Diindolylmethane Research Articles

Abstract 5419: Effects of selective aryl hydrocarbon receptor modulators (SAhRMs) in enhancing activity of tamoxifen against doxorubicin resistant MCF-7 Cells: Role of BRCA1

Abstract Recent reports indicate that SAhRMs such as diindolylmethane (DIM) inhibit α-estrogen receptor (ER) expression and estrogen signaling in human breast cancer cells. There is also increasing evidence that SAhRMs demonstrate anti estrogenic and antitumorigenic activity in ER positive human breast adenocarcinoma (MCF-7) cells via cross talk between aromatic hydrocarbon receptor (AhR) and ER pathways. Additional reports suggest that BRCA1 transcriptional activity is enhanced by interactions with AhR and the levels of BRCA1 expression play a role in tamoxifen sensitivity/resistance in breast cancer cells by altering estrogen receptor-coregulator interactions. However, the effects of AhR and its agonists and antagonists, and their modulatory effect on BRCA1 in resistant human breast cancer cells have not been investigated. We have tested the hypothesis that DIM, an AhR agonist may inhibit cell growth in a concentration and time dependant manner against doxorubicin resistant MCF-7 (MCF-7/dox) cells and may enhance the activity of 4-OH tamoxifen (TAM) against both MCF-7 and MCF-7/dox cells by enhancing the expression of BRCA1. Initially we determined the cytotoxic effect of DIM against the two cell lines by utilizing the MTT assay. Both DIM and TAM were equally effective against MCF-7 and MCF-7/dox cells with the IC50 values of approximately 90 μM and 20 μM, respectively at 72 hr. However, a combination of DIM and TAM resulted in an increase in cytotoxcity and statistically significant lowering of the IC50 concentration to 50 μM and 10 μM for each drug, respectively. We also investigated the effects of AhR antagonist, α-naphthoflavone (NF) which had a similar IC 50 value of approximately 112 μM against both these cell lines, whereas a combination of NF and TAM required only half the concentrations of 50 μM and 10 μM to inhibit 50 % of the cell growth of each cell line. Since BRCA1, a tumor suppressor gene, has been implicated in the mechanism of tamoxifen induced growth inhibition, we determined the expression of this protein upon exposure of these cells to DIM. Results from western blot analysis show that there is a significant increase in BRCA1 and AhR protein levels in these cells upon treatment with DIM when compared to protein levels from untreated MCF-7 and MCF-7 /dox cells. Thus activation of BRCA1 by SAhRMs appears to be one of the mechanisms responsible for enhanced growth inhibitory effect of tamoxifen against these cells. These data suggest that either an AhR agonist or an antagonist can be conveniently employed in combination with TAM to increase the activity of TAM against both MCF-7 cells and doxorubicin resistant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5419.

Abstract 5655: 3,3′-Diindolylmethane selectively induces proteasomal degradation of the class I histone deacetylases

Abstract This study aims to identify the molecular target of 3,3′-Diindolylmethane (DIM), an anti-cancer agent that induces cell cycle arrest and apoptosis through unknown mechanisms. Previously, we have shown that DIM can enhance the efficacy of butyrate in colon cancer prevention in the APCmin/+ mouse model [Cancer Prevention Research, 2(6):581-9, 2009]. In our studies trying to identify the intracellular target of DIM, we found that DIM selectively induced proteasome-mediated degradation of the class I histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC8) in colon cancer cells, without affecting other HDAC proteins (class II or class III HDACs). DIM-induced down-regulation of the class I HDACs was also observed in vivo in tumor xenografts in nude mice. The depletion of the HDACs by DIM caused a reduction of the HDAC activity on the promoters of CDKN1A/WAF1/CIP1 and CDKN1B/KIP1 genes (encoding cyclin-dependent kinase inhibitors p21 and p27, respectively) and significantly increased the expression of p21 and p27, which arrested cells at the G2 phase of the cell cycle. The degradation of the HDACs also caused DNA damage and triggered apoptosis. Based on these data, we conclude that DIM acts by selectively targeting the class I HDACs to promote their degradation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5655.

