Pregnenolone (P(5)), a common precursor of many
steroids, is present in the blood of normal adult men at concentrations of 1-3 nM. In vitro, P(5) was found to stimulate LNCaP-cell proliferation 7-8-fold at a physiological concentration (2 nM), and 3-4-fold at a subphysiological concentration (0.2 nM). Growth stimulation at the 2-nM concentration was comparable with that of the
androgen,
dihydrotestosterone at its physiological concentration (0.5 nM; 9-10-fold increase in cell number). To determine whether P(5) or its metabolites were mediating this growth response, LNCaP cells were incubated with [3H]P(5) and high-performance liquid chromatography (HPLC) was performed. After a 48-h exposure, two unidentified metabolites were detected. Although, the P(5) metabolites slightly increased LNCaP-cell growth in vitro, their effect was significantly less than P(5) alone, suggesting that the growth stimulation was mediated by P(5) itself. We further showed that P(5) sustained its proliferative activity in vivo and stimulated the growth of LNCaP-
tumor xenografts in intact male SCID mice as well as in castrated animals. In order to determine whether P(5) was binding to a specific site in LNCaP cells, receptor binding studies were performed. Scatchard analysis predicted for a single class of binding sites with K(d)=1.4 nM. Studies were performed to determine the effects of P(5) on transcription mediated by wild-type and LNCaP
androgen receptors. P(5) was shown to activate transcription through the LNCaP
androgen receptor (AR), but not the wild-type AR. This implies that P(5) most likely stimulates LNCaP-cell proliferation through binding to the cellular mutated AR present in LNCaP cells. We have also demonstrated that drugs designed to be antagonists of the
androgen,
progesterone and
estrogen receptors, and one of our novel compounds designed to be an inhibitor of
androgen synthesis, were potent inhibitors of the AR-mediated transcriptional activity induced by P(5), and were able to inhibit LNCaP-cell proliferation. These findings suggest that some
prostate cancer patients who appear to become
hormone-independent may have
tumors which are stimulated by P(5) via a mutated AR and that these patients could benefit from treatment with
antiestrogens, antiprogestins, or with some of our novel
androgen synthesis inhibitors.