We showed previously that nuclear localization of the androgen receptor (AR) and expression of the androgen-responsive gene FK506-binding protein 5 (FKBP5) in esophageal adenocarcinoma (EAC) tissues were associated with decreased patient survival, suggesting a role for androgens in this cancer.

To investigate the effect of the AR ligand <em>5α</em>-dihydrotestosterone (<em>DHT</em>) on AR-expressing EAC cell lines in vitro.

In tissue resection specimens from EAC patients, FKBP5 expression was positively associated with proliferation as measured by Ki-67 expression. We stably transduced AR into three AR-negative EAC cell lines, OE33, JH-EsoAd1, and OE19, to investigate androgen signaling in vitro. In the AR-expressing cell lines, 10 nM DHT, the concentration typically used to study AR signaling, induced changes in the expression of androgen-responsive genes and inhibited proliferation by inducing cell cycle arrest and senescence. At lower DHT concentrations near the half maximal inhibitory concentration (IC50), the AR-expressing cell lines proliferated and there were changes in the expression of androgen-responsive genes. In direct co-culture with cancer-associated fibroblast-like PShTert myofibroblasts, 10 nM DHT induced changes in the expression of androgen-responsive genes but did not inhibit proliferation.

This is the first study to show that EAC cell lines respond to androgen in vitro. Proliferation together with the expression of androgen-responsive genes was dependent on the concentration of DHT, or the presence of a permissive microenvironment, consistent with observations in the tissues. These findings are consistent with a role for androgen signaling in EAC.

Deficiency of the <em>5α</em>-reductase 2 enzyme impairs the conversion of testosterone to dihydrotestosterone (<em>DHT</em>) and differentiation of external genitalia, seminal vesicles and prostate in males. The present study describes the phenotype, genotype and gender identity in a large cohort of patients with <em>5α</em>RD2. All patients underwent detailed clinical evaluation, hormonal profile, karyotyping and molecular analysis of the SRD5A2 gene. The molecular analysis of the SRD5A2 gene showed the presence of mutant alleles in 24 patients. We found 6 novel mutations IVS(1-2) T>C, p.A52T, 188-189insTA, 904-905ins A, p.A12T and p.E57X in our patients. All patients had ambiguous genitalia and the degrees of under-virilization ranged from penoscrotal hypospadias and microphallus to clitoromegaly. The position of gonads was variable in patients with same mutation. All the patients with mutations in the SRD5A2 gene had male gender identity. Those reared as female had gender dysphoria and underwent gender reassignment. Though a specific genotype-phenotype correlation could not be established in our patient but confirming the diagnosis of <em>5α</em>RD2 with assessment of the SRD5A2 gene may help in appropriate gender assignment.

Triple-negative breast cancer (TNBC) is an aggressive subtype that lacks effective targeted therapeutics strategy and has poor prognosis. Targeting androgen receptor (AR) in TNBC is thought to be a promising approach. We hypothesized that AR, functioning as a transcription factor, controls cell behavior via regulating the expression of microRNA molecules (miRNAs). The expression of 84 breast cancer-specific miRNAs in MDA-MB-231 cells, a highly invasive TNBC model system, was investigated using PCR arrays following treatment of cells with <em>5α</em>-dihydrotestosterone (<em>DHT</em>). The expression of 33 miRNAs was changed by more than 2 folds including miR-328-3p, which was up-regulated by 13 folds. Transfection of cells with either miR-328-3p mimic or anti-sense molecules decreased cell motility. <em>DHT</em>-mediated effect on the expression and function of CD44, a target of miR-328-3p, was investigated. CD44 expression and cell adhesion to hyaluronic acid (HA) were down-regulated when cells were treated with <em>DHT</em> or transfection with a miR-328-3p mimic. On the other hand, the AR antagonist, bicalutamide, or transfection of cells with miR-328-3p anti-sense molecules had the opposite effect. Cells transfected with miR-328-3p anti-sense molecules reduced the negative effect of <em>DHT</em> on CD44 expression and cell adhesion to HA. In addition, <em>DHT</em> further reduced the expression of CD44 and cell adhesion to HA in cells transfected with miR-328-3p mimic. These results strongly suggest that miRNAs can mediate AR regulation of breast cancer cells and that AR controls the expression of CD44 via miRNA-dependent and independent mechanisms.

