A neuroactive steroid 5alpha-androstane-3alpha,17beta-diol regulates androgen receptor level in astrocytes

Optic nerve head (ONH) astrocytes from patients with glaucomatous optic neuropathy exhibit increased production of 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), a neuroactive metabolite of 5alpha-dihydrotestosterone (5alpha-DHT). To determine whether ONH astrocytes are androgen target cells, and whether 3alpha-diol is capable of regulating astrocyte functions, we studied the response of human ONH astrocytes to 3alpha-diol compared with 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one (R1881), a synthetic 5alpha-DHT agonist. In ONH astrocytes, both 3alpha-diol and R1881 increased protein levels of androgen receptor (AR) and glial fibrillary acidic protein (GFAP), however, only R1881 also increased the AR mRNA level and astrocyte proliferation. Both R1881 and 3alpha-diol rapidly activate the mitogen-activated protein kinase (MAPK) signaling pathway in ONH astrocytes, as confirmed by phosphorylation of extracellular signal-regulated kinase (ERK). 3Alpha-diol also activates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. 3Alpha-diol regulates the increase of AR protein level and the phosphorylation through the PI3K/Akt pathway, whereas R1881 regulates them through the MAPK/ERK pathway. Our findings demonstrate that human ONH astrocytes are androgen target cells and respond to androgens by the rapid activation of cell signaling. The activation of the PI3K/Akt pathway by 3alpha-diol may regulate various properties of astrocytes, including cell motility and survival, and may play a role in the formation and maintenance of the reactive phenotype of ONH astrocytes in glaucoma.     

The impact of cell adhesion changes on proliferation and survival during prostate cancer development and progression

In the normal prostate epithelium, androgen receptor (AR) negative basal epithelial cells adhere to the substratum, while AR expressing secretory cells lose substratum adhesion. In contrast, prostate cancer cells both express AR and adhere to a tumor basement membrane. In this review, we describe the differential expression of integrins, growth factor receptors (GFRs), and AR in normal and cancerous epithelium. In addition, we discuss how signals from integrins, GFRs, and AR are integrated to regulate the proliferation and survival of normal and malignant prostate epithelial cells. While cell adhesion is likely of great importance when considering therapeutic approaches for treatment of metastatic prostate cancer, no data on integrin expression are available from tissues of prostate cancer metastasis. However, several drug targets that are upregulated after androgen ablative therapy regulate cell adhesion and thus novel targeted therapies indirectly interfere with cell adhesion mechanisms in prostate cancer cells.     

AZGP1 is androgen responsive and involved in AR-induced prostate cancer cell proliferation and metastasis

Alpha-2-glycoprotein 1, zinc-binding (AZGP1), known as zinc-alpha-2-glycoprotein (ZAG), is a multifunctional secretory glycoprotein and relevant to cancer metastasis. Little is known regarding the underlying mechanisms of AZGP1 in prostate cancer (PCa). In the present study, we report that AZGP1 is an androgen-responsive gene, which is involved in AR-induced PCa cell proliferation and metastasis. In clinical specimens, the expression of AZGP1 in PCa tissues is markedly higher than that in adjacent normal tissues. In cultures, expression of AZGP1 is upregulated by the androgen-AR axis at both messenger RNA and protein levels. Furthermore, Chip-Seq assay identifies canonical androgen-responsive elements (AREs) at AZGP1 enhancer; and dual-luciferase reporter assays reveal that the AREs is highly responsive to androgen whereas mutations of the AREs abolish the reporter activity. In addition, AZGP1 promotes G1/S phase transition and cell cycle progress by increasing cyclin D1 levels in PCa cells. Functional studies demonstrate that knocking down endogenous AZGP1 expression in LNCaP and CWR22Rv1 cells largely weaken androgen/AR axis-induced cell migration and invasion. In vivo xenotransplantation tumor experiments also show that AZGP1 involves in androgen/AR axis-mediated PCa cell proliferation. Taken together, our study implicates for the first time that AZGP1 is an AR target gene and is involved in androgen/AR axis-mediated cell proliferation and metastasis in primary PCa.     

