Diindolylmethane Research Articles

Effect of diindolylmethane supplementation on low-grade cervical cytological abnormalities: double-blind, randomised, controlled trial

Background:Cervical screening identifies many women with low-grade abnormalities. In vitro and in vivo studies have shown that diindolylmethane (DIM) could potentially halt (cervical) carcinogenesis. We report on a randomised controlled trial of the effect of DIM in women with low-grade cervical cytological abnormalities.Methods:We conducted a pragmatic double-blind, randomised controlled trial of 150 mg DIM (from BioResponse DIM) or placebo daily for 6 months in women with newly diagnosed, low-grade cytological abnormalities. Randomisation was in the ratio 2 (DIM) to 1 (placebo). All women were invited for colposcopy at 6 months with biopsy of any abnormality.Results:Of the 551 randomised women available for analysis, 9% on DIM and 12% on placebo had cervical intraepithelial neoplasia-2 (CIN2) or worse after 6-month supplementation (risk ratio (RR) 0.7 (95% confidence interval (CI): 0.4–1.2)), whereas 4.6% and 5.1%, respectively, had CIN3 or worse (RR 0.9 (95% CI: 0.4–2.0)). A total of 27.3% of women on DIM and 34.3% on placebo had no sign of disease (negative cytology, colposcopy and human papilloma virus (HPV) tests) at 6 months (RR 0.8 (95% CI: 0.6–1.0)). Of those HPV-positive at baseline, 69% (114 out of 166) of the DIM group were positive at 6 months compared with 61% (43 out of 71) of the placebo group: RR 1.1 (95% CI: 0.9–1.4). Diindolylmethane supplementation was well tolerated.Conclusion:The results suggest that short-term DIM supplementation (150 mg day−1) is well tolerated, but is unlikely to have an effect on cytology or HPV infection. Uncertainty remains regarding its effect on CIN2+.

3,3′‐Diindolylmethane Alters Ca2+ Homeostasis and Viability in MG63 Human Osteosarcoma Cells

The effect of the natural product 3,3'-diindolylmethane (DIM) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MG63 human osteosarcoma cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). DIM at concentrations of 40-80 μM induced a [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). DIM-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca(2+)](i) rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca(2+)](i) rise. At concentrations of 10-50 μM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca(2+)](i) rise by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. DIM caused cell death that may involve apoptosis.

B-DIM Inhibits Androgen Receptor Activation and Potentiates Radiation Effect in Hormone Refractory Prostate Cancer

Increased consumption of cruciferous vegetables is associated with decreased risk in prostate cancer (PCa). Two active compounds in cruciferous vegetables are indole-3-carbinol (I3C) and its dimeric derivative, 3,3'-diindolylmethane (DIM). Nutritional grade B-DIM (absorption-enhanced) has proven safe in a Phase I trial for PCa. We have investigated the anti-cancer activity of B-DIM as a new biological approach to improve the clinical outcome of radiotherapy (RT) for hormone refractory PCa. This approach was tested in vitro using human PCa cell lines positive or negative for androgen receptor (AR) expression and in vivo in an orthotopic model of PCa.

Chemopreventive effects of synthetic C-substituted diindolylmethanes originating from cruciferous vegetables in human oral cancer cells

Diindolylmethane (DIM), an isothiocyanate found in cruciferous vegetables, has been shown to have cancer chemopreventive effects. A series of synthetic C-substituted DIMs (C-DIMs) analogs was developed, including DIM-C-pPhtBu and DIM-C-pPhC6H5, which exhibited better inhibitory activity in cancer cells than DIM. This study examined the effects of C-DIMs on the growth of human oral cancer cells. DIM-C-pPhtBu and DIM-C-pPhC6H5 decreased the number of viable KB cells and induced caspase-dependent apoptosis. The apoptotic cell death was accompanied by a change in Bax/Bcl-2 ratio and damage to mitochondrial membrane potential through the induction of death receptor 5 and the cleavage of Bid and caspase 8. Studies on the mechanism of action showed that the apoptotic cell death induced by DIM-C-pPhtBu and DIM-C-pPhC6H5 was mediated by endoplasmic reticulum stress. In addition, C-DIMs inhibited cell proliferation and induced PARP cleavage through death receptor 5 and CHOP in HEp-2 and HN22 cells. This provides the first evidence that synthetic C-DIMs originating from cruciferous vegetables induce apoptosis in human oral cancer cells through the endoplasmic reticulum stress pathway.