Circadian clocks have been described in each tissue of the hypothalamo-pituitary-ovarian axis. Although a role for the clock in the timing of ovulation is indicated, the impact of diseases that disrupt fertility on clock function or the clocks' role in the etiology of these pathologies has yet to be fully appreciated. Polycystic ovary syndrome (PCOS) is a particularly devastating endocrinopathy, affecting approximately 10% of women at childbearing age. Common features of PCOS are a polycystic ovary, amenorrhea, and excess serum androgen. Approximately 40% of these women have metabolic syndrome, including hyperinsulinemia, dyslipidemia, and hyperleptinemia. It has been suggested that excess androgen is a critical factor in the etiology of PCOS. We have examined the effects of androgen excess during puberty on the phase of circadian clocks in tissues of the metabolic and hypothalamo-pituitary-ovarian axes. Female period1-luciferase (per1-luc) rats were exposed to androgen (<em>5α</em>-dihydrotestosterone [<em>DHT</em>]) or placebo for 4-6 weeks (short term) or 9-15 weeks (long term). As expected, <em>DHT</em>-treated animals gained more weight than controls and had disrupted estrous cycles. At the end of treatment, tissues, including the liver, lung, kidney, white adipose, cornea, pituitary, oviduct, and ovarian follicles, were cultured, and per1-luc expression in each was recorded. Analysis of per1-luc expression revealed that <em>DHT</em> exposure increased phase distribution of multiple oscillators, including ovarian follicles, liver, and adipose, and altered phase synchrony between animals. These data suggest that excess androgen during puberty, a common feature of PCOS, negatively affects internal circadian organization in both the reproductive and metabolic axes.

In brainstem slices of male rats, we examined in single neurons of the medial vestibular nucleus (MVN) the effect of exogenous administration of estrogenic (17β-estradiol, E2) and androgenic (<em>5α</em>-dihydrotestosterone, <em>DHT</em>) steroids on the synaptic response to vestibular afferent stimulation. By whole cell patch clamp recordings we showed that E2 induced synaptic long-term potentiation (LTP) that was cancelled by the subsequent administration of <em>DHT</em>. Conversely, <em>DHT</em> induced synaptic long-term depression (LTD) that was partially reversed by E2. The electrophysiological findings were supported by immunohistochemical analysis showing the presence of estrogen (ER: α and β) and androgen receptors (AR) in the MVN neurons. We found that a large number of neurons were immunoreactive for ERα, ERβ, and AR and most of them co-localized ERβ and AR. We also showed the presence of P450-aromatase (ARO) in the MVN neurons, clearly proving that E2 can be locally synthesized in the MVN. On the whole, these results demonstrate a role of estrogenic and androgenic signals in modulating vestibular synaptic plasticity and suggest that the enhancement or depression of vestibular synaptic response may depend on the local conversion of T into E2 or <em>DHT</em>.

Neurosteroids, such as testosterone and their metabolites, are known to modulate neuronal excitability. The enzymes regulating the metabolism of these neurosteroids, thus, may be targeted as a noval strategy for the development of new antiepileptic drugs. The present work targeted two such enzymes i,e aromatase and <em>5α</em>-reductase in order to explore the potential of letrozole (an aromatase inhibitor) on pentylenetetrazole (PTZ)-induced kindling in mice and the ability of finasteride (a <em>5α</em>-reductase inhibitor) to modulate any such effects. PTZ (30 mg/kg, i.p.), when administered once every two days (for a total of 24 doses) induced kindling in Swiss albino mice. Letrozole (1 mg/kg, p.o.), administered prior to PTZ, significantly reduced the % incidence of kindling, delayed mean onset time of seizures and reduced seizure severity score. Letrozole reduced the levels of plasma 17β-estradiol after induction of kindling. The concurrent administration of finasteride and letrozole produced effects similar to letrozole on PTZ-kindling and on estradiol levels. This implies that the ability of letrozole to redirect the synthesis of dihydrotestosterone (<em>DHT</em>) and <em>5α</em>-androstanediol from testosterone doesn't appear to play a significant role in the protective effects of letrozole against PTZ kindling. Letrozole, however, increased the levels of <em>5α</em>-<em>DHT</em> in mice plasma. The aromatase inhibitors, thus, may be exploited for inhibiting the synthesis of proconvulsant (17β-estradiol) and/or redirecting the synthesis of anticonvulsant (<em>DHT</em> and <em>5α</em>-androstanediol) neurosteroids.