Abstract 5683: The dietary anti-estrogenic agent 3,3′-diindolylmethane inhibits metastatic propensity in thyroid cancer cells

Abstract The incidences of thyroid proliferative diseases (TPD) including thyroid cancer and goiter are ever increasing with thyroid cancer being the most common among endocrine cancers. Recent statistics reveal 37,000 new cases diagnosed in US alone in 2008 and worldwide almost 27 million patients are affected. Diet has always been of prime importance in its association with cancer development and prevention. Several studies have found inverse association of cancer risk with consumption of dietary products, such as tomatoes, soy and cruciferous vegetables. We have been studying the effect of DIM (3,3′-diindolylmethane), a chemopreventive agent against hormone responsive cancers. In this study we show that thyroid cells express functional estrogen receptor (ER) -α and ER-β and DIM has anti-cancer effects on estrogen responsive thyroid cancer cells. Interestingly, we found that DIM does not affect the levels of ER but it does target metastatic phenotype of thyroid cells as evidenced by decreased adhesion, invasion and migration in vitro. In an attempt to define the cellular target of this inhibition, we observed that DIM mediates down-regulation of matrix metalloproteinases (MMPs) and cell adhesion proteins, integrins. This correlates with decreased thyroid cell proliferation and inhibition of metastatic propensity by DIM in thyroid cancer cells. These preclinical studies are being extended to the clinic where the anti-estrogenic property of DIM is being examined in goiter patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5683.

3,3′‐Diindolylmethane (DIM) inhibits crosstalk between DU145 prostate cancer cells and THP‐1 monocytes in vitro and in vivo

Tumor‐associated macrophages are known to influence neoplastic growth and progression by providing a rich supply of cytokines, proteases, and growth factors. The present study examined the crosstalk between prostate cancer cells and macrophages and whether DIM inhibits these interactions. Our in vitro results revealed that the migration of DU145 cells through collagen‐coated filter paper was markedly increased when THP‐1 cells were co‐cultured in the well under the filter paper. In addition, the DU145 cell‐conditioned medium stimulated the migration of THP‐1 cells. DIM inhibited these migrations of DU145 and THP‐1 cells. The secretion of IL‐6 and IL‐8 were markedly increased when DU145 cells were co‐cultured with THP‐1 cells and DIM inhibited the secretion of these cytokines. Solid tumor growth was markedly accelerated when nude mice were co‐injected with DU145 plus THP‐1 cells compared to DU145 cells alone, and DIM inhibited the tumor growth in mice co‐injected with both cells. Immunohistochemistry of tumor tissues revealed that the expression of CD31 (an endothelial marker), vascular endothelial growth factor, and Ki67 was increased in the co‐inoculation group, which was inhibited by DIM treatment. The present results indicate that interactions between DU145 and THP‐1 cells induce angiogenesis and the migration and growth of these cells and DIM inhibits these effects.

3,3′-Diindolylmethane (DIM) inhibits the growth and invasion of drug-resistant human cancer cells expressing EGFR mutants

Epidermal Growth Factor Receptor (EGFR) mutants are associated with resistance to chemotherapy, radiation, and targeted therapies. Here we found that the phytochemical 3,3′-Diindolylmethane (DIM) can inhibit the growth and also the invasion of breast cancer, glioma, and non-small cell lung cancer cells regardless of which EGFR mutant is expressed and the drug-resistant phenotype. DIM reduced an array of growth factor signaling pathways and altered cell cycle regulators and apoptotic proteins favoring cell cycle arrest and apoptosis. Therefore, DIM may be used in treatment regimens to inhibit cancer cell growth and invasion, and potentially overcome EGFR mutant-associated drug resistance.

