The 11β-hydroxyandrostenedione pathway and C11-oxy C(21) backdoor pathway are active in benign prostatic hyperplasia yielding 11keto-testosterone and 11keto-progesterone.

In clinical approaches to benign prostatic hyperplasia (BPH) and prostate cancer (PCa), steroidogenesis or the disruption thereof is the main thrust in treatments restricting active androgen production. Extensive studies have been undertaken focusing on testosterone and dihydrotestosterone (DHT). However, the adrenal C11-oxy C19 steroid, 11β-hydroxyandrostenedione (11OHA4), also contributes to the active androgen pool in the prostate microenvironment, and while it has been shown to impact castration resistant prostate cancer, the C11-oxy C19 steroids together with the C11-oxy C21 steroids have not been studied in BPH. The study firstly investigated the metabolism of these adrenal steroids in the BPH-1 model. Comprehensive profiles identified 11keto-testosterone as the predominant active androgen in the metabolism of the C11-oxy C19 steroids, and we identified, for the first time, 11β-hydroxy-5α-androstane-3α,17β-diol, a novel steroid in the 11OHA4-pathway. Analysis of the inactivation and reactivation of the metabolites showed that DHT is more readily inactivated than 11keto-dihydrotestosterone (11KDHT). The conversion of 11β-hydroxyprogesterone (11βOHPROG) yielded 11keto-progesterone (11KPROG), while the latter yielded 11keto-dihydroprogesterone (11KDHPROG). BPH tissue analysis identified high levels of 11β-hydroxyandrosterone (4-14 ng/g) and 11keto-androsterone (9-160 ng/g), together with androstenedione (A4; ∼7.5 ng/g). The major C11-oxy C21 steroids detected were 11βOHPROG (∼46 ng/g), 11KPROG (∼130 ng/g) as well as 11KDHPROG (∼282 ng/g). While circulatory 11βOHPROG was detected below the limit of quantification, 11KPROG and 11KDHPROG were detected at 6 and 8.5 nmol/L, respectively. Glucuronide derivatives of both 11KPROG and pregnanetriol were also detected. 11OHA4 was the major free androgen in circulation at 85.9 nmol/L, ±12-fold higher than A4, together with 5α-androstane-3α,17β-diol quantified at 69.3 nmol/L. Circulatory C11-oxy C19 steroids levels were also significantly higher (8-fold) than the C11-oxy C21 steroid levels, while the former were similar to the C19 steroid levels, in contrast to levels in PCa. The study highlights the contribution of adrenal C11-oxy steroids to the androgen pool in BPH underscoring their limited reactivation and elimination, and significant inter-individual variations regarding steroid levels and conjugation. Targeted steroid metabolome analysis is critical to understanding prostate steroidogenesis and disease progression, and analysis of circulatory C11-oxy C19 and C11-oxy C21 steroids, together with intraprostatic levels, add to our current understanding of BPH.

du Toit T ，Swart AC 《-》

Profiling adrenal 11β-hydroxyandrostenedione metabolites in prostate cancer cells, tissue and plasma: UPC(2)-MS/MS quantification of 11β-hydroxytestosterone, 11keto-testosterone and 11keto-dihydrotestosterone.

Adrenal C19 steroids 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 C19 steroids 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 C19 steroids 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 C19 metabolites at significantly higher levels than the C19 steroids, 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.

du Toit T ，Bloem LM ，Quanson JL ，Ehlers R ，Serafin AM ，Swart AC ... -  《-》

CYP17A1 exhibits 17αhydroxylase/17,20-lyase activity towards 11β-hydroxyprogesterone and 11-ketoprogesterone metabolites in the C11-oxy backdoor pathway.

Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) plays a pivotal role in the regulation of adrenal and gonadal steroid hormone biosynthesis. More recent studies highlighted the enzyme's role in the backdoor pathway leading to androgen production. Increased CYP17A1 activity in endocrine disorders and diseases are associated with elevated C21 and C19 steroids which include 17α-hydroxyprogesterone and androgens, as well as C11-oxy C21 and C11-oxy C19 steroids. We previously reported that 11β-hydroxyprogesterone (11OHP4), 21-deoxycortisol (21dF) and their keto derivatives are converted by 5α-reductases and hydroxysteroid dehydrogenases yielding C19 steroids in the backdoor pathway. In this study the 17α-hydroxylase and 17,20-lyase activity of CYP17A1 towards the unconventional C11-oxy C21 steroid substrates and their 5α- and 3α,5α-reduced metabolites was investigated in transfected HEK-293 cells. CYP17A1 catalysed the 17α-hydroxylation of 11OHP4 to 21dF and 11-ketoprogesterone (11KP4) to 21-deoxycortisone (21dE) with negligible hydroxylation of their 5α-reduced metabolites while no lyase activity was detected. The 3α,5α-reduced C11-oxy C21 steroids-5α-pregnan-3α,11β-diol-20-one (3,11diOH-DHP4) and 5α-pregnan-3α-ol-11,20-dione (alfaxalone) were rapidly hydroxylated to 5α-pregnan-3α,11β,17α-triol-20-one (11OH-Pdiol) and 5α-pregnan-3α,17α-diol-11,20-dione (11K-Pdiol), with the lyase activity subsequently catalysing to conversion to the C11-oxy C19 steroids, 11β-hydroxyandrosterone and 11-ketoandrosterone, respectively. Docking of 11OHP4, 11KP4 and the 5α-reduced metabolites, 5α-pregnan-11β-ol-3,20-dione (11OH-DHP4) and 5α-pregnan-3,11,20-trione (11K-DHP4) with human CYP17A1 showed minimal changes in the orientation of these C11-oxy C21 steroids in the active pocket when compared with the binding of progesterone suggesting the 17,20-lyase is impaired by the C11-hydroxyl and keto moieties. The structurally similar 3,11diOH-DHP4 and alfaxalone showed a greater distance between C17 and the heme group compared to the natural substrate, 17α-hydroxypregnenolone potentially allowing more orientational freedom and facilitating the conversion of the C11-oxy C21 to C11-oxy C19 steroids. In summary, our in vitro assays showed that while CYP17A1 readily hydroxylated 11OHP4 and 11KP4, the enzyme was unable to catalyse the 17,20-lyase reaction of these C11-oxy C21 steroid products. Although CYP17A1 exhibited no catalytic activity towards the 5α-reduced intermediates, once the C4-C5 double bond and the keto group at C3 were reduced, both the hydroxylation and lyase reactions proceeded efficiently. These findings show that the C11-oxy C21 steroids could potentially contribute to the androgen pool in tissue expressing steroidogenic enzymes in the backdoor pathway.

van Rooyen D ，Yadav R ，Scott EE ，Swart AC ... -  《-》

Inefficient UGT-conjugation of adrenal 11β-hydroxyandrostenedione metabolites highlights C11-oxy C(19) steroids as the predominant androgens in prostate cancer.

