifferentiated intestinal

HT29 cells, do not affect the viability of the differentiated CaCo2 cells. The

reversibility of their effects has also been tested and is discussed.

 

PMID: 9510500

 

Biochem Biophys Res Commun. 1996 Nov 1;228(1):153-8.

 

3,3'-Diindolylmethane induces apoptosis in human cancer cells.

 

Ge X, Yannai S, Rennert G, Gruener N, Fares FA.

 

Department of Food Engineering and Biotechnology, Technion-Israel Institute of

Technology, Haifa, Israel.

 

3,3'-Diindolylmethane is a dimer of indole-3-carbinol formed both in vivo and in

vitro. In this study, human cancer cells MCF-7 (with wild-type p53), T47-D

(mutant p53), and Saos-2 (deficient in p53 gene), were used to examine the

anticancer activities of 3,3'-diindolylmethane. The dose-dependent growth

inhibitory effect was found in all these cell lines. Exposure of the cells to 50

microM solution of 3,3'-diindolylmethane for 48 h, apoptosis (programmed cell

death) was evidenced by the characteristic morphology of cell nuclei under

fluorescence microscope and the DNA "ladder" in agarose gel electrophoresis. The

percentage of apoptotic cells in each cell line was found to be 12% for MCF-7,

14% for T47D and 13% for Saos2 cells. Exposure of MCF-7 cells to 100 microM

3,3'-diindolylmethane for 24 h, 19% of apoptotic cells were detected by flow

cytometry analysis. The lowest dose required for induction of apoptosis in MCF-7

cells was found to be 10 microM after 72 h incubation. Western blot showed that

wild-type p53 protein was unchanged after MCF-7 cells had been exposed to 50

microM 3,3'-diindolylmethane for 8 h. This study provides evidences that

3,3'-diindolylmethane induces apoptosis in human cancer cells and that the

induction of apoptosis is independent of p53 pathway.

 

PMID: 8912651

  

Biochem Pharmacol. 1996 Apr 26;51(8):1069-76.

 

Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists

and antagonists in T47D human breast cancer cells.

 

Chen I, Safe S, Bjeldanes L.

 

Veterinary Physiology and Pharmacology, Texas A&M University, College Station

77843-4466, USA.

 

Indole-3-carbinol (I3C) is a major component of Brassica vegetables, and

diindolylmethane (DIM) is the major acid-catalyzed condensation product derived

from I3C. Both compounds competitively bind to the aryl hydrocarbon (Ah) receptor

with relatively low affinity. In Ah-responsive T47D human breast cancer cells,

I3C and DIM did not induce significantly CYP1A1-dependent ethoxyresorufin

O-deethylase (EROD) activity or CYP1A1 mRNA levels at concentrations as high as

125 or 31 microM, respectively. A 1 nM concentration of

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced EROD activity in these cells,

and cotreatment with TCDD plus different concentrations of I3C (1-125 microM) or

DIM (1-31 microM) resulted in a > 90% decrease in the induced response at the

highest concentration of I3C or DIM. I3C or DIM also partially inhibited (< 50%)

induction of CYP1A1 mRNA levels by TCDD and reporter gene activity, using an

Ah-responsive plasmid construct in transient transfection assays. In T47D cells

cotreated with 5 nM [3H]TCDD alone or in combination with 250 microM I3C or 31

microM DIM, there was a 37 and 73% decrease, respectively, in formation of the

nuclear Ah receptor. The more effective inhibition of induced EROD activity by

I3C and DIM was due to in vitro inhibition of enzyme activity. Thus, both I3C and

DIM are partial Ah receptor antagonists in the T47D human breast cancer cell

line.

 

PMID: 8866829

 

Food Chem Toxicol. 1995 Oct;33(10):851-7.

 

Mechanisms of indole-3-carbinol (I3C) anticarcinogenesis: inhibition of aflatoxin

B1-DNA adduction and mutagenesis by I3C acid condensation products.

 

Takahashi N, Dashwood RH, Bjeldanes LF, Williams DE, Bailey GS.

