3βHSD 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 Delta(4)-androstenedione by the enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) or to Delta(5)-androstenediol by 17 beta HSD. 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 Delta(5)-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 3 beta HSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the similar to 10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3 beta HSD-catalyzed conversion to Delta(4)-androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17 beta HSD-catalyzed conversion to Delta(5)-androstenediol. Conclusion: Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3 beta HSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

Automated summary

Conversion of adrenally produced DHEA to DHT is a key mechanism in castration resistance of prostate cancer. This study investigated the kinetics of these reactions in cells and found that low concentrations of DHEA primarily underwent 3 beta HSD-catalyzed conversion to Delta(4)-androstenedione, while high concentrations underwent 17 beta HSD-catalyzed conversion to Delta(5)-androstenediol.