Inactivation of AR/TMPRSS2-ERG/Wnt Signaling Networks Attenuates the Aggressive Behavior of Prostate Cancer Cells

The development of prostate cancer and its progression to castrate-resistant prostate cancer (CRPC) after antiandrogen ablation therapy are driven by persistent biological activity of androgen receptor (AR) signaling. Moreover, studies have shown that more than 50% of human prostate cancers overexpress ERG (v-ets avian erythroblastosis virus E26 oncogene related gene) due to AR-regulated TMPRSS2-ERG fusion gene. However, the reported roles of TMPRSS2-ERG fusion in cancer progression are not clear. In this study, we investigated the signal transduction in the AR/TMPRSS2-ERG/Wnt signaling network for studying the aggressive behavior of prostate cancer cells and further assessed the effects of BR-DIM and CDF [natural agents-derived synthetic formulation and analogue of 3,3'-diindolylmethane (DIM) and curcumin, respectively, with improved bioavailability] on the regulation of AR/TMPRSS2-ERG/Wnt signaling. We found that activation of AR resulted in the induction of ERG expression through TMPRSS2-ERG fusion. Moreover, we found that ERG overexpression and nuclear translocation activated the activity of Wnt signaling. Furthermore, forced overexpression of ERG promoted invasive capacity of prostate cancer cells. More important, we found that BR-DIM and CDF inhibited the signal transduction in the AR/TMPRSS2-ERG/Wnt signaling network, leading to the inactivation of Wnt signaling consistent with inhibition of prostate cancer cell invasion. In addition, BR-DIM and CDF inhibited proliferation of prostate cancer cells and induced apoptotic cell death. On the basis of our findings, we conclude that because BR-DIM and CDF downregulate multiple signaling pathways including AR/TMPRSS2-ERG/Wnt signaling, these agents could be useful for designing novel strategies for the prevention and/or treatment of prostate cancer.

3,3′-Diindolylmethane induces immunotoxicity via splenocyte apoptosis in neonatal mice

3,3′-Diindolylmethane (DIM), a major product of indole-3-carbinol derived from vegetables of the genus Brassica, exhibits chemotherapeutic activity and various immune modulatory effects in animal models and in vitro studies. Although extensive studies have only focused on DIM's beneficial effects, the toxic effects of DIM on the immune systems have not been clearly elucidated. The aim of this study was to explore the immunotoxic effects of DIM in a neonatal mouse and to further evaluate whether DIM administration affects rotavirus (RV)-induced gastroenteritis. Interestingly, multiple immunotoxic effects were observed in the DIM treated group, including decreases in various immune cells (F4/80+, CD11c+, CD19+, and CD3+ cells) in the spleen, induction of splenic white pulp atrophy, an increase in immune cell apoptosis, and decreased expression of various toll-like receptors (TLRs) in the spleen and small intestine. Apoptosis was notably promoted by up-regulating caspase-3 activity and by the change in the ratio of Bcl-2/Bax activities. Finally, oral administration of DIM led to deterioration of RV-induced intestinal disease and delayed viral clearance in the intestine and MLNs. Our results indicate that oral administration of DIM in neonatal mice induces immunotoxicity and hampers efficient RV clearance in the intestine. This new information about the immunotoxic roles of DIM in a newborn mouse model may provide valuable clues for the development of a safe supplement, especially one designed for human infants.