P26 Modulation of mRNA and protein levels of CYP1A1, 1A2, and 1B1 in nontumorigenic breast epithelial cells (MCF10A) by cabbage juice and its active components

3,3′-diindolylmethane (DIM) is a dimer compound converted from Indoly-3-carbinol that had been studied as promising chemo-preventive agent against breast cancer. In this study, we observed that proportion of CD133+Nanog+ subpopulation in MCF-7 cells was significantly increased after DIM administration with up-regulated AKT activity by using CyTOF assay. SPADE analysis revealed this stem-like subpopulation exhibited apoptosis-resistance property against DIM treatment. By combining with AKT inhibitor AZD5363, DIM induced CD133 expression could be suppressed. In addition, a combination treatment of MCF-7 and MDA-MB-231 breast cancer cells with DIM and AZD5363 showed synergistic decreases in cell proliferation and induced apoptosis. Furthermore, results from imaging flow cytometry suggested that FoxO3a nuclear localization and PUMA expression could be improved by combination of AZD5363 with DIM. Taken together, the above observations suggested that the combination of AZD5363 with DIM could be developed as potential therapy for breast cancer.

The p38 MAPK pathway is critical for 5,5′-dibromodiindolylmethane-induced apoptosis to prevent oral squamous carcinoma cells

Cruciferous vegetables contain isothiocyanates including diindolylmethane (DIM) that exhibit cancer chemopreventive effects. We developed a series of synthetic ring-substituted DIM analogs including 5,5'-dibromoDIM that exhibited better inhibitory activity in breast and colon cancer cells than DIM. In this study, we investigated whether 5,5'-dibromoDIM inhibits the proliferation of KB and YD-10B oral squamous carcinoma cell lines. 5,5'-dibromoDIM decreased the cell survival and inhibited the growth of oral cancer cells. Exposure of KB and YD-10B cells to 5,5'-dibromoDIM induced caspase-dependent apoptosis evidenced by poly-ADP ribose polymerase cleavage, accumulation of sub-G1 population, and nuclear condensation and fragmentation. In addition, apoptotic cell death was correlated with damage to the mitochondrial membrane potential through a decrease in the level of Bcl-2 protein expression. Mechanistic studies showed that mitochondria-dependent apoptosis induced by 5,5'-dibromoDIM was mediated by the p38 mitogen-activated protein kinase pathway but not the ERK1/2 and JNK pathway. These results highlight 5,5'-dibromoDIM as an important chemopreventive agent for the clinical treatment of oral cancer through the p38 mitogen-activated protein kinase pathway.

Selective Activation of Estrogen Receptor-β Target Genes by 3,3′-Diindolylmethane

3,3'-Diindolylmethane (DIM) is a natural compound found in cruciferous vegetables that has antiproliferative and estrogenic activity. However, it is not clear whether the estrogenic effects are mediated through estrogen receptor (ER)alpha, ERbeta, or both ER subtypes. We investigated whether DIM has ER subtype selectivity on gene transcription. DIM stimulated ERbeta but not ERalpha activation of an estrogen response element upstream of the luciferase reporter gene. DIM also selectively activated multiple endogenous genes through ERbeta. DIM did not bind to ERbeta, indicating that it activates genes by a ligand-independent mechanism. DIM causes ERbeta to bind regulatory elements and recruit the steroid receptor coactivator (SRC)-2 coactivator, which leads to the activation of ER target genes. Silencing of SRC-2 inhibited the activation of ER target genes, demonstrating that SRC-2 is required for transcriptional activation by DIM. Our results demonstrate that DIM is a new class of ERbeta-selective compounds, because it does not bind to ERbeta, but instead it selectively recruits ERbeta and coactivators to target genes.

Chemopreventive Agent 3,3′-Diindolylmethane Selectively Induces Proteasomal Degradation of Class I Histone Deacetylases

3,3'-Diindolylmethane (DIM) is an anticancer agent that induces cell cycle arrest and apoptosis through unknown mechanisms. Here, we report that DIM can selectively induce proteasome-mediated degradation of class I histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC8) without affecting the class II HDAC proteins. DIM induced downregulation of class I HDACs in human colon cancer cells in vitro and in vivo in tumor xenografts. HDAC depletion relieved HDAC-mediated transcriptional inhibition of the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP2, significantly increasing their expression and triggering cell cycle arrest in the G(2) phase of the cell cycle. Additionally, HDAC depletion was associated with an induction of DNA damage that triggered apoptosis. Our findings indicate that DIM acts to selectively target the degradation of class I HDACs.