Although the adrenal C19 steroids, androstenedione and testosterone, contribute to prostate cancer (PCa) progression the full complement of adrenal androgens, including the C11-oxy C19 steroids, 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) and their androgenic metabolites, 11keto-testosterone (11KT) and 11keto-dihydrotestosterone (11KDHT) have, to date, not been considered. This study investigated the contribution of 11OHA4 and 11OHT to the pool of active androgens in the prostate. Steroid profiles were determined in LNCaP, C4-2B and VCaP cell models, in PCa tissue, and in plasma focussing on the inactivation, reactivation and glucuronidation of 11OHA4, 11OHT and their downstream products using ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). The C11-oxy C19 steroids were the predominant steroids with the production of 11KT and 11KDHT in prostate cell models identifying 11β-hydroxysteroid dehydrogenase type 2 activity. Active:inactive steroid ratios indicated efficient inactivation of dihydrotestosterone (DHT) and 11KDHT by 3α-hydroxysteroid dehydrogenases, while the reactivation of DHT by retinol-like dehydrogenases was greater than the reactivation of 11KDHT. In PCa tissue, inactive C11-oxy C19 steroids ranged from 27 to 30 ng/g, whereas inactive C19 steroids were below 1 ng/g. Steroid glucuronidation was impeded: in VCaP cells, the C11-oxy C19 steroids were unconjugated and the C19 steroids fully conjugated; in C4-2B cells, all steroids were unconjugated, except for DHT of which 50% was conjugated; in LNCaP cells only androsterone, 11KT and 11β-hydroxyandrosterone were unconjugated. In PCa patients' plasma 11KDHT was present only in the unconjugated form, with 11KT also predominantly unconjugated (90-95%). Even though plasma and tissue sample numbers were limited, this study serves to demonstrate the abundance of C11-oxy C19 steroids, with notable differences in their metabolism, dictated by steroidogenic enzymes and hampered conjugation, affecting active androgen levels. Larger cohorts are required to analyse profiles in modulated metabolic pathways, in order to shed light on treatment outcomes. The C11-oxy C19 steroids are involved in PCa, with impeded glucuronidation in PCa ascribing a dominant role to these steroids in disease progression.

du Toit T ，Swart AC 《-》

The in vitro metabolism of 11β-hydroxyprogesterone and 11-ketoprogesterone to 11-ketodihydrotestosterone in the backdoor pathway.

Increased circulating 11β-hydroxyprogesterone (11OHP4), biosynthesised in the human adrenal, is associated with 21-hydroxylase deficiency in congenital adrenal hyperplasia. 17α-hydroxyprogesterone levels are also increased, with the steroid's metabolism to dihydrotestosterone in the backdoor pathway contributing to hyperandrogenic clinical conditions. In this study we investigated the in vitro biosynthesis and downstream metabolism of 11OHP4. Both cytochrome P450 11β-hydroxylase and aldosterone synthase catalyse the biosynthesis of 11OHP4 from progesterone (P4) which is converted to 11-ketoprogesterone (11KP4) by 11β-hydroxysteroid dehydrogenase type 2, while type 1 readily catalysed the reverse reaction. We showed in HEK-293 cells that these C11-oxy C21 steroids were metabolised by steroidogenic enzymes in the backdoor pathway-5α-reductase (SRD5A) and 3α-hydroxysteroid type 3 (AKR1C2) converted 11OHP4 to 5α-pregnan-11β-ol,3,20-dione and 5α-pregnan-3α,11β-diol-20-one, while 11KP4 was converted to 5α-pregnan-3,11,20-trione and 5α-pregnan-3α-ol-11,20-dione (alfaxalone), respectively. Cytochrome P450 17α-hydroxylase/17,20-lyase catalysed the hydroxylase and lyase reaction to produce the C11-oxy C19 steroids demonstrated in the conversion of alfaxalone to 11-oxy steroids demonstrated in the conversion of alfaxalone to 11ketoandrosterone. In LNCaP cells, a prostate cancer cell model endogenously expressing the relevant enzymes, 11OHP4 and 11KP4 were metabolised to the potent androgen, 11-ketodihydrotestosterone (11KDHT), thus suggesting the C11-oxy C21 steroids contribute to the pool of validating the in vitro biosynthesis of C11-oxy C19 steroids from C11-oxy C21 steroids. The in vitro reduction of 11KP4 at C3 and C5 by AKR1C2 and SRD5A has confirmed the metabolic route of the urinary metabolite, 3α,20α-dihydroxy-5β-pregnan-11-one. Although our assays have demonstrated the conversion of 11OHP4 and 11KP4 by steroidogenic enzymes in the backdoor pathway yielding 11KDHT, thus suggesting the C11-oxy C21 steroids contribute to the pool of potent androgens, the in vivo confirmation of this metabolic route remains challenging.

van Rooyen D ，Gent R ，Barnard L ，Swart AC ... -  《-》