DIM and Breast Health

 

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

Ahmad A, Kong D, Wang Z, Sarkar SH, Banerjee S, Sarkar FH.

Department of Pathology, Barbara Ann Karmanos Cancer Center, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.

J Cell Biochem. 2009 Nov 1;108(4):916-25.

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. (c) 2009 Wiley-Liss, Inc.

PMID: 19693769 [PubMed - indexed for MEDLINE]

 

 

Low concentrations of diindolylmethane, a metabolite of indole-3-carbinol, protect against oxidative stress in a BRCA1-dependent manner.

Fan S, Meng Q, Saha T, Sarkar FH, Rosen EM.

Department of Oncology, Georgetown University, Washington, District of Columbia 20057, USA.

Cancer Res. 2009 Aug 1;69(15):6083-91.

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

PMID: 19622773 [PubMed - indexed for MEDLINE]

 

 

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. kmrahman@med.wayne.edu

Cancer Res. 2006 May 1;66(9):4952-60.

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]

 

 

Managing Cyclical Mastalgia with Absorbable Diindolylmethane: A Randomized, Placebo-controlled Trial

Zeligs MA, Brownstone PK, Sharp ME, Westerlind K,Wilsom SM, and Johs S

JANA. 2005; 7(3): 5-14.

This intervention study investigated the efficacy and safety of absorbable diindolylmethane (BioResponse-DIM®) in cyclical mastalgia (recurrent breast pain). Otherwise healthy, pre-menopausal women with cyclical mastalgia were given absorbable diindolylmethane (DIM) or placebo for consecutive three month periods in a randomized, double-blind, crossover study. Breast symptoms were monitored using daily entries on a "breast pain diary" as the assessment tool. Urine and blood samples were collected to confirm safety and the impact of absorbable DIM on estrogen metabolism. Results showed clinical improvement with absorbable DIM. Improvement was not seen with placebo. A statistically significant reduction in duration of breast pain, severity of pain, swelling, and soreness accompanied absorbable DIM use, based on comparison of visual analog pain scores from treatment and placebo periods (p=.001-.03). In addition, absorbable DIM was shown to increase the ratio of 2-hydroxy to 16-hydroxy estrone metabolites in urine (p<.05). Supplementation with absorbable DIM was found to be an effective intervention for cyclical mastalgia. Its use as a dietary supplement deserves further investigation in conditions where modifying estrogen metabolism may be of benefit.

 

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.

Carcinogenesis. 2005 Apr;26(4):771-8. Epub 2005 Jan 20.

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]

 

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.

Cancer Res. 2005 Jan 1;65(1):364-71.

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]

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Effect of 3,3’-diindolylmethane (DIM) supplements on urinary estrogen metabolites in postmenopausal women with breast cancer.

Dalessandri KM, Firestone GL, Fitch MD, Bradlow HL, and Bjeldanes LF

Proc. Am. Assoc. Cancer Res. 2002 Apr; 43, 3198

Dietary indoles, present in Brassica plants such as cabbage, broccoli, and Brussels sprouts have been shown to be protective against hormone dependent cancers. 3,3’-diindolylmethane (DIM) is under study as one of the main protective indole metabolites. Postmenopausal women ages 50-70 from Marin County, California, with a history of early stage breast cancer, were screened for interest and eligibility in this study on the effect of absorbable DIM (BioResponse-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-OHE)), 16-alpha hydroxyestrone (16a-OHE1), DIM, estrone (E1), estradiol (E2), estriol (E3), 6b-hydroxycortisol (6b-OHC), and cortisol in the first morning urine sample before intervention and 31 days after intervention. Nineteen women completed the study, making a total of ten in the treatment group and nine in the placebo group. DIM treated subjects, relative to placebo, showed a significant increase in levels of 2-OHE1 (p=0.020), DIM (p=0.045) and cortisol (p=0.039), and a nonsignificant increase of 47% in the 2-OHE1/16a-OHE1 ratio from 1.46 to 2.14 (p=0.059). These findings could be associated with a lower risk status for breast cancer.

 

 

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.

Carcinogenesis. 2002 Aug;23(8):1297-305.

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]

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The Influence of 3'3-Diindolylmethane on Breast Tumor Growth, Invasion and Metastasis.

Tou J, Hong C, and Bjeldanes LF.

Experimental Biology, 2001 Apr; Orlando, Florida, Meeting Abstract

Diets rich in cruciferous vegetables are associated with protection against various cancers including breast cancer. Indole-3-carbinol (I3C) is an important phytochemical present in cruciferous vegetables. Studies report that oral administration of I3C to carcinogen-treated rodents reduced tumor growth. However, I3C is unstable and under acid conditions in the stomach is rapidly converted to the more potent compound 3',3- diindolylmethane (DIM) suggesting that antitumorigenic activity may not be due entirely to the parent compound but to its digestive metabolite.

This study investigated the effect of DIM on the growth of transplantable human breast tumor cells in female athymic nude mice. Subcutaneous injections of 100 ug DIM/day significantly reduced tumor size. Yet morbidity in breast cancer patients does not arise from the primary tumor but from tumor metastasis. Therefore, we investigated the effect of DIM on tumor cell adhesion, motility and invasiveness which are key processes in the metastatic cascade. The highly metastatic estrogen receptor negative MDA-MB-231 and Hs578T human breast cancer cells were used as well as a poorly metastatic estrogen receptor positive MCF-7 human breast cancer cell line. DIM doses of 10 and 50 microM significantly inhibited adhesion to Matrigel coated wells, motility as measure by wound healing assay and breast tumor cell invasiveness using a modified Boyden chamber.

DIM inhibited key steps in the metastatic process in both the estrogen positive and estrogen negative cell lines suggesting DIM exerts anti-metastatic effects via estrogen-dependent and independent pathways. Furthermore, microarray analysis showed the down regulated expression of a number of genes involved in adhesion, motility, extracellular matrix degradation enzymes and invasiveness in breast tumor cells treated with 10 and 50 microM DIM. Results suggest that DIM may have therapeutic effects against breast tumor growth and tumor spread.

 


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