Glioblastoma (GB) is the most common and aggressive primary brain tumor in adult humans. Therapeutic resistance and tumor recurrence after surgical resection contributes to a poor prognosis for glioblastoma patients. Men are known to be more likely than women to develop an aggressive form of GB. Although the reasons for this disparity remain poorly understood, differences in sex steroids have emerged as a leading explanation. Studies indicate that GB-derived cells express androgen receptors (ARs) and synthesize androgens, suggesting that androgens may have a role in the tumor pathogenesis. Thus, our objective was to investigate the effects of the <em>5α</em>-reductase enzyme inhibitor dutasteride, the AR antagonists cyproterone and flutamide, and combinations of these drugs on the metabolism, proliferation, and invasion capacity of GB-derived U87 cells. We also examined the effects of three natural androgens testosterone, androstenedione and dihydrotestosterone (T, A₄, and <em>DHT</em>) on these cells. Cell metabolism was investigated by MTT assay, proliferation was assessed by the bromodeoxyuridine (BrdU) incorporation assay, and invasion was assessed by Boyden chamber assay. The results revealed that T and especially <em>DHT</em>, but not A₄, increased U87 cell metabolism and proliferation. Following these findings, we examined the effect of adding dutasteride, cyproterone, or flutamide to the culture media and found that they all significantly decreased cell metabolism and proliferation. Dutasteride also significantly reduced cell invasion. Moreover, any combination of these drugs enhanced their inhibitory effects; the combination of dutasteride to flutamide was most effective at decreasing GB cell proliferation. Our results suggest that administering a combination of AR antagonists and enzyme blockers may be a more effective alternative treatment for GB.

Keywords: Glioblastoma; androgens; cancer; cyproterone; dutasteride; finasteride.

In rodents, the hypothalamo-pituitary-adrenal (HPA) axis is controlled by a precise regulatory mechanism that is influenced by circulating gonadal and adrenal hormones. In males, gonadectomy increases the adrenocorticotropic hormone (ACTH) and corticosterone (CORT) response to stressors, and androgen replacement returns the response to that of the intact male. Testosterone (T) actions in regulating HPA activity may be through aromatization to estradiol, or by <em>5α</em>-reduction to the more potent androgen, dihydrotestosterone (<em>DHT</em>). To determine if the latter pathway is involved, we assessed the function of the HPA axis response to restraint stress following hormone treatments, or after peripheral or central treatment with the <em>5α</em>-reductase inhibitor, finasteride. Initially, we examined the timecourse whereby gonadectomy alters the CORT response to restraint stress. Enhanced CORT responses were evident within 48 h following gonadectomy. Correspondingly, treatment of intact male rats with the <em>5α</em>-reductase inhibitor, finasteride, for 48 h, enhanced the CORT and ACTH response to restraint stress. Peripheral injections of gonadectomized male rats with <em>DHT</em> or T for 48 h reduced the ACTH and CORT response to restraint stress. The effects of T, but not <em>DHT</em>, could be blocked by the third ventricle administration of finasteride prior to stress application. These data indicate that the actions of T in modulating HPA axis activity involve <em>5α</em>-reductase within the central nervous system. These results further our understanding of how T acts to modulate the neuroendocrine stress responses and indicate that <em>5α</em> reduction to <em>DHT</em> is a necessary step for T action.

The androgen-directed treatment of prostate cancer (PCa) is fraught with the recurrent profile of failed treatment due to drug resistance and must be addressed if we are to provide an effective therapeutic option. The most singular difficulty in the treatment of PCa is the failure to respond to classical androgen withdrawal or androgen blockade therapy, which often develops as the malignancy incurs genetic alterations and gain-of-function somatic mutations in the androgen receptor (AR). Physical cellular damaging therapeutic agents, such as radiation or activatable heat-generating transducers would circumvent classical "anti-functional" biological resistance, but to become ultimately effective would require directed application modalities. To this end, we have developed a novel AR-directed therapeutic agent by creating bivalent androgen hormone-AF-2 compounds that bind with high affinity to AR within cells. Here, we used molecular modeling and synthetic chemistry to create a number of compounds by conjugating <em>5α</em>-dihydrotestosterone (<em>DHT</em>) to various AF-2 motif sequence peptides, through the use of a glycine and other spacer linkers. Our data indicates these compounds will bind to the AR in vitro and that altering the AF-2 peptide composition of the compound does indeed improve affinity for the AR. We also show that many of these bivalent compounds can readily pass through the plasma membrane and effectively compete against androgens alone.

Polycystic ovary syndrome (PCOS) is a reproductive and metabolic disorder characterized by hyperandrogenism, ovulatory dysfunction, visceral obesity and insulin resistance. We hypothesized that changes in glucocorticoid metabolism and signaling in the visceral adipose tissue may contribute to disturbances of lipid metabolism in the rat model of PCOS obtained by <em>5α</em>-dihydrotestosterone (<em>DHT</em>) treatment of prepubertal female Wistar rats. The results confirmed that <em>DHT</em> treatment caused anovulation, obesity and dyslipidemia. Enhanced glucocorticoid prereceptor metabolism, assessed by elevated intracellular corticosterone and increased 11 beta-hydroxysteroid dehydrogenase type 1 mRNA and protein levels, was accompanied by glucocorticoid receptor (GR) nuclear accumulation. In concert with the increased expression of GR-regulated prolipogenic genes (lipin-1, sterol regulatory element binding protein 1, fatty acid synthase, phosphoenolpyruvate carboxykinase), histological analyses revealed hypertrophic adipocytes. The results suggest that glucocorticoids influence lipid metabolism in the visceral adipose tissue in the way that may contribute to pathogenesis of metabolic disturbances associated with PCOS.

