Our research objective is to understand more, through subjective, self-reports on discussion boards/forums, persons' experiences associated with the use of drugs that alter androgen metabolism, such as finasteride. Finasteride is an orally active, specific inhibitor of <em>5α</em>-reductase, which is localized to many androgen-dependent tissues. Finasteride inhibits the conversion of testosterone (T) to dihydrotestosterone (<em>DHT</em>), and is commonly used to treat benign prostatic hypertrophy (BPH) and male pattern baldness (MPB), both disorders associated with elevated <em>DHT</em> levels and <em>5α</em>-reductase activity in the prostate and hair follicles, respectively. It is now acknowledged that long-term use and discontinuation of finasteride has adverse effects (AEs); however, these claims have not been well documented. In this study, discussion board posts (forums) were analyzed as self-reports of what finasteride users indicate is problematic for them. Reports were categorized by the age of subjects as well as the types of AEs described: antiandrogenic, estrogenic, central, and nonspecific/severe. A total of 244 cases were recorded and analyzed on the discussion forum on propeciahelp.com . Among these, 74 (32%) cases reported antiandrogenic affects, 43 (19%) reported estrogenic effects, 70 (30%) reported central effects, 11 (5%) reported nonspecific/severe AEs, and 31 (14%) reported AEs in all categories. The categorization of AEs may prompt further investigation into the pathophysiology of post-finasteride syndrome (PFS). Also, subjective reports may engender greater understanding of the perceived lasting AEs of finasteride.

BACKGROUND

Testosterone, <em>5α</em>- and 5β-dihydrotestosterone (-<em>DHT</em>) induce an acute in vitro vasorelaxation and in vivo vasodepressor, hypotensive and antihypertensive responses. Our aim was to study whether androgen-induced blood pressure (BP) reduction is involved with a blockade of Ca2+ influx through L-type voltage-operated calcium channels (L-VOCCs) and/or the signaling pathways of α1-adrenoceptors to induce vasoconstriction, which are one of the major mechanisms of BP maintenance.

METHODS

The relaxing potency and efficacy of each androgen in large conduit (thoracic aorta) and resistance (mesenteric) arteries from male hypertensive (SHR) and normotensive (WKY) rats were established. Blood vessels were isometrically recorded and precontracted with KCl or phenylephrine (Phe).

RESULTS

Androgens induced concentration-dependent vasorelaxation in precontracted arteries from SHR and WKY rats. 5β-DHT was always the most potent vasorelaxant in arteries from SHR. The KCl-induced contraction resulted significantly more sensitive to androgen-induced vasorelaxation than the Phe-induced contraction. On Phe-induced contraction, 5β-DHT was more potent in the mesenteric artery than in the thoracic aorta.

CONCLUSIONS

The vasorelaxation induced by androgens is mainly mediated by blocking L-VOCCs and in lesser extent by the blockade of multiple signaling pathways operative during α-adrenoceptor-induced vasoconstriction. 5β-DHT regulates vascular resistance and BP by mainly acting in the mesenteric arterial bed, which may explain its outstanding antihypertensive response previously reported.

This study describes a validated LC-MS/MS method for assaying 23 steroids within a single run from 150 μl of human plasma, serum or prostatic tissue homogenate. Isotope-labeled steroids were used as internal standards. Samples were extracted with toluene, and ketosteroids were derivatized with hydroxylamine prior to LC-MS/MS analysis. The steroids were separated on a C18 column and methanol was used as an organic solvent with the addition of 0.2 mM ammonium fluoride to improve underivatized estradiol (E2) ionization. Certified reference serums as well as plasma samples, and homogenates of prostate tissue were utilized in the method validation. The specificity of the method was inspected with a total of 27 steroids. The validation proved that the method was suitable for the quantitative analysis of a wide panel of androgens (testosterone, T (3.3 pM-13 nM); androstenedione, A4 (3.3 pM-13 nM); <em>5α</em>-androstanedione, DHA4 (13 pM-13 nM); dehydroepiandrosterone, DHEA (67 pM-133 nM); dihydrotestosterone, <em>DHT</em> (33 pM-33 nM); 11-ketodihydrotestosterone, 11K<em>DHT</em> (13 pM-13nM); 11-ketotestosterone, 11KT (33 pM-6.7 nM); 11β-hydroxyandrostenedione, 11bOHA4 (33 pM-13 nM); 11β-hydroxytestosterone, 11OHT (13 pM-33 nM)), as well as estrogens (estrone, E1 (3.3 pM-13 nM)), progestagens (17α-hydroxypregnenolone, 17OHP5 (32 pM-127 nM); 17α-hydroxyprogesterone, 17OHP4 (67 pM-133 nM); progesterone, P4 (3.3 pM-13 nM); pregnenolone, P5 (6.6 pM-13 nM)), and glucocorticoids (cortisol, F (33 pM-134 nM); cortisone E (66 pM-131 nM); corticosterone, B (33 pM-67 nM); 11-deoxycortisol, S (33 pM-66 nM); 21-hydroxyprogesterone, 21OHP4 (32 pM-13 nM)). Furthermore, E2 (335 pM-134 nM) and 11α-hydroxyandrostenedione, 11aOHA4 (33 pM-33 nM) could be analyzed if the concentration in the sample was high enough. In addition, aldosterone, A (128 pM-64 nM) and 11-ketoandrostenedione, 11KA4 (33 pM-13 nM) could be analyzed semiquantitatively. The limits of quantification for all compounds ranged from 0.9 to 91 pg/ml, and from 0.009 to 0.9 pg/mg tissue. Compared to our previous method, this new method also permits the analysis of the more challenging steroids, like <em>DHT</em>, DHEA and P5, and a panel of 11-ketosteroids.

Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme <em>5α</em>-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to <em>DHT</em>. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and <em>DHT</em>) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the <em>5α</em>-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed <em>5α</em>-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux.

The oligodendrocyte density is greater and myelin sheaths are thicker in the adult male mouse brain when compared with females. Here, we show that these sex differences emerge during the first 10 postnatal days, precisely at a stage when a late wave of oligodendrocyte progenitor cells arises and starts differentiating. Androgen levels, analyzed by gas chromatography/tandem-mass spectrometry, were higher in males than in females during this period. Treating male pups with flutamide, an androgen receptor (AR) antagonist, or female pups with <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>), revealed the importance of postnatal androgens in masculinizing myelin and their persistent effect into adulthood. A key role of the brain AR in establishing the sexual phenotype of myelin was demonstrated by its conditional deletion. Our results uncover a new persistent effect of postnatal AR signaling, with implications for neurodevelopmental disorders and sex differences in multiple sclerosis.

Polycystic ovary syndrome (PCOS) is associated with gut microbiota disturbance. Emerging evidence has shown that gut microbiota plays a major role in the development of PCOS. To better understand how the gut microbiota contributes to the development of PCOS, we investigated the influences of high-fat diet and hyperandrogenism, independently or synergistically, have on the gut microbiota in rats. Furthermore, we explored the associations between gut microbiota and hyperandrogenism or other hallmarks of PCOS. Twenty female SD rats were randomized at aged 3 weeks into 4 groups (n = 5, each); HA: PCOS rats fed with ordinary diet; HF: rats with high-fat diet (HFD); HA-HF: PCOS rats fed with HFD; and C: control rats with ordinary diet. PCOS rat model was induced by <em>5α</em>-dihydrotestosterone (<em>DHT</em>) injection for 6 weeks. The fasting blood glucose (FBG), plasma insulin, testosterone, free testosterone, TNF-α, MDA, SOD, LPS, TLR4, TG, TC, HDL-C, and LDL-C levels were measured. The molecular ecology of the fecal gut microbiota was analyzed by 16S rDNA high-throughput sequencing. The results showed that rats in the HA and HA-HF group displayed abnormal estrous cycles with increasing androgen level and exhibited multiple large cysts with diminished granulosa layers in ovarian tissues. Compare with the C group, relative abundance of the Bacteroidetes phylum decreased significantly in the other groups (P < 0.05). The Chao1 was the highest in the group C and significantly higher than the HA-HF group (P < 0.05). T, FT, insulin, MDA, LPS, and TNF-α levels had the negative correlation with the richness of community (Chao1 index) in the gut. The rats in the HF and HA-HF groups tended to have lower Shannon and Simpson indices than the C group (P < 0.01, respectively). However, there were no significant differences between C group and the HA group in the Shannon and Simpson values. Beta diversity analysis was then performed based on a weighted UniFrac analysis. The PCoA plots showed a clear separation of the C group from the other groups. ANOSIM analysis of variance confirmed that there were statistically significant separations between the C group and the HA, HA-HF, and HF groups (P < 0.01, respectively). These results showed that <em>DHT</em> with HFD could lower diversity of the gut microbial community. Both HFD and <em>DHT</em> could shift the overall gut microbial composition and change the composition of the microbial community in gut. Furthermore, our analyses demonstrated that the levels of TG, MDA, TNF-α, LPS, TLR4, T, FT, FINS, and HDL-C were correlated with the changes of in the gut microbiome. HFD and <em>DHT</em> were associated with the development and pathology of PCOS by shaping gut microbial communities.

