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1.
J Steroid Biochem Mol Biol ; 183: 192-201, 2018 10.
Article in English | MEDLINE | ID: mdl-29936123

ABSTRACT

The progression of castration resistant prostate cancer (CRPC) is driven by the intratumoral conversion of adrenal androgen precursors to potent androgens. The expression of aldo-keto reductase 1C3 (AKR1C3), which catalyses the reduction of weak androgens to more potent androgens, is significantly increased in CRPC tumours. The oxidation of androgens to their inactive form is catalysed by 17ß-hydroxysteroid dehydrogenase type 2 (17ßHSD2), but little attention is given to the expression levels of this enzyme. In this study, we show that the 11-oxygenated androgen precursors of adrenal origin are the preferred substrate for AKR1C3. In particular we show that the enzymatic efficiency of AKR1C3 is 8- and 24-fold greater for 11-ketoandrostenedione than for the classic substrates androstenedione and 5α-androstanedione, respectively. Using three independent experimental systems and a computational model we subsequently show that increased ratios of AKR1C3:17ßHSD2 significantly favours the flux through the 11-oxygenated androgen pathway as compared to the classical or 5α-androstanedione pathways. Our findings reveal that the flux through the classical and 5α-androstanedione pathways are limited by the low catalytic efficiently of AKR1C3 towards classical androgens combined with the high catalytic efficiency of 17ßHSD2, and that the expression of the oxidative enzyme therefore plays a vital role in determining the steady state concentration of active androgens. Using microarray data from prostate tissue we confirm that the AKR1C3:17ßHSD2 ratio is significantly increased in patients undergoing androgen deprivation therapy as compared to benign tissue, and further increased in patients with CRPC. Taken together this study therefore demonstrates that the ratio of AKR1C3:17ßHSD2 is more important than AKR1C3 expression alone in determining intratumoral androgen levels and that 11-oxygenated androgens may play a bigger role in CRPC than previously anticipated.


Subject(s)
Aldo-Keto Reductase Family 1 Member C3/metabolism , Androgens/chemistry , Androgens/metabolism , Computational Biology/methods , Estradiol Dehydrogenases/metabolism , Oxygen/metabolism , Prostatic Neoplasms, Castration-Resistant/metabolism , Humans , Male , Prostatic Neoplasms, Castration-Resistant/pathology , Steroids/metabolism , Tumor Cells, Cultured
2.
Mol Cell Endocrinol ; 441: 76-85, 2017 02 05.
Article in English | MEDLINE | ID: mdl-27519632

ABSTRACT

The abundant adrenal C19 steroid 11ß-hydroxyandrostenedione (11OHA4) has been written off as a dead-end product of adrenal steroidogenesis. However, recent evidence has demonstrated that 11OHA4 is the precursor to the potent androgenic 11-oxygenated steroids, 11-ketotestosterone and 11-ketodihydrotestosterone, that bind and activate the human androgen receptor similarly to testosterone and DHT. The significance of this discovery becomes apparent when considering androgen dependent diseases such as castration resistant prostate cancer and diseases associated with androgen excess, e.g. congenital adrenal hyperplasia and polycystic ovary syndrome. In this review we describe the production and metabolism of 11-oxygenated steroids. We subsequently discuss their androgenic activity and highlight the putative role of these androgens in disease states.


Subject(s)
Androgens/metabolism , Oxygen/metabolism , Steroids/metabolism , Adrenal Cortex/metabolism , Adrenal Hyperplasia, Congenital , Animals , Female , Humans , Male , Polycystic Ovary Syndrome/metabolism , Prostatic Neoplasms, Castration-Resistant/metabolism
3.
Article in English | MEDLINE | ID: mdl-27479683

ABSTRACT

11-Oxygenated steroids such as 11-ketotestosterone and 11-ketodihydrotestosterone have recently been shown to play a putative role in the development and progression of castration resistant prostate cancer. In this study we report on the development of a high throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC(2)-MS/MS) method for the analysis of thirteen 11-oxygenated and six canonical C19 steroids isolated from a cell culture matrix. Using an Acquity UPC(2) BEH 2-EP column we found that UPC(2) resulted in superior selectivity, increased chromatographic efficiency and a scattered elution order when compared to conventional reverse phase ultra-performance liquid chromatography (UPLC). Furthermore, there was a significant improvement in sensitivity (5-50 times). The lower limits of quantification ranged between 0.01-10ngmL(-1), while the upper limit of quantification was 100ngmL(-1) for all steroids. Accuracy, precision, intra-day variation, recovery, matrix effects and process efficiency were all evaluated and found to be within acceptable limits. Taken together we show that the increased power of UPC(2)-MS/MS allows the analyst to complete in vitro assays at biologically relevant concentrations for the first time and in so doing determine the routes of steroid metabolism which is vital for studies of androgen responsive cancers, such as prostate cancer, and could highlight new mechanisms of disease progression and new targets for cancer therapy.


Subject(s)
Androgens/analysis , Chromatography, Liquid/methods , Chromatography, Supercritical Fluid/methods , High-Throughput Screening Assays , Tandem Mass Spectrometry/methods , Humans , Reference Standards , Reproducibility of Results
4.
PLoS One ; 11(7): e0159867, 2016.
Article in English | MEDLINE | ID: mdl-27442248

ABSTRACT

Dihydrotestosterone (DHT) is regarded as the most potent natural androgen and is implicated in the development and progression of castration resistant prostate cancer (CRPC). Under castrate conditions, DHT is produced from the metabolism of the adrenal androgen precursors, DHEA and androstenedione. Recent studies have shown that the adrenal steroid 11ß-hydroxyandrostenedione (11OHA4) serves as the precursor to the androgens 11-ketotestosterone (11KT) and 11-ketodihydrotestosterone (11KDHT). In this study we comprehensively assess the androgenic activity of 11KT and 11KDHT. This is the first study, to our knowledge, to show that 11KT and 11KDHT, like T and DHT, are potent and efficacious agonists of the human androgen receptor (AR) and induced both the expression of representative AR-regulated genes as well as cellular proliferation in the androgen dependent prostate cancer cell lines, LNCaP and VCaP. Proteomic analysis revealed that 11KDHT regulated the expression of more AR-regulated proteins than DHT in VCaP cells, while in vitro conversion assays showed that 11KT and 11KDHT are metabolized at a significantly lower rate in both LNCaP and VCaP cells when compared to T and DHT, respectively. Our findings show that 11KT and 11KDHT are bona fide androgens capable of inducing androgen-dependant gene expression and cell growth, and that these steroids have the potential to remain active longer than T and DHT due to the decreased rate at which they are metabolised. Collectively, our data demonstrates that 11KT and 11KDHT likely play a vital, but overlooked, role in the development and progression of CRPC.


Subject(s)
Prostatic Neoplasms, Castration-Resistant/metabolism , Testosterone/analogs & derivatives , Androgens/metabolism , Animals , Biosynthetic Pathways , Cell Line, Tumor , Cell Proliferation , Humans , Male , Protein Binding , Receptors, Androgen/metabolism , Response Elements , Testosterone/metabolism
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