Abstract PR-06: Upregulation of microRNA-21 (miR-21) in human bronchial epithelial cells chronically exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus nicotine and modulation of these effects by diindolylmethane

Abstract MicroRNAs (miRNAs) represent a class of naturally occurring small noncoding RNA molecules that regulate genes by either inducing mRNA degradation or inhibiting translation. The expression of miRNAs is deregulated in several cancers, including lung cancer. However, most studies have been carried out using full-blown cancer tissues and the regulation of miRNAs during early-stage tumorigenesis is unknown. In the present study, we used an in vitro model of lung tumorigenesis to assess the effect of chronic treatment with tobacco compounds on the expression of miR-21, one of the most deregulated miRNAs in lung cancer, in human bronchial epithelial cells (HBEC). Freshly prepared NNK (10 µM) and nicotine (10 µM) were added to the culture media containing NHBC every 3 days for 3 weeks. Beginning the second week of NNK plus nicotine treatment, the dietary chemopreventive agent diindolylmethane (DIM) was added, every 3 days, to the culture media until the termination of the study. Then, the cells were harvested, RNA prepared and the expression level of miR-21 and its target genes PDCD4 and RECK were analyzed by QRT-PCR. Levels of PDCD4 and RECK were also examined by Western immunoblotting. NNK plus nicotine treatment significantly elevated miR-21 levels but markedly decreased expression of PDCD4 and RECK at both gene and protein levels. The effect of the tobacco compounds on miR-21 as well PDCD4 and RECK was reversed by treatment with DIM. This study shows that miR-21 levels are deregulated beginning the early phase of lung tumorigenesis and miR-21 is a valuable target for chemopreventive agents. Citation Information: Cancer Prev Res 2010;3(1 Suppl):PR-06.

Antiangiogenic Effects of Indole-3-Carbinol and 3,3'-Diindolylmethane Are Associated with Their Differential Regulation of ERK1/2 and Akt in Tube-Forming HUVEC1–3

We previously reported that indole-3-carbinol (I3C), found in cruciferous vegetables, suppresses angiogenesis in vivo and in vitro. However, the underlying molecular mechanisms still remain unclear. Antiangiogenic effects of its major metabolite, 3,3′-diindolylmethane (DIM), also have not been fully elucidated. In this study, we investigated the effects of these indoles on angiogenesis and tested a hypothesis that I3C and DIM inhibit angiogenesis and induce apoptosis by affecting angiogenic signal transduction in human umbilical vein endothelial cells (HUVEC). We found that I3C and DIM at 25 μmol/L significantly inhibited tube formation and only DIM induced a significant increase in apoptosis in tube-forming HUVEC. DIM showed a stronger antiangiogenic activity than I3C. At the molecular level, I3C and DIM markedly inactivated extracellular signal-regulated kinase 1/2 (ERK1/2) and the inhibitory effect of DIM was significantly greater than that of I3C. DIM treatment also resulted in activation of the caspase pathway and inactivation of Akt, whereas I3C did not affect them. These results indicate that I3C and DIM had a differential potential in the regulation of the 2 principal survival signals, ERK1/2 and Akt, in endothelial cells. We also demonstrated that pharmacological inhibition of ERK1/2 and/or Akt was enough to inhibit tube formation and induce caspase-dependent apoptosis in tube-forming HUVEC. We conclude that both I3C and DIM inhibit angiogenesis at least in part via inactivation of ERK1/2 and that inactivation of Akt by DIM is responsible for its stronger antiangiogenic effects than those of I3C.

3,3'-Diindolylmethane Inhibits Activation of Akt through Inhibition of Hepatocyte Growth Factor Receptor c-Met Signaling.