Great advances in tissue androgen analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) have made it possible to evaluate the tissue androgen content from a single needle prostate biopsy specimen. In this study, we investigated if pre-treatment androgen content in prostate biopsy specimens could predict their response to primary androgen deprivation therapy (ADT) and future castration-resistant prostate cancer (CRPC). One-hundred and sixty-five prostate cancer patients who received primary ADT were enrolled. They had received multiple core prostate needle biopsy at diagnosis, and an additional one needle biopsy specimen was obtained for tissue androgen determination using LC-MS/MS. The patients' prostate specific antigen (PSA) values were periodically followed during the treatment and patients were determined to have CRPC when their PSA value increased continuously to 25% above the nadir and a 2.0 ng/mL increase. A significant correlation was found between PSA value decline velocity (PSA half-time) after ADT and pre-ADT tissue androgen content. Twenty-three patients were determined to have CRPC. These CRPC patients had a significantly high concentration of tissue T (p < 0.01) and low concentration of tissue <em>5α</em>-dihydrotestosterone (<em>DHT</em>) (p < 0.01), resulting in a higher tissue T/<em>DHT</em> ratio (p < 0.001). A multivariate Cox proportional hazard model revealed the pre-ADT tissue T/<em>DHT</em> ratio and Gleason score as independent predictors for CRPC development. By using the two statistically significant variables, the relative risk of CRPC development could be calculated. The results of this study suggest that the evaluation of prostate androgen content in a single needle biopsy specimen may be useful to predict future CRPC development after primary ADT. Further studies are required for the clinical application of T/<em>DHT</em> ratio evaluation.

BACKGROUND

A common germline variant in HSD3B1(1245A>C) encodes for a hyperactive 3β-hydroxysteroid dehydrogenase 1 (3βHSD1) missense that increases metabolic flux from extragonadal precursor steroids to <em>DHT</em> synthesis in prostate cancer. Enabling of extragonadal <em>DHT</em> synthesis by HSD3B1(1245C) predicts for more rapid clinical resistance to castration and sensitivity to extragonadal androgen synthesis inhibition. HSD3B1(1245C) thus appears to define a subgroup of patients who benefit from blocking extragonadal androgens. However, abiraterone, which is administered to block extragonadal androgens, is a steroidal drug that is metabolized by 3βHSD1 to multiple steroidal metabolites, including 3-keto-<em>5α</em>-abiraterone, which stimulates the androgen receptor. Our objective was to determine if HSD3B1(1245C) inheritance is associated with increased 3-keto-<em>5α</em>-abiraterone synthesis in patients.

METHODS

First, we characterized the pharmacokinetics of 7 steroidal abiraterone metabolites in 15 healthy volunteers. Second, we determined the association between serum 3-keto-<em>5α</em>-abiraterone levels and HSD3B1 genotype in 30 patients treated with abiraterone acetate (AA) after correcting for the determined pharmacokinetics.

RESULTS

Patients who inherit 0, 1, and 2 copies of HSD3B1(1245C) have a stepwise increase in normalized 3-keto-<em>5α</em>-abiraterone (0.04 ng/ml, 2.60 ng/ml, and 2.70 ng/ml, respectively; P = 0.002).

CONCLUSIONS

Increased generation of 3-keto-<em>5α</em>-abiraterone in patients with HSD3B1(1245C) might partially negate abiraterone benefits in these patients who are otherwise more likely to benefit from CYP17A1 inhibition.

BACKGROUND

Prostate Cancer Foundation Challenge Award, National Cancer Institute.

