NUCLEAR TYPE-II [H-3] ESTRADIOL BINDING-SITES IN MCF-7 HUMAN BREAST-CANCER CELLS - BINDING INTERACTIONS WITH BIS-([3,4-DIHYDROXYPHENYL]-METHYLENE)CYCLOHEXANONE ESTERS AND INHIBITION OF CELL-PROLIFERATION

Previous studies from this laboratory demonstrated that -bis-([3,4-dih ydroxyphenyl]methylene)cyclohexanone (BDHPC) and related compounds mim ic methyl p-hydroxyphenyllactate (MeHPLA) as endogenous ligands for nu clear type II [H-3]estradiol binding sites. Occupancy of type II sites by these agents results in the inhibition of malignant cell prolifera tion in vitro and mammary tumor growth in vivo. The present studies we re designed to assess the effects of BDHPC esterification on type II s ite binding interactions in uterine nuclei and in cultured MCF-7 human breast cancer cells in vitro. The results of these experiments demons trate that in rat uterine nuclear fractions BDHPC acetate (Kd approxim ately 100 nM) interacts with type II sites with a 100-fold lower affin ity than BDHPC (Kd approximately 0.9 nM) and BDHPC benzoate failed to inhibit [H-3]estradiol binding under these experimental conditions. Co nversely, BDHPC and BDHPC acetate displayed very similar binding affin ities for type II sites in cultured MCF-7 human breast cancer cells an d there was a direct correlation between nuclear type II site occupanc y and the inhibition of cellular proliferation by these two compounds. BDHPC benzoate failed to interact with type II sites or inhibit MCF-7 cell proliferation. Taken together, these results suggested that BDHP C acetate, but not BDHPC benzoate, was being hydrolyzed by esterases i n MCF-7 cells, releasing the free parent compound. This conclusion was supported by the observations that incubation of BDHPC acetate in mam mary tumor cytosol preparations resulted in essentially quantitative h ydrolysis to BDHPC as determined by thin layer chromatography (TLC) an d by high performance liquid chromatography (HPLC) analysis of tumor c ytosol extracts. Conversely, BDHPC benzoate was not hydrolyzed by tumo r esterases which is consistent with the inability of this compound to bind to type II sites or inhibit MCF-7 human breast cancer cell proli feration. These experiments confirm and extend the hypothesis that est erase hydrolysis of MeHPLA related compounds represents an important b iological step involved in the control of the biological activity of t ype II site agonists which appear to regulate malignant cell prolifera tion through this binding interaction.