Abstract Background: 3,3'-Diindolylmethane (DIM), the major acid condensation product of indole-3-carbinol, is a promising anticancer compound that is bioavailable, non-toxic, and easily obtainable. Although DIM has strong anticancer activity against breast cancer cells in vitro and in vivo, the exact mechanism for this activity is not known. We and others have found that DIM inhibits activation of the Akt pathway in breast cancer cells. Akt is a serine threonine protein kinase involved in many important cellular functions including proliferation and cell survival. Akt is active when phosphorylated at serine-473 and threonine-308. In breast cancer cells, Akt can be activated by various stimuli, including signaling from growth factor receptors like the hepatocyte growth factor (HGF) receptor c-Met, which is often overexpressed in breast cancer. In this project, we show that c-Met is an upstream point of inhibition of the Akt pathway by DIM.Materials and Methods: When MDA-MB-231 breast cancer cells reached 70-80% confluency, we serum-starved them by reducing the fetal bovine serum concentration in the media from 10% to 1% for 20 hours and then treated with DIM or the vehicle control DMSO for 4 hours or the indicated time. When using recombinant growth factors to activate Akt or c-Met, we incubated cells in medium containing no fetal bovine serum and added the growth factors for 10 minutes after incubation with DIM. To analyze protein expression and protein phosphorylation we performed Western blotting. To analyze c-Met kinase activity we used a commercially available kit.Results: DIM does not inhibit activation of Akt downstream of either epidermal growth factor or insulin-like growth factor-1. However, DIM does inhibit Akt activation downstream of HGF and of fetal bovine serum. DIM inhibits tyrosine phosphorylation of c-Met at Y1349 and Y1234/1235. DIM does not affect total c-Met protein or mRNA levels and does not directly inhibit c-Met kinase activity.Discussion: While we have determined that DIM inhibits c-Met phosphorlyation, and therefore HGF signaling, the mechanism of this inhibition requires further study. We hypothesize that DIM induces serine phosphorylation of c-Met, since c-Met can be inhibited by reactive oxygen species (ROS)-induced phosphorylation of serine-985 by protein kinase C (PKC), and DIM increases ROS levels in cells.Components of the Akt pathway are frequent targets for mutations in breast cancer and c-Met and its ligand HGF are associated with lower survival and increased risk of distant metastases in breast cancer. This project represents an important step in determining the mechanism of action for DIM, a promising phytochemical for the treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6117.

Oral Administration of 3,3’-Diindolylmethane Inhibits Lung Metastasis of 4T1 Murine Mammary Carcinoma Cells in BALB/c Mice1–3

3,3’-diindolylmethane (DIM) is the major in vivo product of the acid-catalyzed oligomerization of indole-3-carbinol present in cruciferous vegetables, and it has been shown to exhibit anticancer properties. In this study, we assessed the effects of DIM on the metastasis of 4T1 mouse mammary carcinoma cells. In vitro culture studies showed that DIM dose-dependently inhibited the migration, invasion, and adhesion of 4T1 cells at concentrations of 0–10 μmol/L without attendant changes in cell viability. In an in vivo lung metastasis model, 4T1 cells (2 × 105 cells/mouse) were injected into the tail veins of syngeneic female BALB/c mice. Beginning on the second day, the mice were subjected to gavage with 0–10 mg DIM/(kg body weight · d) for 13 d. Oral DIM administration resulted in a marked reduction in the number of pulmonary tumor nodules. DIM treatment significantly reduced the levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and vascular cell adhesion molecule (VCAM)-1 and increased TIMP-2 levels in the sera and lungs of mice injected with 4T1 cells. Additionally, DIM treatment reduced the serum concentrations of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)a. We have demonstrated that DIM profoundly inhibits the lung metastasis of 4T1 cells, which was accompanied by reduced levels of MMP, adhesion molecules, and proinflammatory cytokines. These results indicate that DIM has potential as an antimetastatic agent for the treatment of breast cancer.