Estrogenic activities of testosterone (T) and <em>5a</em>-dihydrotestosterone (<em>DHT</em>) were detected and measured by using their specific stimulatory effects on alkaline phosphatase (AP) activity in human endometrial adenocarcinoma cells of the Ishikawa Var-1 line. These two physiologic androgens were able to induce, at microM concentrations, estrogenic effect believed to be mediated by the estrogen receptor (ER) since the antiestrogens ICI-164384 and 4-hydroxytamoxifen (OHTam), but not the antiandrogens hydroxyflutamide (OHFl) or cyproterone acetate (CPA), reversed that effect. By using another in vitro bioassay, based on the progestin-specific stimulation of AP activity in cells of the T47D human breast cancer line, progestagenic activity was detected and measured in T, <em>DHT</em> and three synthetic androgens: nandrolone (19-nortestosterone). 7 alpha-methyl 19-nortestosterone (MENT) and mibolerone (7 alpha, 17 alpha-dimethyl 19-nortestosterone) (DMNT). While progestagenic effects of T and <em>DHT</em> required relatively high concentrations (microM levels), the synthetic androgens stimulated AP activity at nM or pM levels. These effects seem to be mediated by the progesterone receptor (PR), since they are completely abolished by the antiprogestins RU-486, ZK-98299 and ZK-112993, but not by the antiandrogen OHFl. These simple in vitro bioassays, expressing biological effects of the test compounds in human cells in culture, revealed dual or multiple hormonal activities coexisting in a single compound and provide quantitative information of considerable pharmacological importance concerning the complex actions of drugs.

Previous studies by our group have shown that low intra-prostatic dihydrotestosterone (<em>DHT</em>) induced BPH epithelial cells (BECs) to recruit CD8+ T cells. However, the influence of the recruited CD8+ T cells on BECs under a low androgen level is still unknown. Here, we found CD8+ T cells have the capacity to promote proliferation of BECs in low androgen condition. Mechanism dissection revealed that interaction between CD8+ T cells and BECs through secretion of CCL5 might promote the phosphorylation of STAT5 and a higher expression of CCND1 in BECs. Suppressed CCL5/STAT5 signals via CCL5 neutralizing antibody or STAT5 inhibitor Pimozide led to reverse CD8+ T cell-enhanced BECs proliferation. IHC analysis from Finasteride treated patients showed PCNA expression in BECs was highly correlated to the level of CD8+ T cell infiltration and the expression of CCL5. Consequently, our data indicated infiltrating CD8+ T cells could promote the proliferation of BECs in low androgen condition via modulation of CCL5/STAT5/CCND1 signaling. The increased secretion of CCL5 from the CD8+ T cells/BECs interaction might help BECs survive in a low <em>DHT</em> environment. Targeting these signals may provide a new potential therapeutic approach to better treat BPH patients who failed the therapy of <em>5α</em>-reductase inhibitors.

OBJECTIVE

Low-circulating testosterone is associated with development of type 2 diabetes in obese men. In this study, we examined the effects of experimental overfeeding and weight gain on serum levels of sex hormones and skeletal muscle expression of steroidogenic enzymes in healthy men with (FH+) and without (FH-) a family history of type 2 diabetes.

METHODS

Following a 3-day lead in energy balanced diet, FH+ (n = 9) and FH- men (n = 11) were overfed by 5200 kJ/day (45% fat) for 28 days. Body weight, fasting glucose, insulin, sex steroid, sex hormone binding globulin (SHBG) levels, insulin sensitivity (hyperinsulinaemic-euglycaemic clamp) and body fat (DXA) were assessed in all individuals at baseline and day 28, and sex steroidogenesis-related enzyme expression in vastus lateralis biopsies was examined in a subset (n = 11).

RESULTS

Body weight, fat mass and fasting insulin levels were increased by overfeeding (P < 0.01) and insulin was increased significantly more in FH+ men (P<0.01). Serum sex hormone binding globulin (SHBG) and <em>5α</em>-dihydrotestosterone (<em>DHT</em>) were reduced with overfeeding (P < 0.05), and serum testosterone and <em>DHT</em> were reduced to a greater extent in FH+ men (P < 0.05). Overfeeding reduced mRNA expression of 3β-hydroxysteroid dehydrogenase (HSD) and 17βHSD (P ≤ 0.007), independently of group. <em>5α</em>-Reductase (SRD5A1) mRNA expression was not changed overall, but a time by group interaction was observed (P = 0.04).

CONCLUSIONS

Overfeeding reduced SHBG and muscle expression of enzymes involved in the formation of testosterone in skeletal muscle. Men with a family history of T2DM were more susceptible to deleterious outcomes of overfeeding with greater reductions in serum testosterone and DHT and greater increases in markers of insulin resistance, which may contribute to increased risk of developing type 2 diabetes.

The normal prostate as well as prostatic diseases are influenced by androgens. The exact reason for an altered and uncontrolled response to androgens, whether benign as in benign prostate hyperplasia (BPH) or malignant as in the case of prostate cancer (PC), is not known in detail. Nevertheless, restriction of androgen receptor activation by reduction of available androgens is of great clinical value in both diseases. In BPH the inhibition of the conversion of testosterone into <em>5α</em>-dihydrotestosterone (<em>DHT</em>) by <em>5α</em>-reductase (5AR) is highly efficient and used in general practice, while the situation in PC is more complex. Specific inhibition of 5AR does not provide as efficient relief of symptoms as general androgen deprivation therapy (ADT), and the use of 5ARI for PC prevention is still under debate. Further, the altered steroid metabolism in castration resistant prostate cancer (CRPC) together with the complex paracrine signalling between different androgen responsive cell types, make the development of more specific drugs targeting androgen receptor signalling both more relevant and challenging.