Mechanisms and Therapeutic Implications of Cell Death Induction by Indole Compounds

Indole compounds, obtained from cruciferous vegetables, are well-known for their anti-cancer properties. In particular, indole-3-carbinol (I3C) and its dimeric product, 3,3′-diindolylmethane (DIM), have been widely investigated for their effectiveness against a number of human cancers in vitro as well as in vivo. These compounds are effective inducers of apoptosis and the accumulating evidence documenting their ability to modulate multiple cellular signaling pathways is a testimony to their pleiotropic behavior. Here we attempt to update current understanding on the various mechanisms that are responsible for the apoptosis-inducing effects by these compounds. The significance of apoptosis-induction as a desirable attribute of anti-cancer agents such as indole compounds cannot be overstated. However, an equally intriguing property of these compounds is their ability to sensitize cancer cells to standard chemotherapeutic agents. Such chemosensitizing effects of indole compounds can potentially have major clinical implications because these non-toxic compounds can reduce the toxicity and drug-resistance associated with available chemotherapies. Combinational therapy is increasingly being realized to be better than single agent therapy and, through this review article, we aim to provide a rationale behind combination of natural compounds such as indoles with conventional therapeutics.

3,3′-Diindolylmethane inhibits breast cancer cell growth via miR-21-mediated Cdc25A degradation

3,3'-Diindolylmethane (DIM) is a potential cancer preventive phytochemical derived from Brassica vegetables. The effects of DIM on cell-cycle regulation in both estrogen-dependent MCF-7 and estrogen receptor negative p53 mutant MDA-MB-468 human breast cancer cells were assessed in this study. DIM inhibited the breast cancer cell growth in vitro and in vivo, and caused cell-cycle arrest by down-regulating protein levels of cell-cycle related kinases CDK1, CDK2, CDK4, and CDK6, as well as Cyclin B1 and Cdc25A. Meanwhile, it was revealed that Ser(124) phosphorylation of Cdc25A is primarily responsible for the DIM-induced Cdc25A degradation. Furthermore, treatment of MCF-7 cells with DIM increased miR-21 expression and down-regulated Cdc25A, resulting in an inhibition of breast cancer cell proliferation. These observations collectively suggest that by differentially modulating cellular signaling pathways DIM is able to arrest the cell-cycle progression of human breast cancer cells.

Inhibition of Fatty Acid Synthase and Sp1 Expression by 3,3′-Diindolylmethane in Human Breast Cancer Cells

The putative cancer preventive agent 3,3′-diindolylmethane (DIM) is formed in the acidic environment of the stomach following consumption of indole-3-carbinol (I3C), which is present in vegetables of the Brassica genus. We have recently shown that the transcription factor Sp1 is involved in the regulation of both proliferation and de novo lipogenesis in cancer cells. Here we show that DIM inhibits the proliferation of 3 human breast cancer cell lines, MCF-7, MDA-MB-231, and SKBr-3, and concomitantly inhibits the expression of Sp1 and fatty acid synthase (FAS). There were no DIM-related effects on the proliferation or expression of Sp1 or FAS in the nontumorigenic human breast epithelial cell line MCF-10A. These results suggest that inhibition of Sp1 and/or FAS expression could contribute to the anticancer properties of the dietary indoles.

Attenuation of multi-targeted proliferation-linked signaling by 3,3′-diindolylmethane (DIM): From bench to clinic