 

Department of Food Science & Technology, Oregon State University, Corvallis

97331, USA.

 

Possible inhibitory mechanisms of indole-3-carbinol (I3C) against aflatoxin B1

(AFB1), a potent hepatocarcinogen, were examined in rainbow trout. In the

Salmonella assay using a trout post-mitochondrial activation system, I3C itself

was not an antimutagen against AFB1. The study also evaluated: the antimutagenic

ability of I3C oligomers; an acid reaction mixture (RXM) of I3C, generated at low

pH to simulate I3C products formed under acidic conditions of the stomach;

3,3-diindolylmethane (I33'), the major derivative of I3C found in trout liver;

and 5,6,11,12,17,18- hexahydrocyclononal [1,2-b:4,5-b':7,8-b"]triindole , the

cyclic trimer of I3C (CT), a derivative of I3C in liver and one of the major

components of RXM. Concentrations of 3.5 microM and greater of I33', CT or RXM

showed about 80% inhibition compared with the control. Higher concentrations (70

microM) of the various I3C oligomers also inhibited (to a maximum of 55%)

mutagenesis of synthetic AFB1-8,9-epoxide added to the Salmonella assay, in the

absence of activating enzymes. I33' inhibited total microsome catalysed AFB1-DNA

binding in vitro in an apparently non-competitive manner (Kis = 27.6 +/- 9.4

microM, Kii = 37.5 +/- 32.2 microM). These results suggest that the

anticarcinogenic effect of I3C against AFB1 in rainbow trout, and perhaps other

species, is due in part to inhibition of AFB1 bioactivation enzymes and to

scavenging of the activated AFB1-8,9-epoxide.

 

PMID: 7590529

  

J Biochem Toxicol. 1995 Aug;10(4):191-201.

 

The anticarcinogen 3,3'-diindolylmethane is an inhibitor of cytochrome P-450.

 

Stresser DM, Bjeldanes LF, Bailey GS, Williams DE.

 

Department of Food Science and Technology, Oregon State University, Corvallis

97331-6602, USA.

 

Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several

mechanisms of inhibition may exist. We reported previously that

3,3'-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent

noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent

ethoxyresorufin O-deethylase with Ki values in the low micromolar range. We now

report a similar potent inhibition by 3,3'-diindolylmethane of rat and human

CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential

activity assays. 3,3'-Diindolylmethane also inhibited in vitro CYP-mediated

metabolism of the ubiquitous food contaminant and potent hepatocarcinogen,

aflatoxin B1. There was no inhibition of cytochrome c reductase. In addition, we

found 3,3'-diindolylmethane to be a substrate for rat hepatic microsomal

monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These

observations indicate that 3,3'-diindolylmethane can inhibit the catalytic

activities of a range of CYP isoforms from lower and higher vertebrates in vitro.

This broadly based inhibition of CYP-mediated activation of procarcinogens may be

an indole-3-carbinol anticarcinogenic mechanism applicable to all species,

including humans.

 

PMID: 8568833

 

Food Chem Toxicol. 1995 Feb;33(2):111-20.

 

Regulation of hepatic cytochrome P4501A by indole-3-carbinol: transient induction

with continuous feeding in rainbow trout.

 

Takahashi N, Dashwood RH, Bjeldanes LF, Bailey GS, Williams DE.

 

Department of Food Science and Technology, Oregon State University, Corvallis

97331.

 