Benign prostatic hyperplasia and prostate cancer can be treated with the <em>5α</em>-reductase inhibitors, finasteride and dutasteride, when pharmacodynamic biomarkers are useful in assessing response. A novel method was developed to measure the substrates and products of <em>5α</em>-reductases (testosterone, <em>5α</em>-dihydrotestosterone (<em>DHT</em>), androstenedione) and finasteride and dutasteride simultaneously by liquid chromatography tandem mass spectrometry, using an ABSciex QTRAP(®) 5500, with a Waters Acquity™ UPLC. Analytes were extracted from serum (500 µL) via solid-phase extraction (Oasis(®) HLB), with (13)C3-labelled androgens and d9-finasteride included as internal standards. Analytes were separated on a Kinetex C18 column (150 × 3 mm, 2.6 µm), using a gradient run of 19 min. Temporal resolution of analytes from naturally occurring isomers and mass +2 isotopomers was ensured. Protonated molecular ions were detected in atmospheric pressure chemical ionisation mode and source conditions optimised for <em>DHT</em>, the least abundant analyte. Multiple reaction monitoring was performed as follows: testosterone (m/z 289 → 97), <em>DHT</em> (m/z 291 → 255), androstenedione (m/z 287 → 97), dutasteride (m/z 529 → 461), finasteride (m/z 373 → 317). Validation parameters (intra- and inter-assay precision and accuracy, linearity, limits of quantitation) were within acceptable ranges and biological extracts were stable for 28 days. Finally the method was employed in men treated with finasteride or dutasteride; levels of <em>DHT</em> were lowered by both drugs and furthermore the substrate concentrations increased.

Human 3-alpha hydroxysteroid dehydrogenase type 3 (3α-HSD3) has an essential role in the inactivation of <em>5α</em>-dihydrotestosterone (<em>DHT</em>). Notably, human 3α-HSD3 shares 97.8% sequence identity with human 20-alpha hydroxysteroid dehydrogenase (20α-HSD) and there is only one amino acid difference (residue 54) that is located in their steroid binding pockets. However, 20α-HSD displays a distinctive ability in transforming progesterone to 20α-hydroxy-progesterone (20α-OHProg). In this study, to understand the role of residue 54 in the steroid binding and discrimination, the V54L mutation in human 3α-HSD3 has been created. We have solved two crystal structures of the 3α-HSD3·NADP(+)·Progesterone complex and the 3α-HSD3 V54L·NADP(+)·progesterone complex. Interestingly, progesterone adopts two different binding modes to form complexes within the wild type enzyme, with one binding mode similar to the orientation of a bile acid (ursodeoxycholate) in the reported ternary complex of human 3α-HSD3·NADP(+)·ursodeoxycholate and the other binding mode resembling the orientation of 20α-OHProg in the ternary complex of human 20α-HSD·NADP(+)·20α-OHProg. However, the V54L mutation directly restricts the steroid binding modes to a unique one, which resembles the orientation of 20α-OHProg within human 20α-HSD. Furthermore, the kinetic study has been carried out. The results show that the V54L mutation significantly decreases the 3α-HSD activity for the reduction of <em>DHT</em>, while this mutation enhances the 20α-HSD activity to convert progesterone.

The role of testosterone in cardiovascular (CV) homeostasis is in controversy, and the exact effects of testosterone on the cardiovascular system remain poorly understood. Testosterone is metabolized by aromatase into 17β-estradiol and by <em>5α</em>-reductase into dihydrotestosterone (<em>DHT</em>). Thus, identification of these metabolites in the heart may help to explain the controversy regarding the cardiovascular effects of testosterone. We analyzed the expression patterns of these testosterone-metabolizing enzymes and assessed the effect of its enzymatic activity inhibition on ischemia (40 min)/reperfusion (4h, I/R) via the left anterior descendent coronary artery in intact and gonadectomized male rats. Myocardial damage was measured as percentage of infarcted area vs. area at risk. Aromatase and <em>5α</em>-reductase protein expression was found in the left ventricle of intact and orchidectomized rats. Exogenous testosterone had no effect on I/R induced myocardial damage in intact male rats, meanwhile exogenous testosterone protects against I/R injury in orchidectomized rats. However, enzymatic inhibition of aromatase increased myocardial damage in the presence of testosterone, while enzymatic inhibition of <em>5α</em>-reductase significantly decreased the level of myocardial damage. Our results also showed that sub-chronic inhibition of <em>5α</em>-reductase resulted in myocardial protection in both groups. Furthermore, in orchidectomized and intact male rats IV treatment with <em>DHT</em> induces a significant increase in the myocardial damage induced by I/R. Thus, the effect of testosterone on cardiovascular pathophysiology could be related, at least in part to changes in the balance of testosterone <em>5α</em>-reduction and aromatization.

