Indole-3-Carbinol Selectively Uncouples Expression and Activity of Estrogen Receptor Subtypes in Human Breast Cancer Cells

Abstract

Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor (ER)-alpha (ERalpha) and ERbeta. Indole-3-carbinol (I3C) strongly down-regulated ERalpha protein and transcript levels, without altering the level of ERbeta protein, in both cell lines. In cells transfected with the ERalpha promoter linked to a luciferase gene reporter, I3C ablated ERalpha promoter activity. Propyl pyrazole triol (PPT) is a highly selective ERalpha agonist, whereas, 17beta-estradiol activates both ERalpha and ERbeta. I3C treatment inhibited the PPT- and 17beta-estradiol-induced proliferation of breast cancer cells, disrupted the PPT and 17beta-estradiol stimulation of estrogen response element (ERE)-driven reporter plasmid activity as well as of endogenous progesterone receptor transcripts. Using an in vitro ERE binding assay, I3C was shown to inhibit the level of functional ERalpha and stimulated the level of ERE binding ERbeta even though the protein levels of this receptor remained constant. In ERalpha-/ERbeta+ MDA-MB-231 breast cancer cells, I3C treatment stimulated a 6-fold increase in binding of ERbeta to the ERE. I3C also induced ERE- and activator protein 1-driven reporter plasmid activities in the absence of an ER agonist, suggesting that ERbeta is activated in indole-treated cells. Taken together, our results demonstrate that the expression and function of ERalpha and ERbeta can be uncoupled by I3C with a key cellular consequence being a significantly higher ERbeta:ERalpha ratio that is generally highly associated with antiproliferative status of human breast cancer cells.

Estrogen receptor α and β in uterine fibroids: a basis for altered estrogen responsiveness

“…Because leiomyomas may depend on an ERato-ERb ratio higher than normal for growth in the presence of estrogen, agents causing down-regulation of ERa and/or up-regulation of ERb could be used against fibroids. Interestingly, indole-3-carbinol, a constituent of Brassica vegetables reportedly endowed with potent anticancer activity in rodent models of carcinogenesis (36), was recently shown to downregulate ERa expression without altering ERb expression of MCF-7 breast cancer cells and to cause the levels of EREbound ERa and ERb to decrease and increase, respectively, and the stimulation of the proliferation of the cells by E 2 to drop as a consequence (37). Thus, agents that can decrease the inherently high ERa-to-ERb ratio of MCF-7 cells may also inhibit the estrogen-dependent proliferation of these cells, apparently by improving promoter occupancy of ER target genes by ERb relative to ERa.…”

N-Alkoxy derivatization of indole-3-carbinol increases the efficacy of the G1 cell cycle arrest and of I3C-specific regulation of cell cycle gene transcription and activity in human breast cancer cells

“…I3C treatment of breast cancer cells also results in an inhibition of CDK2 specific enzymatic activity due to a disruption in composition and subcellular localization of the CDK2 protein complex [30], whereas, CDK4 enzymatic activity and protein levels remained relatively unaffected [34]. We have also shown that I3C, but not DIM, down regulates estrogen receptor-alpha expression [32] and acts synergistically with tamoxifen to inhibit the growth of estrogen responsive MCF7 breast cancer cells and suppress CDK2 specific activity [34].…”

“…In cultured cells a significant portion of I3C is converted into its natural dimerization product DIM [28], and we have shown that the antiproliferative effects of I3C are distinct from and complement the bioactivities of DIM [10,[29][30][31][32]. The I3C mediated G1 cell cycle arrest of human breast cancer cells is mediated in part by an I3C activated transcriptional cascade that leads to the rapid inhibition in expression of the G1-acting cyclin dependent kinase 6 (CDK6) transcripts and protein [27].…”

The dietary phytochemical indole-3-carbinol is a natural elastase enzymatic inhibitor that disrupts cyclin E protein processing

“…I3C down-regulates CDK6 transcription by disrupting the interaction between Sp1 transcription factors with a Sp1-Ets composite DNA element in the CDK6 promoter (11). In estrogen-responsive breast cancer cells, I3C suppresses estrogen responsiveness (13,14), down-regulates expression of estrogen receptor-␣ (13), and synergizes with the antiproliferative effects of tamoxifen, an anti-estrogen widely used in breast cancer therapies (10). In nontumorigenic human mammary epithelial cells, I3C can induce the ATM signaling pathway independent of DNA damage to stabilize an active p53 tumor suppressor protein (15).…”