GLUCURONOSYLTRANSFERASE ACTIVITY IN HUMAN CANCER CELL-LINE LNCAP

LNCaP, a human prostate cancer cell line, metabolizes testosterone int o a variety of 5 alpha-reduced C19 steroids, such as dihydrotestostero ne (DHT), androstane-3 alpha,17 beta-diol (3 alpha-DIOL), androstane-3 beta,17 beta-diol (3 beta-DIOL), and androsterone (ADT). Recent repor ts also suggest that 5 alpha-reduced C19 steroid glucuronides can be d etected in the medium. The purpose of this work was to characterize by liquid chromatograph ion spray mass spectroscopy (LCMS) the metabolit es formed by LNCaP during incubation with testosterone and its 5 alpha -reduced C19 steroids. Time course studies using 10 nM labeled testost erone, 3 alpha-DIOL, 3 beta-DIOL, or ADT showed that a large proportio n of polar steroids were produced by LNCaP. Identification of metaboli tes produced by LNCaP was carried out by LCMS using 1 mu M substrates. Analysis of testosterone metabolism indicated that testosterone glucu ronide was formed at 77 +/- 2% after 96 h of incubation. Using DHT as substrate, 3 alpha-DIOL-G and DHT-G were the major metabolites, accoun ting for 46 +/- 4% and 38 +/- 3%, respectively, of the total radioacti vity in the medium; ADT-G accounted for 8 +/- 1%. Further analysis by LCMS also indicated that the glucuronide group in 3 alpha-DIOL-G was a t position 17-carbon. 3 alpha-DIOL-G (86 +/- 3%) was the prominent met abolite formed from 3 alpha-DIOL, a minor product was detected at 7 1% and identified by mass spectrometry to correspond to a trihydroxyla ted C19 steroid. After a 96-h incubation period, 3 beta-DIOL was trans formed into 3 beta-DIOL-G, DHT-G, and a trihydroxylated metabolite of C19 steroid at 48 +/- 3, 26 +/- 1, and 24 +/- 2%, respectively. When A DT was incubated, ADT-G (89 +/- 1%) and two minor products, 3 alpha-DI OL and 3 alpha-DIOL-G, at less than 10% of total metabolites were dete cted. It was also shown that glucuronosyltransferase activity in homog enized cells was about 8% of that in intact cells, whereas the additio n of the cofactor (2 mM uridine 5'-diphosphoglucuronic acid (UDPGA)) p artially restored the activity to 25%. Our data clearly indicate that extensive glucuronidation of testosterone and its metabolites, namely DHT, 3 alpha-DIOL, 3 beta-DIOL and ADT, occurs in LNCaP cells. Because these polar metabolites are not active and cannot be reconverted into active androgens, it is suggested that glucuronosyltransferase may be a major factor in the regulation of androgen action.