3ßHSD activity saturates at physiological substrate concentrations in intact cells.

BACKGROUND: Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3ß-hydroxysteroid dehydrogenase (3ßHSD) or to Δ5 -androstenediol by 17ßHSD. To better understand this process, we studied the kinetics of these reactions in cells. METHODS: Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. RESULTS: The two reactions displayed very different saturation profiles, with only the 3ßHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3ßHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17ßHSD-catalyzed conversion to Δ5 -androstenediol. CONCLUSION: Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3ßHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.