Fig 1.
Biosynthesis of 11KT and 11KDHT from the adrenal androgen precursor 11OHA4.
Enzymes: 11βHSD2, 11β-hydroxysteroid dehydrogenase; 17βHSD2, 17β-hydroxysteroid dehydrogenase; SRD5A1, steroid 5α-reductase type 1; 3αHSD2, 3α-hydroxysteroid dehydrogenase. Steroids: 11OHA4, 11β-hydroxyandrostenedione; 11KA4, 11-ketoandrostenedione; 11KT, 11-ketotestosterone; 11OH-5α-dione, 11OH-5α-androstanedione; 11K-5α-dione, 11-keto-5α-androstanedione; 11KDHT, 11-ketodihydrotestosterone; 11OHAST, 11β-hydroxyandrosterone; 11KAST, 11-ketoadrenosterone; 11K-3α-adiol, 11-keto-5α-androstane-3α,17β-diol.
Fig 2.
Binding of DHT, T, 11KDHT and 11KT to the human AR (A) and transactivation via an ARE (B and C).
Binding affinities, agonist potencies and efficacies of DHT, 11KDHT, T and 11KT relative to the synthetic AR agonist mibolerone are summarised in (D). Whole cell binding assays (A) were conducted in COS-1 cells transiently transfected with pSVARo. Cells were incubated with 0.2 nM [3H]-Mib in the absence and presence of increasing concentrations of either unlabelled Mib, DHT, 11KDHT, T and 11KT for 16 hours. Results are plotted as % specific binding where the total specific binding of [3H]-Mib only is set to 100% and binding of unlabelled steroid is set as a % binding relative to that. Whole cell binding results are shown as means ± SEM of three independent experiments performed in triplicate. Transactivation assays (B and C) where performed in COS-1 cells transiently transfected with the pSVARo expression vector and the 4xSC ARE1.2-luc reporter. Agonist activity was measured by incubating cells in the presence of increasing concentrations of either Mib, DHT, T, 11KDHT or 11KT for 24 h. Induction is shown as % luciferase activity expressed in relative light units (rlu’s), with the maximal response of Mib (10−5 M) set to 100%. Luciferase assays are shown as means ± SEM of six independent experiments performed in quadruplicate.
Fig 3.
Induction of AR-regulated gene expression in LNCaP cells by DHT, 11KDHT and 11KT.
Cells were incubated with CS-FCS-supplemented media for 24 hours prior to treatment with 1 or 10 nM steroid for an additional 24 hours prior to analysis by qPCR. Gene expression was calculated relative to the geometric mean of the reference genes ALAS and PBGD. Fold change over vehicle was calculated using the method described by Pfaffl et al [48]. Results are shown as means ± SEM of three independent experiments performed in triplicate. Data from individual experiments was all normalized using log transformation and mean-centering prior to analysis [49].
Fig 4.
Induction of AR-regulated gene expression in VCaP cells by DHT, 11KDHT, T and 11KT.
Cells were incubated with CS-FCS supplemented media for 24 hours prior to treatment with 1 or 10 nM steroid for an additional 24 hours prior to analysis by qPCR. Gene expression was calculated relative to the geometric mean of the reference genes ALAS and PBGD and are expressed as the fold change over the vehicle control. Results are shown as means ± SEM of three independent experiments performed in triplicate. Data from individual experiments was all normalized using log transformation and mean-centering prior to analysis [49].
Table 1.
Regulation of AR-regulated proteins by DHT, 11KDHT, T and 11KT in VCaP cells.
Cells were incubated with CS-FCS supplemented media for 48 hours prior to treatment with 1 nM steroid. Proteins were subsequently identified using mass spectrometry. Fold changes were calculated relative to the vehicle control. Statistically significant changes are indicated (P<0.05). Results are representative of three independent experiments.
Fig 5.
Induction of cell proliferation in LNCaP and VCaP cells by DHT, 11KDHT, T and 11KT.
Cells were incubated with media supplemented with CS-FCS for 24 hours prior to treatment with 0.1, 1 or 10 nM steroids. Resazurin assays were carried out on day 7 (LNCaP) or day 10 (VCaP) after treatment. Results are shown as means ± SEM of three independent experiments with eight replicates each.
Fig 6.
Metabolism of DHT, 11KDHT, T and 11KT by LNCaP and VCaP cells.
Steroids were analysed by ultra-performance convergence chromatography-mass spectrometry (UPC2-MS/MS). Results are representative of two independent experiments performed in triplicate.