3βHSD activity saturates at physiological substrate concentrations in intact cells

Abstract

BackgroundConversion 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 3β‐hydroxysteroid dehydrogenase (3βHSD) or to Δ5‐androstenediol by 17βHSD. To better understand this process, we studied the kinetics of these reactions in cells.MethodsProstate 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.ResultsThe two reactions displayed very different saturation profiles, with only the 3βHSD‐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 3βHSD‐catalyzed conversion to Δ4‐androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17βHSD‐catalyzed conversion to Δ5‐androstenediol.ConclusionContrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3βHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

AKR1C3 Converts Castrate and Post-Abiraterone DHEA-S into Testosterone to Stimulate Growth of Prostate Cancer Cells via 5-Androstene-3β,17β-Diol

“…We also found that a major intermediate steroid metabolite that accumulated was 5-Adiol, suggesting that the pathway for DHEA metabolism to T proceeds through 5-Adiol. The slow conversion of 5-Adiol to T is indicative of the expected low activity of HSD3B1 which is considered a rate determining step in prostate cancer androgen metabolism which would also be consistent with its saturation kinetics displayed at high DHEA concentrations ( 49, 50 ). This slow conversion could suggest that 5-Adiol might accumulate without being converted to T. However, accumulation of 5-Adiol would likely cause growth attenuation because 5-Adiol is a substrate for estrogen receptor β which slows prostate cancer cell growth and growth attenuation is not observed ( 51, 52 ).…”