Oral diindolylmethane (DIM): Pilot evaluation of a nonsurgical treatment for cervical dysplasia

Objective Standard surgical treatment for CIN may impair fertility generating a need for alternative treatment options. We tested the efficacy and toxicity of oral DIM in the treatment of CIN 2 or 3 lesions. Methods Patients with biopsy-proven cervical intraepithelial neoplasia (CIN) 2 or 3 scheduled for loop electrosurgical excision procedure (LEEP) were randomized 2:1 to receive diindolylmethane (DIM) (BioResponse-DIM®, BioResponse, Boulder, CO) orally at approximately 2 mg/kg/day for 12 weeks or placebo (defatted rice bran, BioResponse). Subjects were evaluated every 3–4 months for 1 year. Analysis of data up to 1 year was assessed including Pap smear, HPV, colposcopy, biopsy and physical examination were performed at follow-up. Central pathology review confirmed all histology diagnoses. Results To date, 64 subjects (mean age 28 years, range 18–61) have been enrolled (45 in the DIM arm, 19 in the placebo arm), with 60 available for analysis. Average follow-up was 6 months. At enrollment, 58% were diagnosed with CIN 2 and 42% with CIN 3, 57% of subjects were Caucasian, 15% African American, 12% Hispanic and 17% Asian. During treatment 2 subjects (3%) complained of nausea (grade 2) at the 3- to 4-month visit. No systemic toxicities were observed (normal CBC, LFTs, comprehensive metabolic). Forty-six subjects had biopsies at first follow-up (77%). Twenty-one subjects (47%) in the DIM group had improved CIN with a decrease by 1–2 grades or a normal result. Median time to improvement was 5 months. Improved Pap smear was seen in 49% (22/45) with either a less severe abnormality or normal result. Colposcopy improved in twenty-five subjects in the DIM group (56%). Of these 25 subjects, 21 (84%) had improved colposcopic impression, 13 (52%) had a decrease in involved quadrants and 18 (72%) had a decrease in lesion number. Complete colposcopic response was observed in 4 subjects (9%). Stratifying by level of dysplasia, age, race, HPV status, tobacco use, contraceptive used did not alter the results. At median follow-up of 6 months, 85% of subjects have not required LEEP based on routine clinical triage of improving global assessment. There was no statistically significant difference in any outcome between the DIM and placebo group. Conclusion Oral DIM at 2 mg/kg/day is well tolerated with no significant toxicity. We observed a high rate of clinically significant improvement in confirmed CIN 2 or 3 lesions among both treatment groups in this randomized clinical trial.

Diindolylmethane Inhibits Cervical Dysplasia, Alters Estrogen Metabolism, and Enhances Immune Response in the K14-HPV16 Transgenic Mouse Model

This study was designed to establish whether 3,3'-diindolylmethane (DIM) can inhibit cervical lesions, alter estrogen metabolism in favor of C-2 hydroxylation, and enhance immune function in the K14-HPV16 transgenic mouse model. Mice were bred, genotyped, implanted with E(2) pellets (0.25 mg/90-day release) under anesthesia, and divided into groups. Wild-type and transgenic mice were given either AIN76A diet alone or with 2,000 ppm DIM for 12 weeks. Blood and reproductive tracts were obtained. Blood was analyzed for estrogen metabolites and IFN-gamma. The cervical transformation zone was sectioned and stained for histology. Estradiol C-2 hydroxylation and serum IFN-gamma levels were significantly increased over controls in wild-type and transgenic mice receiving DIM. In wild-type mice without DIM, hyperplasia of the squamous epithelium was observed. Wild-type mice fed DIM displayed a normal thin epithelium. In transgenic mice without DIM, epithelial cell projections into the stroma (papillae) were present. An additional degree of nuclear anaplasia in the stratum espinosum was observed. Dysplastic cells were present. Transgenic mice fed DIM displayed some mild hyperplasia of the squamous epithelium. DIM increases estrogen C-2 hydroxylation in this model. Serum INF-gamma was increased, indicating increased immune response in the DIM-fed animals. Histopathology showed a marked decrease in cervical dsyplasia in both wild-type and transgenic mice, indicating that DIM delays or inhibits the progression from cervical dysplasia to cervical cancer. Using the K14-HPV16 transgenic mouse model, we have shown that DIM inhibits the development of E6/E7 oncogene-induced cervical lesions.

