title: Profiling adrenal 11β-hydroxyandrostenedione metabolites in prostate cancer cells, tissue and plasma: UPC2-MS/MS quantification of 11β-hydroxytestosterone, 11keto-testosterone and 11keto-dihydrotestosterone.
abstract: Adrenal C19steroids serve as precursors to active androgens in the prostate. Androstenedione (A4), 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) are metabolised to potent androgen receptor (AR) agonists, dihydrotestosterone (DHT), 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). The identification of 11OHA4 metabolites, 11KT and 11KDHT, as active androgens has placed a new perspective on adrenal C11-oxy C19steroids and their contribution to prostate cancer (PCa). We investigated adrenal androgen metabolism in normal epithelial prostate (PNT2) cells and in androgen-dependent prostate cancer (LNCaP) cells. We also analysed steroid profiles in PCa tissue and plasma, determining the presence of the C19steroids and their derivatives using ultra-performance liquid chromatography (UHPLC)- and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). In PNT2 cells, sixty percent A4 (60%) was primarily metabolised to 5α-androstanedione (5αDIONE) (40%), testosterone (T) (10%), and androsterone (AST) (10%). T (30%) was primarily metabolised to DHT (10%) while low levels of A4, 5αDIONE and 3αADIOL (≈20%) were detected. Conjugated steroids were not detected and downstream products were present at <0.05μM. Only 20% of 11OHA4 and 11OHT were metabolised with the former yielding 11keto-androstenedione (11KA4), 11KDHT and 11β-hydroxy-5α-androstanedione (11OH-5αDIONE) and the latter yielding 11OHA4, 11KT and 11KDHT with downstream products <0.03μM. In LNCaP cells, A4 (90%) was metabolised to AST-glucuronide via the alternative pathway while T was detected as T-glucuronide with negligible conversion to downstream products. 11OHA4 (80%) and 11OHT (60%) were predominantly metabolised to 11KA4 and 11KT and in both assays more than 50% of 11KT was detected in the unconjugated form. In tissue, we detected C11-oxy C19metabolites at significantly higher levels than the C19steroids, with unconjugated 11KDHT, 11KT and 11OHA4 levels ranging between 13 and 37.5ng/g. Analyses of total steroid levels in plasma showed significant levels of 11OHA4 (≈230-440nM), 11KT (≈250-390nM) and 11KDHT (≈19nM). DHT levels (<0.14nM) were significantly lower. In summary, 11β-hydroxysteroid dehydrogenase type 2 activity in PNT2 cells was substantially lower than in LNCaP cells, reflected in the conversion of 11OHA4 and 11OHT. Enzyme substrate preferences suggest that the alternate pathway is dominant in normal prostate cells. Glucuronidation activity was not detected in PNT2 cells and while all T derivatives were efficiently conjugated in LNCaP cells, 11KT was not. Substantial 11KT levels were also detected in both PCa tissue and plasma. 11OHA4 therefore presents a significant androgen precursor and its downstream metabolism to 11KT and 11KDHT as well as its presence in PCa tissue and plasma substantiate the importance of this adrenal androgen.
authors.affiliation: Department of Biochemistry, Stellenbosch University, Stellenbosch 7600, South Africa.
authors.forename: Therina
authors.initials: T
authors.lastname: du Toit
authors.affiliation: Department of Biochemistry, Stellenbosch University, Stellenbosch 7600, South Africa.
authors.forename: Liezl M
authors.initials: LM
authors.lastname: Bloem
authors.affiliation: Department of Biochemistry, Stellenbosch University, Stellenbosch 7600, South Africa.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Quanson
authors.affiliation: Department of Biochemistry, Stellenbosch University, Stellenbosch 7600, South Africa.
authors.forename: Riaan
authors.initials: R
authors.lastname: Ehlers
authors.affiliation: Division of Radiobiology, Department of Medical Imaging and Clinical Oncology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg 7505, South Africa.
authors.forename: Antonio M
authors.initials: AM
authors.lastname: Serafin
authors.affiliation: Department of Biochemistry, Stellenbosch University, Stellenbosch 7600, South Africa. Electronic address: acswart@sun.ac.za.
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Swart
mesh_terms: D000311:Adrenal Glands / Q000378:metabolism*; D000368:Aged; D000728:Androgens / Q000378:metabolism; D000735:Androstenedione / Q000031:analogs & derivatives* / Q000378:metabolism; D045744:Cell Line, Tumor; D013196:Dihydrotestosterone / Q000378:metabolism; D018450:Disease Progression; D015972:Gene Expression Regulation, Neoplastic; D020723:Glucuronic Acid / Q000737:chemistry; D006801:Humans; D006915:Hydroxytestosterones / Q000378:metabolism; D008297:Male; D011471:Prostatic Neoplasms / Q000378:metabolism*; D013256:Steroids / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013739:Testosterone / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: C586666:11-ketodihydrotestosterone; D000728:Androgens; D006915:Hydroxytestosterones; D013256:Steroids; D013196:Dihydrotestosterone; D013739:Testosterone; D000735:Androstenedione; C003582:11-hydroxyandrostenedione; D020723:Glucuronic Acid; C003600:11-ketotestosterone
keywords: 11K-DHT; 11keto-testosterone (11KT); Adrenal 11β -hydroxy-androstenedione (11β OHA4); Castration-resistant prostate cancer (CRPC); LC–MS/MS; LNCaP prostate cancer cell model; PNT2 epithelial prostate cell model