Emerging evidence provide credible support in favor of the potential role of bioactive products derived from ingesting cruciferous vegetables such as broccoli, brussel sprouts, cauliflower and cabbage. Among many compounds, 3,3'-diindolylmethane (DIM) is generated in the acidic environment of the stomach following dimerization of indole-3-carbinol (I3C) monomers present in these classes of vegetables. Both I3C and DIM have been investigated for their use in preventing, inhibiting, and reversing the progression of cancer - as a chemopreventive agent. In this review, we summarize an updated, wide-ranging pleiotropic anti-tumor and biological effects elicited by DIM against tumor cells. It is unfeasible to point one single target as basis of cellular target of action of DIM. We emphasize key cellular and molecular events that are effectively modulated in the direction of inducing apoptosis and suppressing cell proliferation. Collectively, DIM orchestrates signaling through Ah receptor, NF-κB/Wnt/Akt/mTOR pathways impinging on cell cycle arrest, modulation of key cytochrome P450 enzymes, altering angiogenesis, invasion, metastasis and epigenetic behavior of cancer cells. The ability of DIM to selectively induce tumor cells to undergo apoptosis has been observed in preclinical models, and thus it has been speculated in improving the therapeutic efficacy of other anticancer agents that have diverse molecular targets. Consequently, DIM has moved through preclinical development into Phase I clinical trials, thereby suggesting that DIM could be a promising and novel agent either alone or as an adjunct to conventional therapeutics such as chemo-radio and targeted therapies. An important development has been the availability of DIM formulation with superior bioavailability for humans. Therefore, DIM appears to be a promising chemopreventive agent or chemo-radio-sensitizer for the prevention of tumor recurrence and/or for the treatment of human malignancies.

Pharmacodynamics of dietary phytochemical indoles I3C and DIM: Induction of Nrf2-mediated phase II drug metabolizing and antioxidant genes and synergism with isothiocyanates

The antioxidant response element (ARE) is a critical regulatory element for the expression of many phase II drug metabolizing enzymes (DME), phase III transporters and antioxidant enzymes, mediated by the transcription factor Nrf2. The aim of this study was to examine the potential activation and synergism of Nrf2-ARE-mediated transcriptional activity between four common phytochemicals present in cruciferous vegetables; the indoles: indole-3-carbinol (I3C), 3,3'-diindolylmethane (DIM); and the isothiocyanates (ITCs): phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). The cytotoxicity of the compounds was determined in a human liver hepatoma cell line (HepG2-C8). The combination index was calculated to assess the synergistic effects on the induction of ARE-mediated gene expressions. Quantitative real-time polymerase chain reaction (qPCR) was employed to measure the mRNA expressions of Nrf2 and Nrf2-mediated genes. I3C and DIM showed less cytotoxicity than SFN and PEITC. Compared with I3C, DIM was found to be a stronger inducer of ARE. Synergism was observed after combined treatments of 6.25 µm I3C + 1 µm SFN, 6.25 µm I3C + 1 µm PEITC and 6.25 µm DIM + 1 µm PEITC, while an additive effect was observed for 6.25 µm DIM + 1 µm SFN. Induction of endogenous Nrf2, phase II genes (GSTm2, UGT1A1 and NQO1) and antioxidant genes (HO-1 and SOD1) was also observed. In summary, the indole I3C or DIM alone could induce or syngergistically induce in combination with the ITCs SFN or PEITC, Nrf2-ARE-mediated gene expression, which could potentially enhance cancer chemopreventive activity.

Dimer Radical Cations of Indole and Indole-3-carbinol: Localized and Delocalized Radical Cations of Diindolylmethane

Extending our previous study on the title species (J. Phys. Chem. A2010, 114, 6787), we investigated the dimer cations that are formed on oxidation of the glucobrassin derivatives indole-3-carbinol (I3C) and diindolylmethane (DIM) and of parent indole (I). Radiolysis in ionic liquid and Ar matrices shows that, at sufficiently high concentrations and/or on annealing the solid glasses, intense intermolecular charge-resonance (CR) absorption bands in the NIR herald the formation of sandwich-type dimer cations. The molecular and electronic structure of these species is modeled by calculations with the double-hybrid B2-PLYP-D density functional method which yields predictions in good accord with experiment. The radical cation of DIM also shows a CR band, but unlike in the case of I and I3C, its occurrence is not dependent on the concentration but instead on the solvent: in ionic liquid the CR band is initially absent and arises only on annealing, whereas in Ar matrices it is present from the outset and undergoes blue shifting and sharpening on annealing. These puzzling findings are rationalized on the basis of B2-PLYP-D calculations which predict that neutral DIM exists in the form of two conformers, present in different relative amounts in the two experiments, which on vertical ionization form distinct radical cations, a nonsymmetric one where the odd electron is largely localized on one of the two indole moieties and one with C(2) symmetry where charge and spin are completely delocalized over both halves of the molecule, thus giving rise to an intramolecular CR transition. On annealing, the nonsymmetric cation relaxes to a similarly delocalized structure with C(s) symmetry, thus explaining the observed increase and the shift of the CR band. We believe that DIM(•+) represents the first example of a radical cation which can exist under the same conditions as a localized and a delocalized complex cation.