In situ estrogen biosynthesis is considered to play pivotal roles in the development and progression of human endometrial carcinoma. However, the biological roles of androgen have remained virtually unknown. Various epidemiological studies have revealed that elevated serum androgen levels are generally associated with an increased risk of developing endometrial carcinoma; however, studies directly examining androgens in carcinoma tissues are relatively rare and reviews summarizing this information are scarce. Therefore, we summarized recent studies on androgens in endometrial carcinoma, especially focusing androgen actions and in situ androgen biosynthesis. Among the enzymes required for local biosynthesis of androgen, 17β-hydroxysteroid dehydrogenase type 5 (conversion from androstenedione to testosterone) and <em>5α</em>-reductase (reduction of testosterone to dihydrotestosterone (<em>DHT</em>)) are the principal enzymes involved in the formation of biologically most potent androgen, <em>DHT</em>. Both enzymes and androgen receptor were expressed in endometrial carcinoma tissues, and in situ production of <em>DHT</em> has been reported to exist in endometrial carcinoma tissues. However, testosterone is not only a precursor of <em>DHT</em> production, but also a precursor of estradiol synthesis, as a substrate of the aromatase enzyme. Therefore, aromatase could be another key enzyme serving as a negative regulator for in situ production of <em>DHT</em> by reducing amounts of the precursor. In an in vitro study, <em>DHT</em> was reported to exert antiproliferative effects on endometrial carcinoma cells. Intracrine mechanisms of androgens, the downstream signals of AR, which are directly related to anticancer progression, and the clinical significance of <em>DHT</em>-AR pathway in the patients with endometrial carcinoma have, however, not been fully elucidated.

Testosterone (T) synthesised in Leydig cells enters the epididymis and may there be converted into dihydrotestosterone (<em>DHT</em>) by <em>5α</em>-reductase (<em>5α</em>-red) or into 17β-oestradiol (E2) by P450 aromatase (P450-aro). D-aspartate (D-Asp) is known to induce T synthesis in the testis. In this study, we investigated the effects of in vivo D-Asp administration in two major regions of the rat epididymis (Region I: initial segment, caput, corpus; Region II: cauda). The results suggest that exogenous D-Asp was taken up by both regions of rat epididymis. D-Asp administration induced a rapid increase in T, followed by a more gradual decrease in the T:<em>DHT</em> ratio in Region I. In Region II, T levels rapidly decreased and the T:<em>DHT</em> ratio was consistently lower relative to the control. Expression of <em>5α</em>-red and androgen receptor genes showed a good correlation with <em>DHT</em> levels in both regions. D-Asp treatment also induced an increase of both E2 levels and oestradiol receptor-α (ERα) expression in Region I, whereas neither E2 levels nor ERα expression were affected in Region II. The early increase of P450-aro expression in Region I and late increase in Region II suggests a direct involvement of D-Asp modulation in P450-aro gene expression. Our results suggest that D-Asp modulates androgen and oestrogen levels and expression of androgen and oestrogen receptors in the rat epididymis by acting on the expression of <em>5α</em>-red and P450-aro genes.

Ethnopharmacological relevance: Psoralea corylifolia L. seed (PCL), commonly known as "Poguzhi" or "BuguZhi", has been widely used to treat kidney yang deficiency in traditional Chinese medicine (TCM) where tonifying the yang deficiency is a representative understanding for treatment of hormonal deficiency disorders such as enuresis, oliguria, and prostatic diseases. Although PCL has been commonly used to treat problems of the urinary system, its efficacy against benign prostatic hyperplasia (BPH) has not yet been reported.

Aim of the study: In the present study, we aimed to assess the in vitro and in vivo efficacy of PCL against BPH, a condition which negatively impacts quality of life in men.

Materials and methods: Normal human prostate cell lines, RWPE-1 and WPMY-1 cells, were stimulated with 10 nM dihydrotestosterone (DHT) to establish an in vitro BPH model. Subsequently, cells were treated with 100 or 200 μg/ml PCL, which inhibited cell proliferation without cytotoxicity, to evaluate the anti-BPH effect of PCL. Eight-week-old male Wistar rats were castrated, except for those in the control group (Con), and BPH was induced by subcutaneous injection of 10 mg/kg testosterone propionate (TP). Concurrent with daily TP injections, 5 mg/kg of finasteride (Fina) and 50 or 100 mg/kg PCL were orally administrated daily for four weeks, excluding the weekends.

Results: In DHT-stimulated RWPE-1 and WPMY-1 cells, expression of androgen receptor (AR) androgen signaling-related markers such as 5α-reductase 2 (5AR2), AR, and prostate-specific antigen (PSA) was upregulated, whereas 100 or 200 μg/ml of PCL treatment downregulated these markers. Furthermore, PCL significantly reduced the mRNA expression of anti-apoptotic genes and increased the mRNA expression of pro-apoptotic gene. In vivo, administration of PCL reduced prostate size and weight in TP-induced BPH rats. Moreover, histological alterations in epithelium thickness were significantly restored by the administration of PCL. Immunohistochemical analysis revealed increased expression of AR and proliferating cell nuclear antigen (PCNA) in TP-induced BPH prostates; these changes were suppressed by administration of 50 or 100 mg/kg PCL.