BACKGROUND

This study aims to validate pro-oxidant actions of nicotine (N), using hydrogen peroxide (H2O2) and the antioxidant glutathione (G) in an in vitro model of human gingival fibroblasts (HGF) and human oral periosteal fibroblasts (HPF); radiolabelled androgens are used as biomarkers of redox status. Oxidative stress is an important mediator of inflammatory repair. The androgen metabolite <em>5α</em>-dihydrotestosterone (<em>DHT</em>) is an effective biomarker of oxidative stress and healing.

METHODS

6 Cell-lines of HGF and HPF established in confluent monolayer culture were incubated in Eagle's MEM using 14C-testosterone and 14C-4-androstendione as substrate; in conjunction with effective concentrations of N, G and H2O2 established at N250, G3 μg/ml and 3%H2O2 w/w, 0.5 μl/ml. Combinations of H2O2G and H2O2GN were used in order to compare the oxidative effects of N/H2O2 and their responses to glutathione. At 24 h, the medium was solvent extracted, evaporated to dryness and subjected to TLC in a benzene/acetone solvent system 4:1 v/v for the separation of metabolites. The separated metabolites were quantified using a radioisotope scanner.

RESULTS

The mean trends of 6 cell-lines for both substrates and each cell type demonstrated that the yield of the main metabolite DHT was significantly reduced by N and H2O2 alone (2-fold, n=6; p<0.01). The inhibition caused by H2O2 was overcome by the antioxidant glutathione in the combination H2O2G, to values similar to those of controls (n=6; p<0.01). It is relevant that when N was added to this neutralized combination, the decrease in yields of DHT triggered by N were comparable to those induced by H2O2; and retaining the positive effect of G.

CONCLUSIONS

Oxidative stress mediated by H2O2 was overcome by glutathione and recurred when nicotine was added, suggestive of a pro- oxidant role for nicotine. Androgen biomarkers are a sensitive index of oxidative stress which affects wound healing.

Prostate cancer (PCa) initially responds to inhibition of androgen receptor (AR) signaling, but inevitably progresses to hormone ablation-resistant disease. Much effort is focused on optimizing this androgen deprivation strategy by improving hormone depletion and AR antagonism. However we found that bicalutamide, a clinically used antiandrogen, actually resembles a selective AR modulator (SARM), as it partially regulates 24% of endogenously <em>5α</em>-dihydrotestosterone (<em>DHT</em>)-responsive genes in AR(+) MDA-MB-453 breast cancer cells. These data suggested that passive blocking of all AR functions is not required for PCa therapy. Hence, we adopted an active strategy that calls for the development of novel SARMs, which induce a unique gene expression profile that is intolerable to PCa cells. Therefore, we screened 3000 SARMs for the ability to arrest the androgen-independent growth of AR(+) 22Rv1 and LNCaP PCa cells but not AR(-) PC3 or DU145 cells. We identified only one such compound; the 4-aza-steroid, MK-4541, a potent and selective SARM. MK-4541 induces caspase-3 activity and cell death in both androgen-independent, AR(+) PCa cell lines but spares AR(-) cells or AR(+) non-PCa cells. This activity correlates with its promoter context- and cell-type dependent transcriptional effects. In rats, MK-4541 inhibits the trophic effects of <em>DHT</em> on the prostate, but not the levator ani muscle, and triggers an anabolic response in the periosteal compartment of bone. Therefore, MK-4541 has the potential to effectively manage prostatic hypertrophic diseases owing to its antitumor SARM-like mechanism, while simultaneously maintaining the anabolic benefits of natural androgens.

