5alpha-androstane-3alpha,17beta-diol supports human prostate cancer cell survival and proliferation through androgen receptor-independent signaling pathways: implication of androgen-independent prostate cancer progression

Androgen and androgen receptor (AR) are involved in growth of normal prostate and development of prostatic diseases including prostate cancer. Androgen deprivation therapy is used for treating advanced prostate cancer. This therapeutic approach focuses on suppressing the accumulation of potent androgens, testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), or inactivating the AR. Unfortunately, the majority of patients with prostate cancer eventually advance to androgen-independent states and no longer respond to the therapy. In addition to the potent androgens, 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), reduced from 5alpha-DHT through 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), activated signaling may represent a novel pathway responsible for the progression to androgen-independent prostate cancer. Androgen sensitive human prostate cancer LNCaP cells were used to compare 5alpha-DHT and 3alpha-diol activated androgenic effects. In contrast to 5alpha-DHT, 3alpha-diol regulated unique patterns of beta-catenin and Akt expression as well as Akt phosphorylation in parental and in AR-silenced LNCaP cells. More significantly, 3alpha-diol, but not 5alpha-DHT, supported AR-silenced LNCaP cells and AR negative prostate cancer PC-3 cell proliferation. 3alpha-diol-activated androgenic effects in prostate cells cannot be attributed to the accumulation of 5alpha-DHT, since 5alpha-DHT formation was not detected following 3alpha-diol administration. Potential accumulation of 3alpha-diol, as a result of elevated 3alpha-HSD expression in cancerous prostate, may continue to support prostate cancer growth in the presence of androgen deprivation. Future therapeutic strategies for treating advanced prostate cancer might need to target reductive 3alpha-HSD to block intraprostatic 3alpha-diol accumulation.     

Ethanol directly increases dihydrotestosterone conversion to 5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 alpha,17 beta-diol in rat Leydig cells

The direct effect of ethanol on dihydrotestosterone (DHT) conversion to 5 alpha-androstan-3 beta,17 beta-diol (3 beta-diol) and 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) by adult rat Leydig cells was examined. Concentrations of ethanol comparable to blood levels of alcoholic men (2.2 - 65 mM) increased DHT conversion to 3 beta - and 3 alpha-diol, in direct relation to the dose of ethanol added; a 2-fold or greater stimulation was observed. Because this effect was blocked by 4-methylpyrazole or a saturating NADH concentration, these results suggest that this action is mediated by Leydig cell alcohol dehydrogenase activity. These results may have significant impact in the testis and/or other DHT sensitive tissues because ethanol may decrease the availability of the proposed active androgen.     

Concentrations of unconjugated 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol and their precursor in human testicular tissue. Comparison with testosterone, 5 alpha-dihydrotestosterone, estradiol-17 beta, and with steroid concentrations in human epididymis

The concentrations of testosterone and its tissular metabolites were determined in testicular and epididymal tissue obtained from eleven male subjects (aged 65-85 years) after orchiectomy for prostatic cancer. The steroids were measured in different tissular compartments, i.e. testis, caput, corpus and cauda epididymis. The values (mean +/- SD; ng/g wet weight) were: Testosterone 724.0 +/- 286.0, 32.08 +/- 2.56, 41.45 +/- 1.77 and 32.24 +/- 2.14; 5 alpha-dihydrotestosterone 6.95 +/- 1.99, 9.76 +/- 2.33, 16.87 +/- 0.21 and 15.79 +/- 2.67; 5 alpha-androstane-3 alpha, 17 beta-diol 6.07 +/- 2.33, 2.17 +/- 0.24, 1.93 +/- 0.02 and 1.17 +/- 0.20; 5 alpha-androstane-3 beta, 17 beta-diol 56.66 +/- 20.97, 3.55 +/- 0.19, 2.21 +/- 0.27 and 3.34 +/- 0.32; estradiol-17 beta 5.36 +/- 3.0, 1.08 +/- 0.014, 1.44 +/- 0.038 and 1.47 +/- 0.03, respectively. Incubation of human testicular tissue with [3H]androst-5-ene-3 beta, 17 beta-diol or [3H]dihydrotestosterone showed that both androstane-diols were exclusively formed from dihydrotestosterone. Since high concentrations of 5 alpha-androstane-3 beta, 17 beta-diol are found in testicular tissue it is suggested that this steroid may be an index of seminiferous tubular function.     