This study investigated the kinetics of hepatic cytochrome P-4501A (CYP1A)

induction in rainbow trout by indole-3-carbinol (I3C), a natural tumour modulator

from cruciferous vegetables, and its low pH reaction products

3,3'-diindolylmethane (I33'),

5,6,11,12,17,18-hexahydrocyclononal[1,2-b:4,5-b':7,8-b"]triindo le cyclic trimer

(CT), and the unresolved I3C acid reaction mixture (RXM). RXM, CT and I33' were

potent inducers of total embryonic CYP1A following direct microinjection, and of

fingerling hepatic CYP1A following ip exposure, whereas I3C itself produced only

a transient and relatively weak induction. It is also reported for the first time

that dietary I3C induced hepatic CYP1A and its associated ethoxyresorufin

O-deethylase (EROD) activity in trout but, again, the induction was weak and

transient even with continuous I3C feeding. Mechanism studies and mixed exposures

with the Ah agonist beta-naphthoflavone indicated that transient induction by I3C

was not due to diet ageing, but appears to involve inactivation of the Ah

inductive pathway and irreversible inactivation of CYP1A-mediated EROD activity

by I3C-derived metabolites. Thus, I3C derivatives exhibit dual capacities for

CYP1A induction and inhibition in trout.

 

PMID: 7867998

  

J Steroid Biochem Mol Biol. 1993 Dec;46(6):791-8.

 

Influence of indole carbinols and growth hormone on the metabolism of

4-androstenedione by rat liver microsomes.

 

Jellinck PH, Makin HL, Sepkovic DW, Bradlow HL.

 

Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.

 

The effect of indole-3-carbinol (IC), an anticarcinogen present in cruciferous

vegetables, to alter the metabolism of 4-androstenedione (AD) by female rat liver

microsomes was investigated and compared to that of its main gastric conversion

product, diindolylmethane (DIM) as well as other specific cytochrome P450

inducers. DIM was a more potent inducer of the hydroxylase which converts

androsterone to its 6 beta-hydroxylated derivative 3 alpha, 6 beta-dihydroxy-5

alpha-androstan-17-one (A) than IC after either oral or intraperitoneal

administration and was also a better in vitro inhibitor. Isosafrole (ISF), which

like IC and DIM, induces CYP1A2 as well as gestodene, were powerful inhibitors of

the in vitro reaction. Naringenin produced only a weak inhibitory effect while

3-methylcholanthrene was inactive. SKF-525A, a prototypic hydroxylase inhibitor,

or 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androst-1-ene-3-one which

inhibits steroid 5 alpha-reductase, also decreased the formation of A from AD by

liver microsomes. The infusion of human growth hormone by osmotic minipump, which

feminizes hepatic steroid metabolism, increased the ability of male rat liver

microsomes to convert AD to A and to respond to induction by IC. The identity of

A, the main polar derivative of AD, induced by IC, DIM and ISF, was tentatively

assigned by a combination of GC-MS and results from metabolic studies with

intermediates in the pathway leading to its formation. It is proposed that the

protective role of indole carbinols against mammary carcinoma due to decreased

formation of 16 alpha-hydroxyestrone from estrone may be further enhanced by the

diminished availability of AD for aromatization to estrone.

 

PMID: 8274413

 

Biochem Pharmacol. 1993 Mar 9;45(5):1129-36.

 

Ah receptor binding properties of indole carbinols and induction of hepatic

estradiol hydroxylation.

 

Jellinck PH, Forkert PG, Riddick DS, Okey AB, Michnovicz JJ, Bradlow HL.

 

Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.

 

The effect of route of administration on the ability of indole-3-carbinol (13C),

an anticarcinogen present in cruciferous vegetables, to induce estradiol

2-hydroxylase (EH) in female rat liver microsomes was investigated and compared

to that of its main gastric conversion product, 3,3'-diindolylmethane (DIM). This

dimer was more potent than 13C after either oral or intraperitoneal

administration and was also a better in vitro inhibitor of EH in control and

13C-induced hepatic microsomes. The induction of both CYP1A1 and 1A2 in about

equal amounts by 13C and DIM as well as of CYP2B1/2 was demonstrated using

monoclonal antibodies. DIM, isosafrole, beta-naphthoflavone, 3-methylcholanthrene