Conclusions: We demonstrated the effect of PCL against BPH, mediated by the regulation of prostate cell proliferation and apoptosis, in DHT-stimulated normal human prostate cell lines and TP-induced BPH rats. These findings suggest that PCL could be a potential therapeutic agent against BPH.

Keywords: Androgen receptor; Apoptosis; Benign prostatic hyperplasia; Dihydrotestosterone; Psoralea corylifolia L.

According to current knowledge, two steroid <em>5α</em>-reductases, designated type 1 (SRD5A1) and type 2 (SRD5A2), are present in all species examined to date. These isozymes play a central role in steroid hormone physiology by catalyzing the reduction of 3-keto-4-ene-steroids into more active <em>5α</em>-reduced derivatives, including the conversion of testosterone (T) to dihydrotestosterone (<em>DHT</em>). A third <em>5α</em>-reductase (SRD5A3, -type 3), which is overexpressed in hormone-refractory prostate cancer cells, has been identified; however, its enzymatic characteristics are practically unknown. Here, we isolated a cDNA encoding hamster Srd5a3 (hSrd5a3) and performed functional metabolic assays to investigate its biochemical properties. The cloned cDNA encodes a 330 amino acid protein that is 87% identical to the homologous protein in mice and 78% to that in humans. However, hSrd5a3 exhibits low sequence homology with its counterparts hSrd5a1 (19%) and hSrd5a2 (17%). A fusion protein consisting of hSrd5a3 and green fluorescent protein provided evidence for cytoplasmic localization in transfected mammalian cells. Real-time PCR analysis revealed that, Srd5a3 mRNA was present in nearly all hamster tissues, with high expression in the cerebellum, Harderian gland and testis. Functional assays expressing hSrd5a3 cDNA in HEK-293 cells revealed that this isozyme is unable to reduce T into <em>DHT</em>. Further expression assays confirmed that similar to testosterone, progesterone, androstenedione and corticosterone are not reduced by hSrd5a3 or human SRD5A3. Together, these results indicate that hSrd5a3 lacks the catalytic activity to transform 3-keto-4-ene-compounds; therefore <em>5α</em>-reductase type 3 may not be involved in <em>5α</em>-reduction of steroids.

T is converted to a more potent androgen, <em>DHT</em> by the action of microsomal membrane enzyme <em>5α</em> reductase 2. Defects in <em>5α</em> reductase 2 isozyme results in incomplete virilisation of external male genitalia. Mutations in SRD5A2 gene leads to diminished enzyme activity, thus hampering <em>DHT</em> synthesis from T. We describe two unrelated patients from India with <em>5α</em>RD2 due to novel insertion of nucleotides in the exon 1 of SRD5A2 gene that lead to premature termination of protein. Master S (case 1; III.8) was 3 years old at initial evaluation, had perineoscrotal hypospadias, microphallus and both testes were palpable in the inguinal region. Master P (case 2; III.9) was born as normal full term baby. He had primary complaint of microphallus, penoscrotal hypospadias and gonads in the inguinal region. Diagnosis of <em>5α</em>RD2 was made, as T/<em>DHT</em> ratio in the two cases was 41 and 131.2 respectively. Sequence analysis of SRD5A2 gene showed an insertion of nucleotides TA in exon 1 (c.188_189). This resulted in premature termination of the protein due to stop codon at amino acid position 7. The protein formed is drastically truncated and inadequate protein synthesized explains the phenotypic characteristics of our patients.

Female pattern hair loss (FPHL) is an important hair disorder, especially when young women are affected. However, pharmacological treatments are not successful in all women. Androgens, especially dihydrotestosterone (<em>DHT</em>), may play a role in FPHL, but many women with this disorder have normal serum androgen levels. It therefore appears that hair follicle levels of <em>DHT</em> depend on in situ testosterone (T) metabolism. Because T can be converted to <em>DHT</em> or estradiol (E2) by <em>5α</em>-reductase (<em>5α</em>-R) and aromatase, respectively, these enzymes would determine <em>DHT</em> and E2 concentrations and their ratio. We propose and apply a low-invasive, sensitive and precise method for the absolute quantification of mRNA levels of aromatase and <em>5α</em>-R isozymes (type 1, type 2 and type 3) in plucked hair from young women with FPHL. Normoandrogenic women with FPHL and controls were studied. Plucked hair samples were obtained by trichogram from vertex scalp and mRNA levels quantified by real-time RT-PCR. We revealed for the first time the presence of <em>5α</em>-R3 mRNA in human hair. Interestingly, one, two, or even three <em>5α</em>-R isozymes were increased in some women with FPHL but not in others, which may explain the lack of response to <em>5α</em>-R inhibitors in some FPHL cases. Aromatase mRNA levels were significantly lower in women with FPHL than in controls. It may therefore produce a reduction in oestrogen levels and an increase in the androgen/oestrogen ratio in hair. The proposed low-invasive technique offers a molecular aetiologic diagnosis of FPHL for the selection of more appropriate pharmacological treatments with early predicted effectiveness.