An alternate pathway for androgen regulation of brain function: activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol

The complexity of gonadal steroid hormone actions is reflected in their broad and diverse effects on a host of integrated systems including reproductive physiology, sexual behavior, stress responses, immune function, cognition, and neural protection. Understanding the specific contributions of androgens and estrogens in neurons that mediate these important biological processes is central to the study of neuroendocrinology. Of particular interest in recent years has been the biological role of androgen metabolites. The goal of this review is to highlight recent data delineating the specific brain targets for the dihydrotestosterone metabolite, 5alpha-androstane, 3beta,17beta-diol (3beta-Diol). Studies using both in vitro and in vivo approaches provide compelling evidence that 3beta-Diol is an important modulator of the stress response mediated by the hypothalmo-pituitary-adrenal axis. Furthermore, the actions of 3beta-Diol are mediated by estrogen receptors, and not androgen receptors, often through a canonical estrogen response element in the promoter of a given target gene. These novel findings compel us to re-evaluate the interpretation of past studies and the design of future experiments aimed at elucidating the specific effects of androgen receptor signaling pathways.     

A high-performance liquid chromatography-tandem mass spectrometry assay of the androgenic neurosteroid 3alpha-androstanediol (5alpha-androstane-3alpha,17beta-diol) in plasma

The testosterone metabolite 3alpha-androstanediol (5alpha-androstane-3alpha,17-diol) is a potential GABA(A) receptor-modulating neurosteroid with anticonvulsant properties and hence could act as a key neuromodulator in the central nervous system. However, there is no specific and sensitive assay for quantitative determination of the androgenic neurosteroid 3alpha-androstanediol in biological samples. We have established a liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay to measure 3alpha-androstanediol in rat plasma. Standard 3alpha-androstanediol added to rat plasma has been successfully analysed with excellent linearity, specificity, sensitivity, and reproducibility. The sensitivity of the method was < 10 ng/ml with a detection limit of 2 ng/ml (6.8 nmol/l) and a linear range of 10-2000 ng/ml. The method was used for the analysis of testosterone-induced increase in plasma 3alpha-androstanediol levels in rats. Testosterone produced a dose-dependent elevation in plasma 3alpha-androstanediol, which was almost completely prevented by pretreatment with the 5alpha-reductase inhibitor finasteride, indicating that 3alpha-androstanediol is synthesized from testosterone via a 5alpha-reductase pathway. This LC-MS-MS method allows accurate, high-throughput analysis of 3alpha-androstanediol in small amounts (200 microl) of plasma and possibly other biological samples.     

Increase in plasma 5 alpha-androstane-3 alpha,17 beta-diol glucuronide as a marker of peripheral androgen action in hirsutism: a side-effect induced by cyclosporine A

Dose-dependent hypertrichosis is a common dermatological side-effect affecting the majority of patients treated with cyclosporine A (CSA). Previous studies have not demonstrated the influence of CSA on specific sex hormone levels. The aim of this study is to investigate whether CSA increases the activity of 5 alpha-reductase, an enzyme which transforms androgens into dihydrotestosterone in peripheral tissues. The metabolite which best reflects this activity is 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (Adiol G). The study was carried out on 49 insulin-dependent diabetes patients participating in the double-blind "Cyclosporine-Diabète-France" clinical trial, of which 28 were treated with CSA (16 males and 12 females), and 21 received only placebo (10 males and 11 females). All patients underwent extensive clinical and laboratory evaluations prior to and during the present study. In addition to Adiol G, testosterone (T), dehydroepiandrosterone sulfate (DHEA S) and sex hormone-binding globulin (SHBG) were assayed. Levels of Adiol G increased significantly in CSA-treated groups: males, 11.86 +/- 2.58 vs 7.83 +/- 2.30 nmol/l; females, 4.48 +/- 2.70 vs 2.10 +/- 1.22 nmol/l; P less than 0.02 (comparison of means). There were no significant differences in this parameter before and during treatment in either the male or female placebo groups (paired t-test). During the treatment period, T, DHEA S, SHBG and the T/SHBG ratio did not significantly change with respect to their baseline values in any of the groups studied (comparison of means). Comparison (using paired t-test) showed a significant increase of DHEA S in CSA-treated groups: males, delta = 3.08 +/- 3.33 nmol/l, P less than 0.01; females, delta = 0.98 +/- 1.13 nmol/l, P less than 0.05. In conclusion, it is possible that CSA induces hypertrichosis or hirsutism by increasing 5 alpha-reductase activity in peripheral tissues. Nevertheless the role of increased DHEA S as a possible Adiol G precursor cannot be excluded.     