and naringenin added in vitro inhibited EH strongly in induced microsomes but

gestodene was a better inhibitor of estrogen 2-hydroxylation in liver microsomes

from untreated female rats. The binding affinities of 13C and DIM to the Ah

receptor were compared to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by

competition studies, and the IC50 values were shown to be 2.0 x 10(-9) M, 5.0 x

10(-5) M and 2.3 x 10(-3) M for TCDD, DIM and 13C, respectively. The ability of

13C or DIM to cause in vitro transformation of the Ah receptor to a form able to

bind to the dioxin-responsive element-3 (DRE3) was compared to that of TCDD and

shown to parallel their abilities to compete for binding of [3H]TCDD to the Ah

receptor. These experiments confirm and extend the proposals that dietary indoles

induce specific cytochrome P450s in rat liver by a mechanism possibly involving

the Ah receptor. The induced monooxygenases, in turn, increase the synthesis of

2-hydroxylated estrogens in the competing pathways of 2- and 16

alpha-hydroxylation which decreases the levels of 16 alpha-hydroxyestrone able to

form stable covalent adducts with proteins including the estrogen receptor. Such

steroid-protein interaction has been correlated with mammary carcinogenesis.

 

PMID: 8384853

 

Food Chem Toxicol. 1992 Jul;30(7):589-99.

 

Effects of indole-3-carbinol on biotransformation enzymes in the rat: in vivo

changes in liver and small intestinal mucosa in comparison with primary

hepatocyte cultures.

 

Wortelboer HM, van der Linden EC, de Kruif CA, Noordhoek J, Blaauboer BJ, van

Bladeren PJ, Falke HE.

 

UTOX, c/o Research Institute of Toxicology (RITOX), University of Utrecht, The

Netherlands.

 

Groups of male Wistar rats were fed semi-synthetic diets containing 0, 200 or 500

mg indole-3-carbinol (13C)/kg for 2, 7, 14 or 28 days. After 2 days, P-450

activities were already induced, but the isoenzyme pattern induced was different

in the liver and the small intestine. Hepatic P4501A1, P4501A2 and P4502B1

apoprotein levels were dose-relatedly enhanced, whereas in the small intestine

induced levels of P4502B1 and P4501A1 were detected but P4501A2 was not induced.

Pentoxy- and ethoxyresorufin dealkylation (PROD and EROD) were dose-relatedly

enhanced in the liver (5- and 7-fold, respectively, in the higher dose group) as

well as in the small intestine (8- and 13-fold, respectively, at 500 mg 13C/kg

diet). Testosterone 16 alpha- and 16 beta-hydroxylation in the small intestine

were enhanced (6-9-fold) from day 2 onwards, but in the liver these activities

were only slightly enhanced from day 7 onwards. Thus, the major forms induced in

the liver appear to be P4501A1, P4501A2, P4502B1 and, to a lesser extent, P4503A,

whereas in the small intestine all of the effects that were found are associated

with only one cytochrome P-450, P4502B1. After 2 days I3C (500 mg/kg) induced

glutathione S-transferase in the liver (1.3-fold) and small intestine (1.5-fold).

Hepatic glucuronyl transferase (GT1) was induced (about 1.6-fold) after 7, 14 and

28 days. DT-diaphorase was induced in the liver (2.7-fold) and small intestine

(1.5-fold) after 14 days of exposure to 500 mg I3C/kg diet. Treatment of rat

hepatocytes with indole-3-acetonitrile and 3,3'-diindolylmethane, but not I3C and

indole-3-carboxaldehyde, enhanced EROD activity and halved testosterone 16 alpha-

and 2 alpha-hydroxylation. All four indoles slightly induced glutathione

S-transferase in cultured hepatocytes. Thus, the in vitro studies suggest that

the in vivo effects of I3C have to be attributed to indole-condensation products,

such as 3,3'-diindolylmethane, but not to I3C itself.

 

PMID: 1521833

  

Chem Biol Interact. 1991;80(3):303-15.

 

Structure elucidation of acid reaction products of indole-3-carbinol: detection

in vivo and enzyme induction in vitro.

 

De Kruif CA, Marsman JW, Venekamp JC, Falke HE, Noordhoek J, Blaauboer BJ,

Wortelboer HM.

 

UTOX, University of Utrecht, The Netherlands.

