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Activation of Estrogen Receptor-Mediated Gene Transcription by the Equine Estrogen Metabolite, 4-Methoxyequilenin, in Human Breast Cancer Cells

Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612

Address all correspondence and requests for reprints to: Gregory R. J. Thatcher, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612. E-mail: thatcher{at}uic.edu.

4-Methoxyequilenin (4-MeOEN) is an O-methylated metabolite in equine estrogen metabolism. O-methylation of catechol estrogens is considered as a protective mechanism; however, comparison of the properties of 4-MeOEN with estradiol (E₂) in human breast cancer cells showed that 4-MeOEN is a proliferative, estrogenic agent that may contribute to carcinogenesis. 4-MeOEN results from O-methylation of 4-hydroxyequilenin, a major catechol metabolite of the equine estrogens present in hormone replacement therapeutics, which causes DNA damage via quinone formation, raising the possibility of synergistic hormonal and chemical carcinogenesis. 4-MeOEN induced cell proliferation with nanomolar potency and induced estrogen response element (ERE)-mediated gene transcription of an ERE-luciferase reporter and the endogenous estrogen-responsive genes pS2 and TGF-. These estrogenic actions were blocked by the antiestrogen ICI 182,780. In the standard radioligand estrogen receptor (ER) binding assay, 4-MeOEN showed very weak binding. To test for alternate ligand-ER-independent mechanisms, the possibility of aryl hydrocarbon receptor (AhR) binding and ER-AhR cross talk was examined using a xenobiotic response element-luciferase reporter and using AhR small interfering RNA silencing in the ERE-luciferase reporter assay. The results negated the possibility of AhR-mediated estrogenic activity. Comparison of gene transcription time course, ER degradation, and rapid activation of MAPK/ERK in MCF-7 cells demonstrated that the actions of 4-MeOEN mirrored those of E₂ with potency for classical and nonclassical estrogenic pathways bracketing that of E₂. Methylation of 4-OHEN may not represent a detoxification pathway because 4-MeOEN is a full, potent estrogen agonist.