5alpha-Androstane-3beta,17beta-diol (3beta-diol), an estrogenic metabolite of 5alpha-dihydrotestosterone, is a potent modulator of estrogen receptor ERbeta expression in the ventral prostrate of adult rats

Prostate is one of the major targets for dihydrotestosterone (DHT), however this gland is also recognized as a nonclassical target for estrogen as it expresses both types of estrogen receptors (ER), especially ERbeta. Nevertheless, the concentrations of aromatase and estradiol in the prostate are low, indicating that estradiol may not be the only estrogenic molecule to play a role in the prostate. It is known that DHT can be metabolized to 5alpha-androstane-3beta,17beta-diol (3beta-diol), a hormone that binds to ERbeta but not to AR. The concentration of 3beta-diol in prostate is much higher than that of estradiol. Based on the high concentration of 3beta-diol and since this metabolite is a physiological ERbeta ligand, we hypothesized that 3beta-diol would be involved in the regulation of ERbeta expression. To test this hypothesis, adult male rats were submitted to castration followed by estradiol, DHT or 3beta-diol replacement. ERbeta and AR protein levels in the prostate were investigated by immunohistochemistry and Western blotting assays. The results showed that after castration, the structure of the prostate was dramatically changed and ERbeta and AR protein levels were decreased. Estradiol had just minor effects on the parameters analyzed. DHT-induced partial recovery of ERbeta while it was the most effective inductor of AR expression. Replacement with 3beta-diol-induced the highest levels of ERbeta, but was comparatively less effective in recovering the AR expression and the gland structure. These results offer evidence that one functional role of 3beta-diol in the prostate may be autoregulation of its natural receptor, ERbeta.     

Na,K-ATPase alpha3 subunit in the goldfish retina during optic nerve regeneration

The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up-regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the Na,K-ATPase catalytic subunit alpha3 isoform by high- sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5-20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50-100 nm ) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up-regulation of the Na,K-ATPase alpha3 subunit is involved in the regrowth of ganglion cell axons after axotomy.     

Neuronal nicotinic acetylcholine receptor alpha3 subunit protein in rat brain and sympathetic ganglion measured using a subunit-specific antibody: regional and ontogenic expression

A synthetic peptide corresponding to the C-terminus of the alpha 3 subunit of the rat neuronal nicotinic acetylcholine receptor (nAChR) was used to generate a rabbit polyclonal alpha 3 antibody. The specificity of this antibody was characterized by immunoblotting, immunohistochemical and immunoprecipitation techniques. Using this antibody, the relative densities of the alpha 3 subunit were quantitatively determined in different brain regions and in superior cervical ganglion (SCG). Among these regions, SCG, interpeduncular nucleus (IPN) and pineal gland showed the highest levels of alpha 3 protein expression. Habenula and superior colliculi had intermediate levels of expression. Low levels were found in cerebral cortex, hippocampus and cerebellum. The ontogenic profile of the alpha 3 subunit in the SCG was also determined. The alpha 3 protein level is low at postnatal day (P 1), but increases rapidly during the first seven postnatal days. This level then plateaus and remains stable through postnatal day 35. These findings suggest that neuronal nAChRs containing the alpha 3 subunit participate in important roles in specific regions of the rat brain and the SCG.     

Improved gating of a chimeric alpha7-5HT3A receptor upon mutations at the M2-M3 extracellular loop

Acetylcholine-evoked currents of the receptor chimera alpha7-5HT3A V201 expressed in Xenopus oocytes are strikingly small when compared to the amount of alpha-bungarotoxin binding sites detected at the oocyte membrane. Since the chimeric receptor is made of the extracellular N-terminal region of the rat alpha7 nicotinic acetylcholine receptor and the C-terminal region of the mouse 5-HT3A receptor, which includes the ion channel, we hypothesized that communication between these two regions was not optimal. Here, we show that mutating to aspartate several adjacent positions in the M2-M3 extracellular linker increases current amplitudes to different extents, thus confirming the important role of this region on receptor gating.     

Concordant up-regulation of cytochrome P450 Cyp3a11, testosterone oxidation and androgen receptor expression in mouse brain after xenobiotic treatment

Inactivation of testosterone by specific hydroxylations is a main function of cytochrome P450 (P450, CYP) in the brain. Recent data imply that induction of brain P450s by neuroactive drugs alters steroid hormone levels and endocrine signalling, giving rise to endocrine disorders. In this study, we investigated this drug–hormone crosstalk in mouse brain. Phenytoin led to a significant increase of 2α-, 2β-, 6β-, 16α- and 16β-hydroxytestosterones, while 6α- and 15α-hydroxytestosterones showed no significant alteration of their metabolism compared with untreated controls. Inhibition of testosterone hydroxylation using the chemical inhibitors orphenadrine, chloramphenicol, ketoconazole and nifedipine as well as antibodies against CYP3A- and 2B-isoforms pointed to major role of Cyp3a11 and an only minor function of Cyp2b9/10 in mouse brain. Cyp3a11 revealed to be the major isoform affected by phenytoin. There was considerable overlap of Cyp3a11 and AR expression in neuronal structures of the limbic system, namely the hippocampus, amygdala, hypothalamus and thalamus. Phenytoin treatment led to an increase of both, Cyp3a11 and AR expression in the limbic system. Additionally, the coherence between CYP3A and AR expression was analysed in PC-12 cells. Inhibition of phenytoin-induced endogenous CYP3A2 and AR by ketoconazole led a reduction of their expression to basal levels. We conclude that Cyp3a11 plays a crucial role in directing drug action to hormonal response within the limbic system of mouse brain in a so-called drug–hormone crosstalk.     