 

The potency of indole-3-carbinol (I3C) to form condensation products under acidic

aqueous conditions was studied. After identifying a known dimer,

3,3'-diindolylmethane (DIM), we elucidated the structures of two trimers also

found in acid reaction mixtures:

5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b"]tri-indole (CTI), and

2,3-bis[3-indolylmethyl] indole (BII). The formation of these indole oligomers

was shown to be pH dependent. The highest amounts of DIM and BII were formed in

aqueous solutions having a pH value ranging from 4 to 5. No CTI could be detected

at pH values above 4.5. In rats that received an oral dose of I3C we could detect

DIM and BII in gastric contents, stomach tissue, small intestine and liver. No

CTI could be detected in vivo after oral exposure to I3C. In in vitro

experiments, using rat hepatocytes, the cytochrome P-450IA1 apoprotein level,

7-ethoxyresorufin O-deethylation activity (EROD) and DT-diaphorase activity (DTD)

were markedly enhanced by DIM and CTI as well as BII.

 

PMID: 1954658

 

Food Chem Toxicol. 1989 Jun;27(6):385-92.

 

In vivo disposition of the natural anti-carcinogen indole-3-carbinol after po

administration to rainbow trout.

 

Dashwood RH, Uyetake L, Fong AT, Hendricks JD, Bailey GS.

 

Department of Food Science and Technology, Oregon State University, Corvallis

97331.

 

Indole-3-carbinol (I3C), a compound found naturally as a glucosinolate in

cruciferous vegetables such as broccoli and cabbage, has been shown to modulate

the carcinogenic process in a number of animal species. The lack of detailed

information on the disposition of I3C in vivo provided the main impetus for the

study reported here, in which the distribution and metabolic fate of I3C was

assessed in selected tissues and excreta after po administration to rainbow trout

(Salmo gairdneri). Animals were fasted for 3 days and given [5-3H]I3C either in

the diet or by single oral gavage (40 mg/kg body weight; 15 muCi/kg body weight).

Following administration, 75% of the initial 3H-dose was detected within the

stomach between 0.5 and 12 hr, after which it was released to distal regions of

the gut for subsequent uptake, distribution and elimination. At the end of the

study (72 hr) 25% of the administered dose was recovered from the water which

reflected excretion through the gills and urinary tract. Significant excretion

also occurred in the bile, with approximately 5% of the initial 3H-dose recovered

from the bile sacs at 72 hr. Further analyses of the radioactive components in

the bile indicated that one or more derivatives of I3C, but not the parent

compound itself, are excreted as glucuronide conjugates using this route.

Radioactivity accumulated in the liver throughout most of the study, reaching

levels of 1-1.5% between 48 and 72 hr of the administered dose. High-performance

liquid chromatography analyses indicated the presence of four main radiolabelled

species in these livers, one of which co-eluted with the parent compound, I3C.

The major radiolabelled species recovered from the liver was tentatively

identified as the dimer, 3,3'-diindolylmethane (I33'), which comprised 40% of the

total hepatic radiolabel. This dimer, I33', was also found to accumulate in the

diet containing I3C, which reflected a time-dependent dimerization of the parent

compound in vitro. These findings are discussed in view of recent postulates of a

role for I3C condensation products such as I33' in the mechanism of I3C

anti-carcinogenesis.

  

PMID: 2792968

 

Food Chem Toxicol. 1987 May;25(5):363-8.

 

Differential induction of mixed-function oxidase (MFO) activity in rat liver and

intestine by diets containing processed cabbage: correlation with cabbage levels

of glucosinolates and glucosinolate hydrolysis products.

 

McDanell R, McLean AE, Hanley AB, Heaney RK, Fenwick GR.

 

Both white and Savoy-type cabbage added to a semi-purified diet at 25% dry weight

and fed to rats ad lib. for 5 days significantly induced ethoxyresorufin (ERR)

deethylation in the small and large intestine. Savoy cabbage also induced hepatic

activity and, in general, exhibited a greater inducing effect than white cabbage.

These enzyme-inducing effects were altered when the cabbage had been processed.