3,3′-Diindolylmethane attenuates experimental arthritis and osteoclastogenesis

3,3′-Diindolylmethane (DIM) is a natural compound formed during the autolysis of glucobrassicin present in Brassica food plants. This study aimed to investigate the therapeutic efficacies of DIM on experimental arthritis. The effects of DIM on experimental arthritis were examined on a rat model of adjuvant-induced arthritis (AIA), with daily AIA paw swelling observation and histological/radiographic analysis. To elucidate the possible mechanisms of its action, serum cytokine levels as well as the expression of receptor activator for nuclear factor κ B ligand (RANKL) in infected tissues were subsequently analyzed. The impact of DIM on osteoclastogenesis was further investigated on a mouse model of endotoxin-induced bone resorption (EIBR) and in vitro cultures of fibroblast-like cells and osteoblasts, with RANKL expression being evaluated with great interest. The administration of DIM was demonstrated to attenuate AIA in animal models, as judged by clinical and histologic indices of inflammation and tissue damage. On the one hand, DIM could reduce the expression of several inflammatory cytokines, which was, however, not adequate to prevent the development of the arthritis. On the other hand, DIM was shown to effectively inhibit the expression of RANKL, leading to the blockade of osteoclastogenesis and consequently an alleviation of experimental arthritis. Further in vitro and in vivo studies confirmed the inhibition of RANKL by DIM. DIM has shown anti-arthritis activity in animal models via inhibiting the expression of RANKL, and thus may offer potential treatments for arthritis and associated disorders.

Modulation of Aflatoxin B1–Mediated Genotoxicity in Primary Cultures of Human Hepatocytes by Diindolylmethane, Curcumin, and Xanthohumols

This study employed cultured human primary hepatocytes to investigate the ability of the putative chemopreventive phytochemicals curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), or 8-prenylnaringenin (8PN) to reduce DNA adduct formation of the hepatocarcinogen aflatoxin B1 (AFB). Following 48 h of pretreatment, DIM and 8PN significantly increased AFB-DNA adduct levels, whereas CUR and IXN had no effect. DIM greatly enhanced the transcriptional expression of cytochrome P450 (CYP) 1A1 and CYP1A2 mRNA. Glutathione S-transferase mRNAs were not increased by any of the treatments. In vitro enzyme activity assays demonstrated that 8PN and DIM, but not CUR or IXN, inhibited human CYP1A1, CYP1A2, and CYP3A4 activities. To distinguish between treatment effects on transcription versus direct effects on enzyme activity for DIM, we evaluated the effects of pretreatment alone (transcriptional activation) versus cotreatment alone (enzyme inhibition). The results demonstrated that effects on gene expression, but not catalytic activity, are responsible for the observed effects of DIM on AFB-DNA adduct formation. The increase in AFB-DNA damage following DIM treatment may be explained through its substantial induction of CYP1A2 and/or its downregulation of GSTM1, both of which were significant. The increase in DNA damage by DIM raises potential safety risks for dietary supplements of DIM and its precursor indole-3-carbinol.

Down‐regulation of uPA and uPAR by 3,3′‐diindolylmethane contributes to the inhibition of cell growth and migration of breast cancer cells