Coincident signalling between the Gi/Go-coupled delta-opioid receptor and the Gq-coupled m3 muscarinic receptor at the level of intracellular free calcium in SH-SY5Y cells

In SH-SY5Y cells, activation of delta-opioid receptors with [D-Pen(2,5)]-enkephalin (DPDPE; 1 microM) did not alter the intracellular free Ca(2+) concentration [Ca(2+)](i). However, when DPDPE was applied during concomitant Gq-coupled m3 muscarinic receptor stimulation by carbachol or oxotremorine-M, it produced an elevation of [Ca(2+)](i). The DPDPE-evoked increase in [Ca(2+)](i) was abolished when the carbachol-sensitive intracellular Ca(2+) store was emptied. There was a marked difference between the concentration-response relationship for the elevation of [Ca(2+)](i) by carbachol (EC(50) 13 microM, Hill slope 1) and the concentration-response relationship for carbachol's permissive action in revealing the delta-opioid receptor-mediated elevation of [Ca(2+)] (EC(50) 0.7 mM; Hill slope 1.8). Sequestration of free G protein beta gamma dimers by transient transfection of cells with a beta gamma binding protein (residues 495-689 of the C terminal tail of G protein-coupled receptor kinase 2) reduced the ability of delta opioid receptor activation to elevate [Ca(2+)](i). However, DPDPE did not elevate either basal or oxotremorine-M-evoked inositol phosphate production indicating that delta-opioid receptor activation did not stimulate phospholipase C. Furthermore, delta-opioid receptor activation did not result in the reversal of muscarinic receptor desensitization, membrane hyperpolarization or stimulation of sphingosine kinase. There was no coincident signalling between the delta-opioid receptor and the lysophosphatidic acid receptor which couples to elevation of [Ca(2+)](i) in SH-SY5Y cells by a PLC-independent mechanism. In SH-SY5Y cells the coincident signalling between the endogenously expressed delta-opioid and m3 muscarinic receptors appears to occur in the receptor activation-Ca(2+) release signalling pathway at a step after the activation of phospholipase C.     

Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A–F. Here we examine the 5-HT₃ receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [³H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC₅₀ when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in K_d and EC₅₀ values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT₃ receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT₃ receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.     

An alternate pathway for androgen regulation of brain function: Activation of estrogen receptor beta by the metabolite of dihydrotestosterone, 5α-androstane-3β,17β-diol

The complexity of gonadal steroid hormone actions is reflected in their broad and diverse effects on a host of integrated systems including reproductive physiology, sexual behavior, stress responses, immune function, cognition, and neural protection. Understanding the specific contributions of androgens and estrogens in neurons that mediate these important biological processes is central to the study of neuroendocrinology. Of particular interest in recent years has been the biological role of androgen metabolites. The goal of this review is to highlight recent data delineating the specific brain targets for the dihydrotestosterone metabolite, 5α-androstane, 3β,17β-diol (3β-Diol). Studies using both in vitro and in vivo approaches provide compelling evidence that 3β-Diol is an important modulator of the stress response mediated by the hypothalmo–pituitary–adrenal axis. Furthermore, the actions of 3β-Diol are mediated by estrogen receptors, and not androgen receptors, often through a canonical estrogen response element in the promoter of a given target gene. These novel findings compel us to re-evaluate the interpretation of past studies and the design of future experiments aimed at elucidating the specific effects of androgen receptor signaling pathways.     

Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Background information. In endocrine cells, IP₃R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺ concentration. There are three subtypes of IP₃R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP₃R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP₃R‐3 in RINm5F cells. Results. We show that immunoprecipitated IP₃R‐3 is a good substrate for PKA. Using a back‐phosphorylation approach, we show that endogenous PKA phosphorylates IP₃R‐3 in intact RINm5F cells. [³H]IP₃ (inositol 1,4,5‐trisphosphate) binding affinity and IP₃‐induced Ca²⁺ release activity were enhanced in permeabilized cells that were pre‐treated with forskolin or PKA. The PKA‐induced enhancement of IP₃R‐3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. Conclusion. The results of the present study reveal a converging step where the cAMP and the Ca²⁺ signalling systems act co‐operatively in endocrine cell responses to external stimuli.