Men are at a higher risk of developing bladder cancer than women. Although the urinary bladder is not regarded as an sex organ, it has the potential to respond to androgen signals. The mechanisms responsible for the gender differences remain unexplained. Androgen receptor (AR) after binding with <em>5α</em>-dihydrotestosteron (<em>DHT</em>) undergoes a conformational change and translocates to nucleus to induce transcriptional regulation of target genes. However androgen/AR signaling can also be activated by interacting with several signaling molecules and exert its non-genomic function. The aim of present study was to explain whether the progression of bladder cancer in men is dependent on androgen/AR signaling. Studies were carried out on human bladder cancer cell lines: HCV29, T24, HT1376 and HTB9. Bladder cancer cells were treated for 48 h with 10 nM <em>DHT</em> or not, with replacement after 24 h. Expression of cell signaling proteins, was analyzed using Western Blot and RT-PCR. Subcellular localization of protein was studied using the ProteoExtract Subcellular Proteome Extraction Kit and Western blot analysis. We showed that <em>DHT</em> treatment significantly increased AR expression in bladder cell line HCV29. We also observed <em>DHT</em>-mediated activation of Akt/GSK-3β signaling pathway which plays a central role in cancer progression. Presented results also show that androgen/AR signaling is implicated in phosphorylation of eIF4E which can promote epithelial-mesenchymal transition (EMT). We indicate that AR plays an essential role in bladder cancer progression in male patients. Therefore, androgen-activated AR signaling is an attractive regulatory target for the inhibition or prevention of bladder cancer incidence in men.

Prostate cancer (PCa) growth and progression rely on the interaction between the androgen receptor (AR) and the testicular ligands, testosterone and dihydrotestosterone (<em>DHT</em>). Almost all men with advanced PCa receive androgen deprivation therapy (ADT). ADT lowers circulating testosterone levels, which impairs AR activation and leads to PCa regression. However, ADT is palliative and PCa recurs as castration-recurrent/resistant PCa (CRPC). One mechanism for PCa recurrence relies on intratumoral synthesis of <em>DHT</em>, which can be synthesized using the frontdoor or primary or secondary backdoor pathway. Androgen metabolism inhibitors, such as those targeting <em>5α</em>-reductase, aldo-keto-reductase family member 3 (AKR1C3), or cytochrome P450 17A1 (CYP17A1) have either failed or produced only modest clinical outcomes. The goal of this review is to describe the therapeutic potential of combined inhibition of <em>5α</em>-reductase and 3α-oxidoreductase enzymes that facilitate the terminal steps of the frontdoor and primary and secondary backdoor pathways for <em>DHT</em> synthesis. Inhibition of the terminal steps of the androgen metabolism pathways may be a way to overcome the shortcomings of existing androgen metabolism inhibitors and thereby delay PCa recurrence during ADT or enhance the response of CRPC to androgen axis manipulation.

Endocrine disrupting substances (EDSs) have the potential to disturb sensitive hormone pathways, particularly those involved in development and reproduction. Both fresh and estuarine water bodies receive inputs of EDSs from a variety of sources, including sewage effluent, industrial effluent and agricultural runoff. Based on current literature, freshwater species appear to respond to lower levels of EDSs than estuarine or marine species. Therefore, effects elicited by EDSs in freshwater teleosts may not be an accurate representation of how EDSs affect teleosts in estuarine and marine environments. To address this potential difference, a short-term reproductive bioassay was conducted under conditions of low and high salinity using mummichog (Fundulus heteroclitus), a euryhaline species that is native to the east coast of North America. The goals of this study were to determine the response of mummichog when exposed to an androgenic EDS and whether salinity affected the response. A model androgen, <em>5α</em>-dihydrotestosterone (<em>DHT</em>), was selected for this experiment. Impacts on reproduction were evaluated at multiple biological levels, including physiological (sex steroid levels), organismal (gonad size and gonad morphology), and functional (egg production) endpoints. Under conditions of high salinity, egg production was significantly reduced at all exposure concentrations. Under conditions of low salinity, there were no significant differences based on <em>DHT</em> treatment; however, egg production in all treatment groups including the control were significantly reduced relative to the high salinity control group. Other reproductive endpoints, such as sex steroid production, showed stronger correlation to fecundity in females than males. This study demonstrates that mummichog fecundity is sensitive to androgenic endocrine disruption while also underscoring the importance of how changes in salinity, an environmental variable, can impact reproduction.

