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Screening for Phytoestrogens using a Cell-based Estrogen Receptor β Reporter Assay
Published on: June 7, 2020
Indole-3-carbinol selectively uncouples expression and activity of estrogen receptor subtypes in human breast cancer
Shyam N Sundar1, Vaishali Kerekatte, Caterina N Equinozio
1Department of Molecular and Cell Biology, 591 LSA, University of California at Berkeley, Berkeley, California 94720-3200 USA.
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.
Insights
Indole-3-carbinol (I3C) selectively reduces estrogen receptor-alpha (ERalpha) while enhancing estrogen receptor-beta (ERbeta) activity in breast cancer cells. This shift in ERbeta:ERalpha ratio demonstrates an antiproliferative effect, offering a potential therapeutic strategy.
Area of Science:
- Molecular Endocrinology
- Cancer Biology
- Pharmacology
Background:
- Estrogen receptors (ERalpha and ERbeta) play critical roles in the proliferation of estrogen-responsive breast cancer cells.
- Selective modulation of ER subtypes is a key strategy in breast cancer therapy.
Purpose of the Study:
- To investigate the effect of Indole-3-carbinol (I3C) on ERalpha and ERbeta expression and function in breast cancer cells.
- To determine if I3C can uncouple ERalpha and ERbeta activity and its impact on cell proliferation.
Main Methods:
- Utilized MCF7, T47D, and MDA-MB-231 breast cancer cell lines.
- Assessed ERalpha and ERbeta protein and transcript levels following I3C treatment.
- Employed luciferase reporter assays to measure ERalpha promoter activity and estrogen response element (ERE)-driven transcription.
- Performed in vitro ERE binding assays to quantify ER subtype binding affinity.
Main Results:
- I3C significantly down-regulated ERalpha protein and transcript levels in MCF7 and T47D cells, without affecting ERbeta protein levels.
- I3C inhibited ERalpha promoter activity and blocked ERalpha-mediated transcriptional activity induced by agonists like PPT and 17beta-estradiol.
- I3C enhanced ERbeta binding to the ERE in both ERalpha/ERbeta+ and ERalpha-/ERbeta+ cells, indicating ERbeta activation and a higher ERbeta:ERalpha functional ratio.
Conclusions:
- I3C effectively uncouples ERalpha and ERbeta expression and function in breast cancer cells.
- The increased ERbeta:ERalpha functional ratio induced by I3C is associated with an antiproliferative status.
- I3C represents a potential therapeutic agent for breast cancer by selectively modulating estrogen receptor signaling.