The content of intact glucosinolate was greater in Savoy than in white cabbage.

The indole glucosinolate (glucobrassicin) content of both types of cabbage was

approximately halved by cooking but was unaffected by fermentation, whilst

homogenization of Savoy cabbage led to the total disappearance of intact

glucosinolates. Levels of the indole glucosinolate breakdown products ascorbigen

and indole-3-carbinol were highest in homogenized cabbage, and ascorbigen levels

were also higher in cooked than in fresh cabbage of either type. When added to

the semi-purified diet and fed ad lib. to rats for 5 days, indole-3-carbinol was

a potent inducer of hepatic ERR deethylation and cytochrome P-450 activity, but

had much less effect in the intestine. Other glucobrassicin metabolites,

diindolylmethane and indole-3-acetonitrile, also had some inducing effect in the

liver but no effect in the intestine, while ascorbigen significantly induced ERR

deethylation in the small and large intestine but had no effect on hepatic MFO

activity.

  

PMID: 3609976

 

J Toxicol Environ Health. 1987;21(3):311-23.

 

Structure-activity relationships of dietary indoles: a proposed mechanism of

action as modifiers of xenobiotic metabolism.

 

Bradfield CA, Bjeldanes LF.

 

In an effort to understand the mechanism by which dietary indoles inhibit

chemically initiated tumorigenesis in experimental animals, we have investigated

the potency of 3-substituted and 1,3-disubstituted indoles on the induction of

intestinal and hepatic cytochrome P-448-dependent monooxygenases in the rat. Oral

intubation with indole-3-carbinol (13C), 1-methoxyindole-3-carbinol (N13C),

1-methoxyindole-3-carboxaldehyde (NCHO), and 3,3'-diindolylmethane (133') at 31

mumol/animal led to significant increases in hepatic ethoxyresorufin O-deethylase

activity (EROD; 15, 7, 6, and 5-fold over control, respectively), while

intubation with indole (IND), 3-methylindole (3MI), indole-3-carboxaldehyde

(13CHO), and indole-3-acetonitrile (IAN) did not increase this monooxygenase

activity over control levels. For the eight indoles tested, there was a strong

relationship between instability in acidic solution, as indicated by the

generation of insoluble products, and capacity to induce hepatic EROD. Further

experiments indicated that 13C did not induce hepatic EROD when dosed ip (thus

bypassing the acidity of the stomach). Acid treatment of 13C generated a reaction

mixture (RXM) that induced EROD after ip or po dosing. Chromatographic

fractionation of the RXM indicated that there exist at least four different 13C

acid-condensation products in the RXM with the ability to induce EROD. The

results presented strongly support the hypothesis that dietary indoles influence

the levels of monooxygenase activities via a series of acid-condensation products

generated upon introduction of the indole into the acidic environment of the

stomach.

 

PMID: 3495667

 

Cancer Res. 1978 May;38(5):1410-3.

 

Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally

occurring indoles.

 

Wattenberg LW, Loub WD.

 

Indole-3-carbinol, 3,3'-diindolylmethane, and indole-3-acetonitrile, three

indoles occurring in edible cruciferous vegetables, have been studied for their

effects on 7,12-dimethylbenz(a)anthracene-induced mammary tumor formation in

female Sprague-Dawley rats and on benzo(a)pyrene-induced neoplasia of the

forestomach in female ICR/Ha mice. When given by p.o. intubation 20 hr prior to

7,12-dimethylbenz(a)anthracene administration, indole-3-carbinol and

3,3'-diindolylmethane had an inhibitory effect on mammary tumor formation, but

indole-3-acetonitrile was inactive. Indole-3-carbinol when added to the diet for

8 days prior to challenge with 7,12-dimethylbenz(a)anthracene inhibited mammary

tumor formation, whereas indole-3-acetonitrile did not. Dietary administration of

all three indoles inhibited benzo(a)pyrene-induced neoplasia of the forestomach

in ICR/Ha mice. The identification of dietary constituents that can inhibit

chemical carcinogens ultimately may be of value in understanding the balance of

factors that determines the neoplastic response to these cancer-producing agents

in the environment.