Mounting experimental evidence demonstrate that sex neuroactive steroids (neurosteroids) are essential for memory formation. Neurosteroids have a profound impact on the function and structure of neural circuits and their local synthesis is necessary for the induction of both long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission and for neural spine formation in different areas of the central nervous system (CNS). Several studies demonstrated that in the hippocampus, 17β-estradiol (E2) is necessary for inducing LTP, while <em>5α</em>-dihydrotestosterone (<em>DHT</em>) is necessary for inducing LTD. This contribution has been proven by administering sex neurosteroids in rodent models and by using blocking agents of their synthesis or of their specific receptors. The general opposite role of sex neurosteroids in synaptic plasticity appears to be dependent on their different local availability in response to low or high frequency of synaptic stimulation, allowing the induction of bidirectional synaptic plasticity. The relevant contribution of these neurosteroids to synaptic plasticity has also been described in other brain regions involved in memory processes such as motor learning, as in the case of the vestibular nuclei, the cerebellum, and the basal ganglia, or as the emotional circuit of the amygdala. The rapid effects of sex neurosteroids on neural synaptic plasticity need the maintenance of a tonic or phasic local steroid synthesis determined by neural activity but might also be influenced by circulating hormones, age, and gender. To disclose the exact mechanisms how sex neurosteroids participate in finely tuning long-term synaptic changes and spine remodeling, further investigation is required.

<strong class="sub-title"> Keywords: </strong> 17β-estradiol; <em>5α</em>-dihydrotestosterone; <em>5α</em>-reductase; LTD; LTP; P450 aromatase; synaptic plasticity; testosterone.

Fibroblasts express androgen receptor (AR) in the normal prostate and during prostate cancer development. We have reported that loss of AR expression in prostate cancer-associated fibroblasts is a poor prognostic indicator. Here we report outcomes of direct and indirect co-cultures of immortalised AR-positive (PShTert-AR) or AR-negative (PShTert) myofibroblasts with prostate cancer cells. In the initial co-cultures the AR-negative PC3 cell line was used so AR expression and signalling were restricted to the myofibroblasts. In both direct and indirect co-culture with PShTert-AR myofibroblasts, paracrine signalling to the PC3 cells slowed proliferation and induced apoptosis. In contrast, PC3 cells proliferated with PShTert myofibroblasts irrespective of the co-culture method. In direct co-culture PC3 cells induced apoptosis in and destroyed PShTerts by direct signalling. Similar results were seen in direct co-cultures with AR-negative DU145 and AR-positive LNCaP and C4-2B prostate cancer cell lines. The AR ligand <em>5α</em>-dihydrotestosterone (<em>DHT</em>) inhibited the proliferation of the PShTert-AR myofibroblasts, thereby reducing the extent of their inhibitory effect on cancer cell growth. These results suggest loss of stromal AR would favour prostate cancer cell growth in vivo, providing an explanation for the clinical observation that reduced stromal AR is associated with a poorer outcome.

BACKGROUND

Androgens play a crucial role in prostate cancer, hence the androgenic pathway has become an important target of therapeutic intervention. Previously we discovered that gene fusions between the 5'-untranslated region of androgen regulated gene TMPRSS2 and the ETS transcription factor family members were present in a majority of the prostate cancer cases. The resulting aberrant overexpression of ETS genes drives tumor progression.

METHODS

Here, we evaluated the expression levels of <em>5α</em>-reductase isoenzymes in prostate cancer cell lines and tissues. We tested the effect of dutasteride, a <em>5α</em>-reductase inhibitor, in TMPRSS2-ERG fusion-positive VCaP cell proliferation and cell invasion. We also evaluated the effect of dutasteride on the TMPRSS2-ERG fusion gene expression. Finally, we tested dutasteride alone or in combination with an anti-androgen in VCaP cell xenografts tumor model.

RESULTS

Our data showed that <em>5α</em>-reductase SRD5A1 and SRD5A3 isoenzymes that are responsible for the conversion of testosterone to DHT, are highly expressed in metastatic prostate cancer compared to benign and localized prostate cancer. Dutasteride treatment attenuated VCaP cell proliferation and invasion. VCaP cells pre-treated with dutasteride showed a reduction in ERG and PSA expression. In vivo studies demonstrated that dutasteride in combination with the anti-androgen bicalutamide significantly decreased tumor burden in VCaP cell xenograft model.

CONCLUSIONS

Our findings suggest that dutasteride can inhibit ERG fusion-positive cell growth and in combination with anti-androgen, significantly reduce the tumor burden. Our study suggests that anti-androgens used in combination with dutasteride could synergistically augment the therapeutic efficacy in the treatment of ETS-positive prostate cancer.