3,3'-Diindolylmethane (DIM) is a known anti-tumor agent against breast and other cancers; however, its exact mechanism of action remains unclear. The urokinase plasminogen activator (uPA) and its receptor (uPAR) system are involved in the degradation of basement membrane and extracellular matrix, leading to tumor cell invasion and metastasis. Since uPA-uPAR system is highly activated in aggressive breast cancer, we hypothesized that the biological activity of B-DIM could be mediated via inactivation of uPA-uPAR system. We found that B-DIM treatment as well as silencing of uPA-uPAR led to the inhibition of cell growth and motility of MDA-MB-231 cells, which was in part due to inhibition of VEGF and MMP-9. Moreover, silencing of uPA-uPAR led to decreased sensitivity of these cells to B-DIM indicating an important role of uPA-uPAR in B-DIM-mediated inhibition of cell growth and migration. We also found similar effects of B-DIM on MCF-7, cells expressing low levels of uPA-uPAR, which was due to direct down-regulation of MMP-9 and VEGF, independent of uPA-uPAR system. Interestingly, over-expression of uPA-uPAR in MCF-7 cells attenuated the inhibitory effects of B-DIM. Our results, therefore, suggest that B-DIM down-regulates uPA-uPAR in aggressive breast cancers but in the absence of uPA-uPAR, B-DIM can directly inhibit VEGF and MMP-9 leading to the inhibition of cell growth and migration of breast cancer cells.

Up-regulation of miR-200 and let-7 by Natural Agents Leads to the Reversal of Epithelial-to-Mesenchymal Transition in Gemcitabine-Resistant Pancreatic Cancer Cells

Pancreatic cancer is the fourth most common cause of cancer death in the United States, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Emerging evidence also suggests that the processes of EMT are regulated by the expression status of many microRNAs (miRNA), which are believed to function as key regulators of various biological and pathologic processes during tumor development and progression. In the present study, we compared the expression of miRNAs between gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells and investigated whether the treatment of cells with "natural agents" [3,3'-diindolylmethane (DIM) or isoflavone] could affect the expression of miRNAs. We found that the expression of miR-200b, miR-200c, let-7b, let-7c, let-7d, and let-7e was significantly down-regulated in gemcitabine-resistant cells, which showed EMT characteristics such as elongated fibroblastoid morphology, lower expression of epithelial marker E-cadherin, and higher expression of mesenchymal markers such as vimentin and ZEB1. Moreover, we found that reexpression of miR-200 by transfection studies or treatment of gemcitabine-resistant cells with either DIM or isoflavone resulted in the down-regulation of ZEB1, slug, and vimentin, which was consistent with morphologic reversal of EMT phenotype leading to epithelial morphology. These results provide experimental evidence, for the first time, that DIM and isoflavone could function as miRNA regulators leading to the reversal of EMT phenotype, which is likely to be important for designing novel therapies for pancreatic cancer.

Low Concentrations of Diindolylmethane, a Metabolite of Indole-3-Carbinol, Protect against Oxidative Stress in a BRCA1-Dependent Manner

The indole-3-carbinol (I3C) metabolite 3,3'-diindolylmethane (DIM) is a proposed cancer prevention agent for various tumor types, including breast cancer. Here, we show that DIM up-regulates expression of the tumor suppressor protein BRCA1 in carcinoma and normal cell types. Up-regulation of BRCA1 was dose and time dependent, and it was observed at physiologically relevant micromolar and submicromolar DIM concentrations when cells were exposed for 72 hours. Treatment with the parent compound (I3C) or DIM (1 micromol/L) protected against cell killing due to H(2)O(2) and other oxidants, and the protection was abrogated by knockdown of BRCA1. DIM stimulated signaling by the antioxidant transcription factor NFE2L2 (NRF2) through the antioxidant response element in a BRCA1-dependent manner. We further showed that DIM rapidly stimulated phosphorylation of BRCA1 on Ser (1387) and Ser (1524) and that these phosphorylations are required for protection against oxidative stress. DIM-induced phosphorylation of BRCA1 on Ser (1387) was dependent on ataxia-telangiectasia mutated. Finally, in our assay systems, H(2)O(2)-induced cell death was not due to apoptosis. However, a significant component of cell death was attributable to autophagy, and both DIM and BRCA1 inhibited H(2)O(2)-induced autophagy. Our findings suggest that low concentrations of DIM protect cells against oxidative stress via the tumor suppressor BRCA1 by several distinct mechanisms.