Human 17â-hydroxysteroid dehydrogenase (17â-HSD) type 1 is an enzyme that acts at the pre-receptor level. It catalyzes the NADPH-dependent reduction of the weak estrogen estrone into the most potent estrogen 17â-estradiol, which exerts proliferative effects via estrogen receptors. Overexpression of 17â-HSD type 1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis. 17â-HSD type 1 thus represents an attractive target for development of new drugs. Recently, we discovered that substituted coumarin derivatives potently and selectively inhibit 17â-HSD type 1. In the present study, we have performed additional biochemical and biological evaluation of the most promising coumarin derivative. First, we used an efficient method for isolation and purification of the active, soluble recombinant human 17â-HSD type 1 from Escherichia coli. This 17â-HSD type 1 showed a specific activity of 0.64±0.08 ìmol min(-1) mg(-1) for estrone reduction in the presence of NADPH at pH 6.5, and a K(m) of 62 nM for estrone. Next, we evaluated the best of the coumarin-derivative inhibitors, showing its reversible and competitive inhibition of 17â-HSD type 1 reductive activity with a K(i) of 53 nM. We confirmed that this coumarin inhibitor acts not only in a cell-free assay, but also decreases endogenous 17â-HSD type 1 activity in human T-47D breast cancer cells. This inhibitor also reduced estrone dependent growth of T-47D cells after 48 h of incubation.
17-Hydroxysteroid Dehydrogenases Cell Line, Tumor