3,3′-Diindolylmethane but not indole-3-carbinol activates Nrf2 and induces Nrf2 target gene expression in cultured murine fibroblasts

There is increasing interest in the gene-regulatory activity of Brassica vegetable derived phytochemicals such as 3,3′-diindolylmethane (DIM) and indole-3-carbinol (I3C). DIM is formed under acidic conditions by dimerization of I3C. This study compared the Nrf2 activating potential of DIM and I3C in murine fibroblasts (NIH3T3). In contrast to its precursor I3C, DIM induces the transactivation of Nrf2. Furthermore, Nrf2 targets such as HO-1, γGCS and NQO1 were increased on the mRNA and protein levels following DIM treatment. DIM was less potent than sulforaphane (used as positive control) in inducing Nrf2-dependent gene expression. The present data suggest that the dimerization of I3C to DIM increases its Nrf2 inducing activity.

Broccoli‐derived phytochemicals indole‐3‐carbinol and 3,3′‐diindolylmethane exerts concentration‐dependent pleiotropic effects on prostate cancer cells: Comparison with other cancer preventive phytochemicals

In the present studies, we utilized prostate cancer cell culture models to elucidate the mechanisms of action of broccoli-derived phytochemicals 3,3'-diindolylmethane (DIM) and indole-3-carbinol (I3C). We found DIM and I3C at 1-5 µM inhibited androgen and estrogen-mediated pathways and induced xenobiotic metabolism pathway. By contrast, DIM and I3C induced cyclin inhibitors, indicators of stress/DNA damage, only at ≥25 µM. We also demonstrated that an inhibitory effect of DIM and I3C on cell growth involves inhibition of insulin-like growth factor-1 receptor expression. More importantly, we showed that differences in efficacies and mechanisms existed between DIM and I3C. These included differences in effective concentrations, a differential effect on androgen receptor binding, and a differential effect on xenobiotic metabolic pathway through aryl hydrocarbon receptor-dependent and -independent mechanism. Furthermore we determined that several other diet-derived cancer protective compounds, similar to DIM and I3C, exhibited pleiotrophic effects on signaling pathways that included proliferation, cell cycle, and nuclear receptors-mediated pathways. However, the efficacies and mechanisms of these compounds vary. We also showed that some cellular pathways are not likely to be affected by DIM or I3C when circulating concentration of orally ingested DIM or I3C is considered. Based on our results, a model for cancer protective effects of DIM and I3C was proposed.

Abstract 3704: Diindolylmethane induces ER stress mediated autophagy in ovarian cancer cells