To establish association between two main lysophosphatidic acid (LPA) receptors (LPAR2 and LPAR1) with the synthesis of estrogens and androgens in type-1 endometrial carcinoma (EC), we evaluated correlation of LPARs expression with expression of steroid 5 alpha-reductase 2 - aromatase (SRD5A2), or cytochrome P450 family 19 subfamily A member 1-<em>5α</em>-reductase (CYP19A1) in EC. Moreover, we aimed to investigate SRD5A2 and CYP19A1 expression in type 1 endometrial cancer and normal endometrium with its correlation to selected clinicopathological features. The studied cancerous samples showed higher CYP19A1 and SRD5A2 expression comparing to normal endometria. We also documented positive correlations between LPAR1 and LPAR2 with responsible for proliferation SRD5A2 in EC tissue which suggests that intratumoral estrogen metabolism and synthesis are pivotal in endometrial carcinoma progression, with the involvement of LPA in this process. However, positive correlation between CYP19A1 and LPAR1 accounts for supporting role of LPA acting via LPAR1 in intratumoral <em>DHT</em> concentration and the ethiology of endometrial cancer progression. Also, owing to the highest expression of LPARs, CYP19A1 and SRD5A2 as well as their association with depth of myoinvasion and FIGO stage LPAR2 and LPAR1 seem to be the efficient candidate prognostic markers in the individual, targeted therapies for EC.

Purpose: Resistance to androgen-deprivation therapies and progression to so-called castrate-resistant prostate cancer (CRPC) remain challenges in prostate cancer (PCa) management and treatment. Among other alterations, CRPC has been associated with metabolic reprogramming driven by androgens. Here, we investigated the role of androgens in regulating glutaminolysis in PCa cells and determined the relevance of this metabolic route in controlling the survival and growth of androgen-sensitive (LNCaP) and CRPC (DU145 and PC3) cells.

<strong class="sub-title"> Methods: </strong> PCa cells (LNCaP, DU145 and PC3) and 3-month old rats were treated with <em>5α</em>-dihydrotestosterone (<em>DHT</em>). Alternatively, LNCaP cells were exposed to the glutaminase inhibitor BPTES, alone or in combination with the anti-androgen bicalutamide. Biochemical, Western blot and extracellular flux assays were used to evaluate the viability, proliferation, migration and metabolism of PCa cells in response to <em>DHT</em> treatment or glutaminase inhibition.

Results: We found that DHT up-regulated the expression of the glutamine transporter ASCT2 and glutaminase, both in vitro in LNCaP cells and in vivo in rat prostate cells. BPTES diminished the viability and migration of PCa cells, while increasing caspase-3 activity. CRPC cells were found to be more dependent on glutamine and more sensitive to glutaminase inhibition. BPTES and bicalutamide co-treatment had an additive effect on suppressing LNCaP cell viability. Finally, we found that inhibition of glutaminolysis differentially affected glycolysis and lipid metabolism in both androgen-sensitive and CRPC cells.

Conclusion: Our data reveal glutaminolysis as a central metabolic route controlling PCa cell fate and highlight the relevance of targeting glutaminase for CRPC treatment.

<strong class="sub-title"> Keywords: </strong> <em>5α</em>-dihydrotestosterone; ASCT2; BPTES; Bicalutamide; Castrate resistance; Glutamine; Glutaminolysis; Prostate cancer.

BACKGROUND

Simultaneous measurement of testosterone (T) and <em>5α</em>-dihydrotestosterone (<em>DHT</em>) is important for diagnosing androgen deficiency states and hyperandrogenism in males and females, respectively. However, immunoassays used for T and <em>DHT</em> determination suffer from inadequate specificity and sensitivity, while tandem mass spectrometry is expensive and demanding in use.

RESULTS

We developed a selective gas chromatography-mass spectrometry (GC-MS) method for parallel T and DHT measurement. The assay showed a linear response up to 46.5nmol/L, intra- and interassay imprecision and inaccuracy <15% and recoveries in spiked samples >90% for both analytes. The limit of quantitation was 0.117nmol/L for T and 0.168nmol/L for DHT. Comparison with immunoassays revealed good agreement for T in males, but a bias in favour of immunoassays at low concentrations for T in females and DHT in both sexes. We established reference ranges for T and DHT and suggest interval partitioning for T according to age in men and menstrual cycle in women. Assay validation in a clinical setting suggests that measuring DHT or T/DHT ratio may help identify patients with polycystic ovary syndrome.

CONCLUSIONS

We developed a selective, simple and inexpensive GC-MS method for parallel measurement of T and DHT with potential use in the clinical laboratory.