 

PMID: 416908

 

Fed Proc. 1976 May 1;35(6):1327-31.

 

Dietary constituents altering the responses to chemical carcinogens.

 

Wattenberg LW, Loub WD, Lam LK, Speier JL.

 

This paper deals with two categories of compounds having the capacity to inhibit

the neoplastic effects of chemical carcinogens on the host. The first are

inducers of increased microsomal mixed function oxidase activity. An increasing

number of these inducers are being found in natural products. Cruciferous

vegetables including brussels sprouts, cabbage, and cauliflower contain such

compounds. Recently indole-3-acetonitrile, indole-3-carbinol and

3,3'-diindolylmethane have been identified as inducers in these three plants.

Other naturally occurring inducers include flavones, safrole, isosafrole,

beta-ionone, and oxidized sterols. Since previous work has shown that synthetic

inducers may protect against chemical carcinogens, the composition of the diet

could play a role in inhibiting the neoplastic response to these carcinogenic

agents. The second category of inhibitors comprises the antioxidants. Several of

these compounds have been found to inhibit the carcinogenic effects of a variety

of chemical carcinogens. Considerable work of this nature has been done with

butylated hydroxyanisole and butylated hydroxytoluene two antioxidants

extensively used as food additives. Other antioxidants having carcinogen

inhibiting capacities include ethoxyquin, disulfiram, and

dimethyldithiocarbamate.

 

PMID: 1261715

 

Cancer Res. 1975 Nov;35(11 Pt. 2):3326-31.

 

Effects of dietary constituents on the metabolism of chemical carcinogens.

 

Wattenberg LW.

 

Dietary constituents of 2 types have been shown to affect the metabolism of

chemical carcinogens by the microsomal mixed-function oxidase system. Naturally

occurring inducers of increased activity of this system are present in plants.

Cruciferous vegetables including Brussels sprouts, cabbage, and cauliflower are

relatively potent in this regard. From these vegetables, three indoles with

inducing activity have been identified. These are indole-3-acetonitrile,

indole-3-carbinol, and 3,3'-diindolylmethane. A 2nd type of dietary constituent

affecting the microsomal mixed-function oxidase system is added phenolic

antioxidant, i.e., butylated hydroxyanisole (BHA) and butylated hydroxytoluene.

Studies of the effect of BHA on metabolism of bezo(a)-pyrene by liver microsomes

have been carried out. BHA feeding results in microsomal changes. The cytochrome

P-450 shows altered spectral characteristics, and the aryl hydrocarbon

hydroxylase system of these microsomes has an increased sensitivity to inhibition

by alpha-naphthoflavone. In addition, there is a decrease in binding of

metabolites of benzo(a)pyrene to DNA upon incubation of these microsomes of

induction of increased mixed function oxidase activity have shown that increased

levels of activity protect against administration of chemical carcinogens. BHA

and butylated hydroxytoluene also have been found to exert a protective effect

against chemical carcinogens. Thus the constituents of the diet could be of

consequence in the neoplastic response to exposure to carcinogens in the

environment.

 

PMID: 1104144

 

J Natl Cancer Inst. 1975 Apr;54(4):985-8.

 

Aryl hydrocarbon hydroxylase induction in rat tissues by naturally occurring

indoles of cruciferous plants.

 

Loub WD, Wattenberg LW, Davis DW.

 

A phytochemical investigation to identify inducers of increased aryl hydrocarbon

hydroxylase (AHH) activity from three cruciferous vegetables, Brussels sprouts,

cabbage, and cauliflower, resulted in the identification of

indole-3-acetonitrile, indole-3-carbinol, and 3,3'-diindolylmethane as naturally

occurring inducers. These compounds are produced during the hydrolysis of

indolyl-methyl glucosinolate by the plant enzyme myrosinase.

 

PMID: 1127728

 

 

 

© 2007-2021 Diindolylmethane Information Resource Center