Abstract Endoplasmic reticulum (ER) is responsible for biosynthesis, folding assembly and modification of various soluble and membrane proteins. ER stress is the imbalance in protein folding resulting in the accumulation of misfolded proteins in ER lumen. In our study, we observed that Diindolylmethane (DIM), a compound present in cruciferous vegetables induces ER stress in ovarian cancer cells. Treatment of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations of DIM for 24hours resulted in the upregulation of Bip, a molecular chaperone. Bip or Grp78 binds to IRE1α, ATF6 and PERK under normal condition, however, during ER stress, Bip dissociates and releases all the three proteins. DIM treatment also significantly increased the expression of IRE1α, ER oxidase (ERO1) and CHOP. CHOP translocate to nucleus and initiate apoptotic stimuli in cells undergoing ER stress. Moreover, DIM induced ER stress also lead to the activation of eIF2α by phosphorylation. Activation of eIF2α inhibits protein translation to avoid further protein accumulation in ER. The release of calcium by DIM treatment in ovarian cancer cells was evaluated by Accuri C6 flowcytometer. The advantage with Accuri C6 is that calcium levels can be monitored without interruption. DIM treatment increased the cytosolic calcium levels in SKOV3 cells. Increase in cytosolic calcium is known to cause ER stress. In addition, our flowcytometry results indicate that DIM induces autophagy in all the ovarian cancer cells in a concentration dependent manner. Exposure of SKOV-3, OVCAR-3 and TOV-21G cells to DIM upregulated the expression of LC3B, a potent indicator of autophagy. DIM induced autophagy was blocked by bafilomycin, an autophagy inhibitor. Treatment of cells with mithramycin (ER stress inhibitor) blocked ER stress proteins activated by DIM. Interestingly, we also observed that blocking DIM-induced ER stress by mithramycin blocked autophagy in ovarian cancer cells. Taken together these results suggest that induction of autophagy in ovarian cancer cells was associated with ER stress caused by DIM. Further mechanistic studies are in progress. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3704. doi:10.1158/1538-7445.AM2011-3704

Abstract 4651: Analysis of 3,3′-diindolylmethane in urine after consumption of cruciferous vegetables in the Singapore Cohort Study

Abstract Background: Indole-3-carbinol (I3C) may be a chemopreventive agent against cancer. Until recently, there has not been a method to quantitatively assess human dietary I3C exposure or dietary exposure to its specific metabolites in biologic samples collected in a non-invasive manner. We have developed a LC-ESI-MS/MS-SRM analysis to quantify 3,3′-diindolylmethane (DIM), a specific metabolite of I3C in urine. This assay has not been validated as a biomarker of I3C intake in free-living people. Methods: We conducted a small feeding study involving 3 subjects. Subjects consumed approximately 300∼500 g of broccoli and collected 24 h urine samples. The developed LC-ESI-MS/MS-SRM analysis on urinary DIM assay showed the expected m/z 247 → 130 in all 24 h urine samples collected after consumption of broccoli; d2-DIM was used as an internal standard. The assay was then used to quantify urinary DIM in 246 subjects of the Singapore Chinese Health Study, a prospective, population-based cohort of middle aged Chinese men and women residing in Singapore. Random, non-fasting, non-timed urine samples were collected from the cohort between 1994 and 1999. Detailed dietary information and urinary levels of isothiocyanates (ITC) were available for these 246 subjects. The dietary information included intake of 8 cruciferous vegetables that had been previously assayed for glucobrassicins, the precursor of I3C.Estimated dietary intake of glucobrassicins was calculated for each individual based on reported intake of each cruciferous vegetable. Spearman correlation coefficients were calculated to determine the correlation between urinary DIM and reported intake of cruciferous vegetables. Results: Among the 246 subjects, urinary DIM levels ranged from undetectable to 167.8 pmol/mg Cr with a mean (sd) of 14.5 (9.59) pmol/mg Cr. The average intake of total cruciferous vegetables was 40.6 g/day and mean estimated intake of glucobrassicins was 6.5 µmol/day. The Spearman correlation coefficients (p-values) for urinary DIM and dietary intake of total cruciferous vegetables and estimated glucobrassicins were 0.09 (0.18) and 0.10 (0.10) respectively. The coefficients for urinary DIM and intake of each cruciferous vegetable were as follows: kaichoi, 0.01 (0.87); watercress, 0.09 (0.15); choisum, 0.04 (0.52); pakchoi, 0.01 (0.84); kailan, 0.02 (0.74), cabbage/wong nga pak, 0.14 (0.03); broccoli, 0.04 (0.58) and cauliflower, 0.14 (0.03). Urinary DIM also was statistically significantly, albeit weakly, associated with urinary total ITC (coefficient = 0.17, P=0.008), a validated biomarker for consumption of dietary ITC. Conclusion: Although lacking of a significant relationship with intake of total cruciferous vegetables, urinary DIM was statistically significantly associated with intake of cabbage/wong nga pak and cauliflower, two cruciferous vegetables with relatively high contents of I3C precursor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4651. doi:10.1158/1538-7445.AM2011-4651