Prostate cancer (PCa) is one of the most prevalent malignancies in men. However, the molecular mechanism controlling the transformation of androgen-dependent PCa (ADPC) to castration-resistant PCa (CRPC) is largely unknown. Androgen receptor (AR) signaling has been reported to play a key role in this process; thus, searching for the novel AR co-activator is important for identifying the mechanism underlying PCa progression. In this study, we focused on the function of mixed lineage leukemia-<em>5α</em> (MLL<em>5α</em>), an epigenetic regulator that exhibits aberrant expression in PCa. MLL<em>5α</em> was the primary expressed form of MLL5 protein in PCa cells and it significantly suppressed proliferation, invasion, and migration in PCa cell lines. Upon stimulation with dihydrotestosterone (<em>DHT</em>), knockdown of MLL<em>5α</em> significantly suppressed N-myc downstream regulated gene 1 (NDRG1) and Kallikrein-related peptidase 3 (KLK3) expression. MLL<em>5α</em> directly bound with AR on the androgen response elements (AREs) and recruited H3K4me3 to the promoters of NDRG1 and KLK3. Downregulation of NDRG1 partially restored the cell invasion and migration suppressed by MLL<em>5α</em>. As evaluated by the proliferation of PCa cells, overexpression of MLL<em>5α</em> synergistically promoted sensitivity to enzalutamide (ENZ) treatment. In PCa patients, MLL<em>5α</em> expression was lower in the high Gleason score (GS) (GS > 7) group than in the low GS (GS < 7) group. In conclusion, suppression of AR/NDRG1 signaling via androgen deprivation therapy (ADT) may be a potential mechanism of CRPC progression. MLL<em>5α</em> significantly suppressed PCa progression by promoting AR/NDRG1 signaling, indicating that regulating MLL<em>5α</em> expression may be a potential treatment approach for patients with advanced PCa.

<strong class="sub-title"> Keywords: </strong> AR; MLL<em>5α</em>; NDRG1; Prostate cancer; enzalutamide; histone methylation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting motor neurons which shows sexual dimorphism in its incidence, age of onset and progression rate. All steroid hormones, including androgens, estrogens and progestogens, have been implicated in modulating ALS. Increasing evidence suggests that steroid hormones provide neuroprotective and neurotrophic support to motor neurons, either directly or via surrounding glial cell interactions, by activating their respective nuclear hormone receptors and initiating transcriptional regulatory responses. The SOD1G93A transgenic mouse also shows sex-specific differences in age of onset and progression, and remains the most widely used model in ALS research. To provide a more comprehensive understanding of the influences of steroid hormone signalling in ALS, we systemically characterised sex hormone receptor expression at transcript and protein levels, cellular localisation and the impact of disease course in lumbar spinal cords of male and female SOD1G93A mice. We found that spinal motor neurons highly express nuclear AR, ERα, ERβ and PR with variations in glial cell expression. AR showed the most robust sex-specific difference in expression and was downregulated in male SOD1G93A mouse spinal cord, in association with depletion in <em>5α</em>-reductase type 2 isoform which primarily metabolises testosterone to <em>DHT</em>. ERα was highly enriched in reactive astrocytes of SOD1G93A mice and ERβ was strongly upregulated. The <em>5α</em>-reductase type 1 isoform was upregulated with disease progression and may influence local spinal cord hormone levels. In conclusion, steroid hormone receptor expression is dynamic and cell-type specific in SOD1G93A mice which may provide targets to modulate progression in ALS.

Women with polycystic ovary syndrome (PCOS) are at increased risk of cardiovascular diseases (CVD); however, the independent role of PCOS in the incident CVD remains unknown. There are reports that hyperhomocysteinemia (HHcy), a potential cause of CVD, is frequently associated with PCOS. The present study investigates the independent attributes of hyperandrogenemia (HA), the integral associate of PCOS, and HHcy in causing atherogenic dyslipidemia. Twenty-five-day old rats were treated with homocysteine (Hcy) at 50 mg/kg/day dose level for 12 weeks. The HepG2 cell lines transfected with siRNA directed to PCSK9 were challenged with Hcy, homocysteine thiolactone (HTL), testosterone, <em>5α</em>-dihydroxytestosterone (<em>5α</em>-<em>DHT</em>), or estradiol for 24 h. Rats administered with Hcy developed HHcy and displayed PCOS-like phenotypes with adversely altered lipid homeostasis and attenuated PI3K-AKT and Wnt signalling cascade. Overexpression of steroidogenic acute regulatory protein (StAR) and down-regulated expression of Aromatase together with elevated testosterone level marked the state of HA. In culture, the HepG2 cells responded independently to Hcy, HTL, testosterone, and <em>5α</em>-<em>DHT</em> by an overt expression of PCSK9 and down-regulated expression of LDLR. The effect was magnified under the combined influence of Hcy and androgen(s). Estradiol, by contrast, exhibited the reverse effect. The findings suggest that HA may independently attribute to an increased cardiovascular risk in PCOS; however, the coexistence of HHcy catalyzes the risk further.