A Fucus vesiculosus extract inhibits estrogen receptor activation and induces cell death in female cancer cell lines.

BACKGROUND: We previously reported the anti-estrogenic activity of the brown seaweed, Fucus vesiculosus. The present study aimed to further investigate its anti-estrogenic modes of action and to assess other potentially biologically relevant anti-tumorigenic effects in estrogen receptor (ER)-dependent and -independent female cancer cell lines. METHODS: The CALUX® assay was used to determine the effect of a F. vesiculosus extract (FVE) on activation of the ER. Aromatase enzymatic activity was measured to determine the potential effect of FVE on estradiol (E2) biosynthesis. Transcriptional activity profiling of 248 genes involved in cancer, immunity, hormonal regulation, protein phosphorylation, transcription, metabolism, and cellular structure was conducted using the NanoString nCounter® analysis system in FVE-treated breast, ovarian and endometrial cancer cell lines. The effects of FVE on cell viability, morphology, membrane integrity, mitochondrial toxicity, induction of apoptotic and autophagic markers, and cell signaling were also analyzed. RESULTS: In co-treatments with 12.5 pM (EC50) E2, FVE (2 %) reduced ER activation by 50 %, exhibiting potent ER antagonistic effects. FVE inhibited aromatase activity in an in vitro assay (IC50 2.0 %). ER-dependent and -independent cancer cell lines showed significantly decreased viability that correlated with increasing FVE concentrations and altered morphological features suggestive of apoptosis and autophagy. Expression of genes that were significantly altered by FVE (p < 0.05) revealed predominantly apoptotic, autophagic and kinase signaling pathways. FVE also effectively inhibited the phosphorylation of Akt, resulting in reduced mTORC1 activities to stimulate autophagy in cells. Concentration-dependent cleavage of PARP and induction of caspase-3 and -7 activities were observed in MDA-MB-231 cells supporting a role for FVE in the promotion of apoptosis. CONCLUSIONS: Our study provides new insights into the anti-estrogenic activity of F. vesiculosus. Moreover, the induction of autophagy and apoptosis on breast, endometrial and ovarian cancer cell lines suggests additional anti-tumorigenic actions of FVE that are independent of ER status in female cancers.

3,3'-diindolylmethane reduces levels of HIF-1alpha and HIF-1 activity in hypoxic cultured human cancer cells.

3,3'-diindolylmethane (DIM) is a chemopreventive and chemotherapeutic phytochemical derived from the metabolism of indoles found at high concentrations in cruciferous vegetables. We have previously shown that DIM exhibits anti-angiogenic properties in cultured vascular endothelial cells and in Matrigel plug assays in rodents. In the present study, we demonstrate that DIM reduces the level of hypoxia-inducible factor (HIF)-1alpha in hypoxic tumor cell lines, as well as HIF-1 transcriptional activity as measured by a reporter assay. Moreover, DIM inhibited the expression of HIF-1-responsive endogenous genes, resulting in the reduced expression of key hypoxia responsive factors, VEGF, furin, enolase-1, glucose transporter-1 and phosphofructokinase. DIM reduced the level of HIF-1alpha in hypoxic cells by increasing the rate of the prolylhydroxylase- and proteasome-mediated degradation of HIF-1alpha, and by decreasing the rate of HIF-1alpha transcription. Using enzyme kinetics studies, we established that DIM interacts with the oligomycin-binding site on the F0 transmembrane component of mitochondrial F1F0-ATPase. The contributions of the resulting increases in levels of ROS and O2 in hypoxic cells to the inhibitory effects of DIM on HIF-1alpha expression are discussed. These studies are the first to show that DIM can decrease the accumulation and activity of the key angiogenesis regulatory factor, HIF-1alpha, in hypoxic tumor cells.

Activation and potentiation of interferon-gamma signaling by 3,3'-diindolylmethane in MCF-7 breast cancer cells.

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.

3,3'-Diindolylmethane inhibits angiogenesis and the growth of transplantable human breast carcinoma in athymic mice.

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.

Indole-3-carbinol stimulates transcription of the interferon gamma receptor 1 gene and augments interferon responsiveness in human breast cancer cells.

Indole-3-carbinol (I3C), a naturally occurring compound of Brassica vegetables, has promising anticancer properties and activates an anti-proliferative pathway that induces a G1 cell cycle arrest of human breast cancer cells. A microarray analysis of I3C treated versus untreated MCF-7 breast cancer cells revealed that I3C increased expression of the interferon gamma receptor 1 (IFNgammaR1). Western blot and RT-PCR analysis demonstrated that I3C strongly and rapidly stimulated IFNgammaR1 gene expression. Transfection of a series of 5' deletion constructs of the IFNgammaR1 reporter plasmids revealed that I3C significantly stimulates the promoter activity of the IFNgammaR1 and uncovered an I3C-responsive region between -540 and -240 bp of the IFNgammaR1 promoter. I3C stimulation of the IFNgammaR1 expression suggests that indole treatment should enhance IFNgamma responsiveness in breast cancer cells. A combination of I3C and IFNgamma synergistically activated STAT1 proteins by increasing phosphorylation at the Tyr-701 site. In addition, I3C and IFNgamma together more effectively induced a G1 cell cycle arrest and stimulated expression of the p21(Waf1/Cip1) cell cycle inhibitor, compared with the effects of either agent alone. Our results suggest that one mechanism by which I3C mediates these anticancer effects is by stimulating expression of the IFNgammaR1 and augmenting the IFNgamma response in MCF-7 human breast cancer cells.

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.

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.