Acute dehydroepiandrosterone (DHEA) administration improves hyperglycemia in rats with streptozotocin (STZ)-induced type 1 diabetes mellitus. Diosgenin, a steroid structurally similar to DHEA (dehydroepiandrosterone), is contained highly levels in dioscorea; however, it is still unclear whether this natural product improves hyperglycemia in the type 1 diabetes model rats through an increase muscular GLUT4 signaling. After 1 week of STZ injection, fasting glucose level was measured in blood taken from the tail vein every 30 min for 150 min after injection of diosgenin or dioscorea (3mg/kg). On another day, muscle was resected 150 min after diosgenin or dioscorea injections. Serum DHEA level increased significantly 120 min after diosgenin or dioscorea injections; concomitantly, blood glucose level decreased significantly. Moreover, GLUT4 translocation, as well as phosphorylation of Akt and PKC ζ/λ, increased significantly by diosgenin or dioscorea administration. However, these effects of diosgenin and dioscorea were blocked by a <em>5α</em>-reductase inhibitor that inhibits synthesizing dehydrotestosterone (<em>DHT</em>) from testosterone. Additionally, significant correlations were observed between blood glucose level, GLUT4 translocation level, and muscular sex steroid hormone level 150 min after the administrations. These results suggest that the diosgenin-induced increase in the DHEA level may contribute to the improvement of hyperglycemia by activating the muscular GLUT4 signaling pathway in type 1 diabetes model rats.

Unconjugated testosterone (T), <em>5a</em>-androst-16-en-3-one (delta-16), dihydrotestosterone (<em>DHT</em>), unconjugated (u Oe) and conjugated oestrogens (c Oe) were determined weekly in the peripheral blood plasma and seminal plasma of 8 boars for a period of 15 months. Four of the boars were kept under natural daylength fluctuations (8-17 h), and 4 boars on a light programme which shifted these fluctuations by half a year. In peripheral plasma of all boars the highest concentrations were found for c Oe (17 ng/ml) followed by delta-16 (7.3), T (2.2) <em>DHT</em> (0.75) and u Oe (0.24). In seminal plasma of all boars (with an average volume of 172 ml) the highest concentrations were again found for c Oe (5.9 ng/ml) followed by delta-16 (1.1), u Oe (0.73), T (0.28) and <em>DHT</em> (0.1). This shows that u Oe reach higher concentrations in seminal plasma than in blood plasma. In boars under the influence of natural daylight steroids fluctuated with season. Maximum concentrations were present from October till December. Minimum concentrations were about 1/10 of the maximum concentrations. All steroids measured in blood and semen were similarly affected and all were highly correlated. In boars on an artificial light programme the off-season depression during the summer was completely restored. This indicates that daylight fluctuations are the main reason for seasonal changes of testicular function in the domestic boar.

Dimethandrolone (DMA: 7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone (MNT) are potent androgens in development for hormonal therapy in men. As <em>5α</em>-reduced androgens, such as <em>5α</em>-dihydrotestosterone (<em>DHT</em>), may raise the risk of benign prostate hyperplasia, accelerate the development of prostate carcinoma, and increase male pattern baldness and acne, we investigated the role of <em>5α</em>-reduction in the androgenic activity of DMA and MNT. The authentic <em>5α</em>-reduced metabolites, <em>5α</em>-dihydroDMA (<em>5α</em>-DHDMA) and <em>5α</em>-dihydroMNT (<em>5α</em>-DHMNT), were prepared by chemical synthesis and compared in vitro and in vivo to the parent compounds. Both <em>5α</em>-reduced androgens bound with high affinity to the rat androgen receptor (AR) and were potent inducers of transactivation of 3XHRE-LUC in CV-1 cells cotransfected with a human AR expression plasmid. To examine in vivo androgenic (stimulation of ventral prostate [VP] and seminal vesicle [SV] weights) and anabolic (stimulation of levator ani [LA] muscle weights) activity, 22-day-old castrate male rats were treated sc for 7 days with various doses of DMA, <em>5α</em>-DHDMA, or testosterone (T) or MNT, <em>5α</em>-DHMNT, or T and necropsied on day 8. <em>5α</em>-DHDMA was at least threefold more potent than T in stimulating growth of the VP but only 30-40% as potent as DMA. <em>5α</em>-DHMNT was four- to eightfold more potent than T, whereas MNT was approximately equipotent to T. To assess the possible role of <em>5α</em>-reduction in VP and SV growth, castrate immature rats were treated with maximally effective doses of T, <em>DHT</em>, DMA, MNT, or the related 19-norandrogen, 7α-methyl-19-nortestosterone (MENT), or vehicle, with or without dutasteride (DUT), an inhibitor of <em>5α</em>-reductases types 1 and 2. In rats treated with T+DUT, serum T was significantly higher (P<0.05) than in rats treated with T alone, and serum <em>DHT</em> was decreased (P<0.001) to levels observed in castrate vehicle-treated rats. DUT significantly reduced both VP and SV weights in T-treated rats, whereas there was no significant effect of DUT on weights of these accessory sex glands in rats treated with DMA, MNT, <em>DHT</em>, or MENT. These results indicate that inhibition of <em>5α</em>-reductase activity in vivo does not affect the androgenic potency of DMA, MNT, or MENT.