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Scientific References on Diindolylmethane Dating Back to 1975

This section is updated on a monthly basis. Paper abstracts are presented in chronological order from most recent.

J Nutr. 2008 Jan;138(1):17-23.

3,3'-Diindolylmethane 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 [PubMed - indexed for MEDLINE]

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 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 [PubMed - indexed for MEDLINE]

J Nutr Biochem. 2007 Aug 16 [Epub ahead of print]

3,3'-Diindolylmethane 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 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 [PubMed - indexed for MEDLINE]

Mol Med. 2007 Jan-Feb;13(1-2):69-78.

Interplay of genes regulated by estrogen and diindolylmethane 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 [PubMed - indexed for MEDLINE]

Cancer Res. 2007 Apr 1;67(7):3310-9.

Inhibition of angiogenesis and invasion by 3,3'-diindolylmethane 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 [PubMed - indexed for MEDLINE]

Carcinogenesis. 2007 Jul;28(7):1471-7. Epub 2007 Feb 28.

Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane 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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

Am J Pathol. 2006 Nov;169(5):1833-42.

Fas-mediated apoptosis in cholangiocarcinoma cells is enhanced by 3,3'-diindolylmethane 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 [PubMed - indexed for MEDLINE]

Cancer Res. 2006 Oct 15;66(20):10064-72.

Down-regulation of androgen receptor by 3,3'-diindolylmethane 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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

Cancer Res. 2006 May 1;66(9):4880-7.

3,3'-Diindolylmethane 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 [PubMed - indexed for MEDLINE]

J Surg Res. 2006 May 15;132(2):208-13. Epub 2006 Mar 31.

3,3'-diindolylmethane 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 [PubMed - indexed for MEDLINE]

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.

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 [PubMed - indexed for MEDLINE]

Chem Res Toxicol. 2006 Mar;19(3):436-42.

Fate of 3,3'-diindolylmethane 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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

Mol Pharmacol. 2006 Apr;69(4):1320-7. Epub 2005 Dec 29.

3,3'-diindolylmethane 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 [PubMed - indexed for MEDLINE]

Biochem Biophys Res Commun. 2006 Feb 10;340(2):718-25. Epub 2005 Dec 20.

3,3'-Diindolylmethane 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 [PubMed - indexed for MEDLINE]

Mol Cancer Ther. 2005 Dec;4(12):1972-81.

Down-regulation of c-FLIP contributes to the sensitization effect of 3,3'-diindolylmethane 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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

Biochem Biophys Res Commun. 2005 Nov 25;337(3):1019-25. Epub 2005 Oct 3.

3,3'-Diindolylmethane, 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 [PubMed - indexed for MEDLINE]

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.

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 [PubMed - indexed for MEDLINE]

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, 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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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 [PubMed - indexed for MEDLINE]

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