Plasma Membrane Receptors for the Cancer-Regulating Progesterone Metabolites, 5α-Pregnane-3,20-dione and 3α-Hydroxy-4-pregnen-20-one in MCF-7 Breast Cancer Cells

Abstract

Recent observations indicate that the progesterone metabolite, 5α-pregnane-3,20-dione (5αP), which is produced at higher levels in tumorous breast tissue, promotes cell proliferation and detachment, whereas 3α-hydroxy-4-pregnen-20-one (3αHP), which is produced at higher levels in nontumorous breast tissue, suppresses proliferation and detachment of MCF-7 breast cancer cells. The objective of the current study was to determine the presence and characteristics of binding sites for these endogenous putative cancer-regulating steroid hormones. Radiolabeled 5αP and 3αHP were used in radioligand binding assays on MCF-7 cell (membrane, cytosolic, and nuclear) fractions. Binding of [3H]5αP and [3H]3αHP was observed only in the plasma membrane fraction, whereas estradiol binding sites were confirmed in the cytosolic and nuclear fractions. The respective membrane binding sites exhibited specificity for the 5αP and 3αHP ligands with no appreciable displacement at 200- to 500-fold excess by other steroids. The association rate constants were calculated as 0.107/min and 0.0089/min and the dissociation rate constants were 0.049 9 and 0.011 for 5αP and 3αHP, respectively. Saturation analyses indicated single classes of molecules with dissociation constants of 4.5 and 4.87 nM and receptor densities of 486 and 629 fmol/mg protein, respectively, for 5αP and 3αHP. Exposure of MCF-7 cells to estradiol for 1, 24, 48, and 72 h resulted in 2.3, 4.2-, 2.99-, and 1.7-fold increases, respectively, in 5αP receptor density. 3αHP resulted in partial suppression of the estradiol-mediated increase in 5αP receptor density. This is the first report of receptors for the progesterone metabolites, 5αP and 3αHP, of their occurrence in breast cancer cell membranes, and of the induction of 5αP receptors by estradiol. The results provide further support for the potential importance of progesterone metabolites in breast cancer.