Abstract 3535: Diindolylmethane potentiates the effect of cisplatin in ovarian cancer cells by inhibiting STAT-3 and VEGF

Abstract Ovarian cancer remains the second leading gynecological cancer in United States. It is usually detected in late stages with poor prognosis. Cisplatin is a chemotherapeutic drug currently used to treat patients with ovarian cancer. However, ovarian tumors develop resistance and cisplatin is associated with systemic toxicity. We previously reported that Diindolylmethane (DIM), a natural compound present in cruciferous vegetables inhibits the proliferation of ovarian cancer cells [Kandala and Srivastava, Mol. Pharmacol. 2010, 78, 297-309]. In this study, we evaluated the effect of DIM with cisplatin in ovarian cancer cells. Exposure of SKOV-3 cells to 50μM DIM for 24 hours followed by treatment with 10µM cisplatin resulted in about 70% reduction in the survival of cells as compared to 30% by cisplatin treatment alone. Western blot analyses indicate that combination (DIM + cisplatin) treatment block the activation of STAT-3 by inhibiting the phosphorylation at Tyr 705 and Ser 727 as compared to cisplatin or DIM treatment alone. The STAT-3 protein level was reduced by 90% by combination treatment. Mcl-1and survivin, which are regulated by STAT-3 were also down-regulated by combination treatment. Interestingly, survivin was up-regulated by cisplatin treatment but completely blocked by combination treatment. Increased apoptosis by combination treatment was evident by caspase-3 and PARP cleavage. In addition, we tested the effects of this combination treatment on ovarian tumor angiogenesis and metastatsis. The results of aortic ring assay suggested that the combination treatment completely suppressed the formation of microvessel sprouts. Furthermore, combination treatment blocked invasion of cells by approximately 50% as evaluated by Boyden's chamber. Combination of DIM and cisplatin suppressed VEGF secretion by 50% as compared to 20% by cisplatin treatment in SKOV-3 cells. Taken together, our results suggest that DIM potentiates the effect of cisplatin by blocking the activation of STAT-3 and inhibiting angiogenesis and metastasis. Further mechanistic and in vivo testing of this combination treatment is under progress. [Supported in part by R01 grants CA106953 and CA 129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3535. doi:10.1158/1538-7445.AM2011-3535

Abstract 2005: Modulation of tumor suppressor gene DNA-methylation by quercetin and dietary indoles

Abstract Epidemiological and preclinical research provide strong evidence for the anticancer properties of bioactive dietary compounds abundantly found in fruits, vegetables and other fiber-rich food stuff. However, the mechanisms by which nutritional intake of such dietary compounds prevent oncogenesis is largely unknown. We hypothesize that synergistic activities of different dietary compounds through epigenetic mechanisms underlie some of the chemopreventive functions of the dietary compounds. We undertook a preliminary study to examine the effects of Quercetin, Indole-3-Carbinol (I3C) and diindolylmethane (DIM) on promoter methylation profiles of selected genes known to be suppressed by methylation in colorectal cancer. Quercetin is a polyphenolic flavonoid, while I3C and DIM are dietary indoles, DIM being a derivative of I3C. The genes queried in this study were p16INK4a, E-cadherin (CDH1), and IGFBP7. p16INK4a is a cell cycle regulator silenced in up to 40-50% of colorectal carcinomas. E-cadherin is a tumor suppressor and metastasis regulator protein the loss of which promotes wnt-signaling, which is dysregulated in the majority of colorectal cancers. IGFBP7 has recently been identified as a p53-responsive gene frequently silenced in colorectal and gastric cancers. In this study, RKO colorectal cancer cells were treated with the three dietary compounds, vehicle (DMSO), or the demethylating compound 5-azacytidine. DNA was harvested and bisulfite converted. Methylation of the gene promoters was examined by methylation-specific PCR using primer sets that amplify only methylated DNA. Gel electrophoresis and Real-time PCR were used to assess the relative abundance of methylated DNA in control and treated samples. Preliminary results show that the dietary compounds can reverse the methylated-promoter phenotype, with the indole compounds having higher potency compared to quercetin especially in demethylating the CDH1 gene promoter. Further experiments will elucidate the molecular mechanisms through which the compounds modulate gene methylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2005. doi:10.1158/1538-7445.AM2011-2005

