Characterising The 11β‐hydroxyandrostenedione‐pathway in In Vitro Models

Abstract

ObjectiveThe 11β‐hydroxyandrostenedione (11OHA4)‐pathway has been primarily characterised, in vitro, in prostate cells, following the quantification of 11OHA4 as a major adrenal C19 steroid. Importantly, 11keto‐testosterone (11KT) and 11keto‐dihydrotestosterone (11KDHT) are downstream active androgens of 11OHA4 —with 11β‐hydroxysteroid dehydrogenases (HSD), 17β‐HSDs and 5α‐reductases catalysing their production, and 3α‐HSDs and uridine‐diphosphate glucuronosyltransferases inactivating them [1]. Progress has been made to characterise the C11‐oxy metabolic pathways by these steroidogenic enzymes in prostate cancer cells, however, other in vitro models should be characterised in terms of their metabolism of 11OHA4 and its metabolites to determine enzymatic conversions and the production of androgens.MethodsThe metabolism of the C19 and C11‐oxy C19 steroids (1 μM) was investigated in JEG‐3 placenta choriocarcinoma, and MCF‐7 BUS and T‐47D breast cancer cells. Steroid glucuronidation and sulfation was investigated in the above mentioned cell models and in benign‐prostatic hyperplasia (BPH) cells. Ultra‐performance convergence chromatography tandem mass spectrometry (UPC2‐MS/MS) enabled the high‐throughput accurate quantification of steroid metabolites.ResultsThe metabolism of testosterone (T), 11KT, dihydrotestosterone (DHT) and 11KDHT were firstly assayed in BPH‐1 cells. Time course analysis of steroids show negligible DHT levels after 96 h, followed by 11KDHT (±0.2 μM) and T (±0.4 μM), while 11KT was present predominantly in its unchanged form throughout the assay. Steroid conjugate (glucuronidated + sulfated) levels were not detected in BPH‐1 cells. Similarly, JEG‐3 cells did not show conjugation, however DHT (0.6 μM remaining) and 11KDHT (0.9 μM remaining) were metabolised to downstream 5α‐androstane‐3α,17β‐diol and androsterone (AST), and 11OHAST and 11KAST, respectively. 11OHA4 was converted to 11keto‐androstenedione (12%) and 11KT (2.5%); and 11KT produced 11KDHT (14%). In MCF‐7 BUS cells, DHT was significantly glucuronidated (p<0.001), whereas 11KDHT was not. Furthermore, 11KAST was the only steroid assayed in the MCF‐7 BUS and T‐47D cells that was significantly sulfated (p<0.05).ConclusionsThe data highlight the production of active androgens and in the case of 11KT, the prolonged presence of a potent androgen in the cellular microenvironment. In addition, decreased inactivation of 11KDHT emphasise the assessment of 11KDHT levels and the inactivation/conjugation of the C11‐oxy steroids in clinical conditions. As evidence, we have identified higher levels of 11KDHT compared to 11KT in prostate cancer tissue and in BPH tissue and serum [2, 3]. This study also identifies, for the first time, the 11OHA4‐pathway in placenta and breast cancer cells, and the sulfation of 11KAST. Ultimately, the characterisation of in vitro pathways could shed light on active in vivo pathways in clinical disorders and diseases.Support or Funding InformationThe South African National Research Foundation (IFR170125217588; CSUR160414162143; 116776) and Stellenbosch University.