Abstract 1320: Estrogen as a modulator of thyroid proliferative disease: Implications for disease etiology and therapy

Abstract Metastasis of cancer is a multifactorial disease and is responsible for over 90% of all cancer related deaths. Thyroid cancer is the most common endocrine related cancer with well differentiated thyroid cancer metastasizing to the nearby lymph nodes in upto 50% of the cases. Interestingly, according to American Thyroid Association (ATA), the incidences of thyroid cancer are four to five times more in women than in men with one in eight women developing thyroid disorder at least once in her lifetime. In this study, we explored the possible etiological contribution of estrogen in thyroid proliferative disease using an in vitro experimental model system. We observed that thyroid cancer cells express functionally active estrogen receptors (ER), ER-α and ER-β. Furthermore, estrogen enhances the metastatic propensity of thyroid cells as evidenced by increased migration, adhesion and invasion, which is reversed by anti-estrogens. These observations were further supported by ER-α down-regulation (siRNA) studies. Western blot and zymography analyses provide direct evidence for this estrogen mediated enhancement of metastasis associated events by enhanced secretion of the proteolytic enzymes, matrix metalloproteinases (MMP), MMP-2 and MMP-9. We also observed that conditioned medium from estrogen treated thyroid cells contains enhanced levels of vascular endothelial growth factor (VEGF) and modulates angiogenesis associated events, such as migration, proliferation and tubulogenesis of endothelial cells. Our studies provide evidence that the higher incidence of thyroid cancer in females is potentially attributed to the presence of a functional ER that participates in cellular processes contributing to enhanced mitogenic, migratory, invasive and angiogenic properties of thyroid cells, which is responsive to anti-estrogens. Our findings formed the basis of an ongoing clinical trial in patients with thyroid proliferative diseases using dietary anti- estrogenic compound, 3, 3’-diindolylmethane (DIM). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1320. doi:10.1158/1538-7445.AM2011-1320

Differential effects of broccoli‐derived phytochemicals indole‐3‐carbinol and its dimer 3, 3’‐diindolylmethane on liver X receptor‐responsive genes in human prostate cancer cells

This study examined the effects of indole‐3‐carbinol (I3C) and 3, 3’‐diindolylmethane (DIM) on liver X receptor (LXR)‐dependent pathways. DIM, but not I3C, induced transcription of ABCG1 and ABCA1 mRNA, two LXR‐responsive ATP‐binding cassette transporters involved in cholesterol efflux, in the androgen responsive LNCaP cells, but not the androgen non‐responsive PC3 cells. Exposure of LNCaP cells to 5 microM DIM for 48 hrs led to a 5‐fold increase in ABCG1 and ABCA1 mRNA levels. Knockdown of LXR alpha and beta isoforms using siRNA abrogated the ability of DIM to induce ABCG1 but not ABCA1 mRNA. Furthermore, knockdown of androgen receptor (AR) expression in LNCaP cells with AR siRNA blocked DIM induction of ABCA1 and ABCG1 mRNAs. The results suggest that DIM modulates cholesterol metabolism through both androgen‐mediated and LXR‐dependent pathways in prostate cancer cells. Furthermore, these effects are unique to DIM but not I3C suggesting that these two dietary components exert cancer preventive effects through different mechanisms.