Interaction of IGF signaling and the androgen receptor in prostate cancer progression

The insulin-like growth factor type I receptor (IGF-IR) has been suggested to play an important role in prostate cancer progression and possibly in the progression to androgen-independent (AI) disease. The term AI may not be entirely correct, in that recent data suggest that expression of androgen receptor (AR) and androgen-regulated genes is the primary association with prostate cancer progression after hormone ablation. Therefore, signaling through other growth factors has been thought to play a role in AR-mediated prostate cancer progression to AI disease in the absence of androgen ligand. However, existing data on how IGF-IR signaling interacts with AR activation in prostate cancer are conflicting. In this Prospect article, we review some of the published data on the mechanisms of IGF-IR/AR interaction and present new evidence that IGF-IR signaling may modulate AR compartmentation and thus alter AR activity in prostate cancer cells. Inhibition of IGF-IR signaling can result in cytoplasmic AR retention and a significant change in androgen-regulated gene expression. Translocation of AR from the cytoplasm to the nucleus may be associated with IGF-induced dephosphorylation. Since fully humanized antibodies targeting the IGF-IR are now in clinical trials, the current review is intended to reveal the mechanisms of potential therapeutic effects of these antibodies on AI prostate cancers.     

Radioimmunoassays for androsterone, 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol

Androsterone (3alpha-hydroxy-5alpha-androstan-17-one), 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol were conjugated at C-16 through sulfur to bovine and human serum albumin. Rabbits injected with these conjugates produced antibodies suitable for radioimmunoassays of these hormone metabolites. Samples were purified on Sephadex LH-20 columns. Levels of these steroids were measured in a rat blood serum pool and in ovarian tissue extract pools.     

Determination of 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol in human plasma by radioimmunoassay

5 alpha-Androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) were measured in human peripheral plasma by radioimmunoassay using celite microcolumn purification. The antisera used for the assay were obtained by immunization of rabbits with 3 alpha,17 beta-dihydroxy-5 alpha-androstane-6-(O-carboxymethyl) oxime: BSA for 3 alpha-diol and 3 beta,17 beta-dihydroxy-5 alpha-androstane-15 alpha-carboxymethyl: BSA for 3 beta-diol. The concentrations (pg/ml +/- SD) of the two diols in normal male and female plasma are respectively: 216 +/- 51 and 49 +/- 32 for 3 alpha-diol, 239 +/- 76 and 82 +/- 45 for 3 beta-diol. Comparison of these results with published ones shows that 3 beta diol concentrations were significantly lower. The high specificity of the assay is due to chromatography on celite microcolumns, allowing elimination of 5-androstene-3 beta,17 beta-diol from the plasma sample.     

Inhibition of Plk1 represses androgen signaling pathway in castration-resistant prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in males in the United States. Majority of prostate cancers are originally androgen-dependent and sensitive to androgen-deprivation therapy (ADT), however, most of them eventually relapse and progress into incurable castration-resistant prostate cancer (CRPC). Of note, the activity of androgen receptor (AR) is still required in CRPC stage. The mitotic kinase polo-like kinase 1 (Plk1) is significantly elevated in PCa and its expression correlates with tumor grade. In this study, we assess the effects of Plk1 on AR signaling in both androgen-dependent and androgen-independent PCa cells. We demonstrate that the expression level of Plk1 correlated with tumorigenicity and that inhibition of Plk1 caused reduction of AR expression and AR activity. Furthermore, Plk1 inhibitor BI2536 down-regulated SREBP-dependent expression of enzymes involved in androgen biosynthesis. Of interest, Plk1 level was also reduced when AR activity was inhibited by the antagonist MDV3100. Finally, we show that BI2536 treatment significantly inhibited tumor growth in LNCaP CRPC xenografts. Overall, our data support the concept that Plk1 inhibitor such as BI2536 prevents AR signaling pathway and might have therapeutic potential for CRPC patients.     

Metabolism and binding in vitro of 5 alpha-androstane-3 beta, 17 beta-diol and of 5 alpha-androstane-3 alpha, 17 beta-diol in cell fractions of rat ventral prostate and liver.

Rat ventral prostate and liver were investigated for the binding in vitro to particulate fractions and for the metabolism of 5 alpha-androstane-3 beta, 17 beta-diol. Comparative investigations were carried out on the metabolism of 5 alpha-androstane-3 alpha, 17 beta-diol. Preparations of the liver were investigated in order to establish the organ specificity of the method. In the prostate, the bulk of the metabolites of 5 alpha-androstane-3 beta, 17 beta-diol was present as steroids of high polarity. Of the less polar metabolites, 17 beta-hydroxy-5 alpha-androstan-3-one, 3 beta-hydroxy-5 alpha-androstan, 17-one and 5 alpha-androstane-3 alpha, 17 beta-diol were detectable. The binding of a 5 alpha-androstane-3 beta, 17 beta-diol to mitochondria and microsomes was unspecific. In the liver, among the less polar metabolites, 3 beta-hydroxy-5 alpha-androstan-17-one was the main metabolite, and the binding was unspecific. The main metabolite in the prostate homogenate of 5 alpha-androstane-3 alpha, 17 beta-diol was 17 beta-hydroxy-5 alpha-androstan-3-one. The portion of highly polar steroids was very low. The portion of unmetabolized hormone was distributed almost equally among the different cell preparations except the nuclei, in which 17 beta-hydroxy-5 alpha-androstan-3-one was higher and 5 alpha-androstane-3 alpha, 17 beta-diol was lower than in the remaining cell fractions.     

Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer cells

Ferruginol, a bioactive compound isolated from a Chilean tree (Podocarpaceae), attracts attention as a consequence of its pharmacological properties, which include anti-fungal, anti-bacterial, cardioprotective, anti-oxidative, anti-plasmodial and anti-ulcerogenic actions. Nevertheless, the molecular basis for these actions remains only partly understood and hence we investigated the effects of ferruginol on androgen-independent human prostate cancer cells (PC3), a known model for solid tumor cells with an exceptional resistance to therapy. The results show that ferruginol induces PC3 cell death via activation of caspases as well as apoptosis-inducing factor (AIF) as confirmed by its translocation into the nucleus. In order to clarify the biochemical mechanism responsible for the anti-tumor activity of ferruginol, we analyzed a set of molecular mediators involved in tumor cell survival, progression and aggressiveness. Ferruginol was able to trigger inhibition/downregulation of Ras/PI3K, STAT 3/5, protein tyrosine phosphatase and protein kinases related to cell cycle regulation. Importantly, the toxic effect of ferruginol was dramatically impeded in a more reducing environment, which indicates that at least in part, the anti-tumoral activity of ferruginol might be related to redox status modulation. This study supports further examination of ferruginol as a potential agent for both the prevention and treatment of prostate cancer.     

Synthesis of 5 alpha-androstane-3 alpha,17 beta-diol 3- and 17-glucuronides

The glucuronidation of 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) was carried out by three different methods: The Koenigs-Knorr reaction using methyl-2,3,4-tri-O-acetyl-1 alpha-bromo-1-deoxy-beta-D-glucuronate, by employing methyl-2,3,4-tri-O-acetyl-1-O-(trichloroacetimidoyl)-alpha-D-gl ucopyranuronate (imidate procedure), and by the reaction of 2,3,4-tri-O-acetyl-alpha-D-glucopyranuronate catalyzed by trimethylsilyl trifluoromethanesulfonate (triflate method). The Koenigs-Knorr method gave the beta-anomers of both the 3- and 17-glucuronides. The imidate procedure also resulted in the beta-anomers of the 3- and 17-glucuronides, but in lower yield. The triflate method, however, yielded only the alpha-anomers of the 3- and 17-glucuronides. The structural assignments of these compounds were made from NMR spectral data obtained with a 500 mHz instrument.     

Synthesis of deuterium-labeled 5 alpha-androstane-3 alpha, 17 beta-diol and its 17 beta-glucuronide.

Using unlabeled androsterone as starting material, 5 alpha-[16,16-2H2]androstan-3 alpha-ol-17-one was synthesized by exchange using deuterated potassium methoxide. This labeled androsterone product was reduced by sodium borodeuteride, which gave predominantly trideuterated 5 alpha-androstane-3 alpha, 17 beta-diol. The labeled androstanediol was conjugated with glucuronide by using the Koenig-Knorr reaction with methyl-1-bromo-1-deoxy-2,3,4-tri-O-acetyl-alpha-D-glucopyranosuronate . The dominant product was identified by thermospray high-performance liquid chromatography/mass spectrometry (MS) and electrospray MS as 5 alpha-[16,16,17-2H3]androstane-3 alpha, 17 beta-diol, 17 beta-glucuronide.     

Pituitary metabolism of 5alpha-androstane-3beta-17beta-diol: intense and rapid conversion into 5alpha-androstane-3beta,6alpha,17beta-triol and 5alpha-androstane-3beta,7alpha, 17beta-triol.

In the male rat pituitary, 5alpha-androstane-3beta, 17beta-diol (3beta-diol) is extensively metabolized into polar steroids. They were identified as 5alpha-androstane-3beta, 6alpha-17beta-triol (6alpha-triol) and 5alpha-androstane-3beta, 7alpha, 17beta-triol (7alpha-triol). 6-alpha-Triol represents 53% and 7alpha-Triol 28% of the total 3beta-diol metabolites. The remaining percentage is related to 6beta and 7beta isomers. The biological role of triols is still unknown.     

Age-related changes in conversion of 5 alpha-androstan-17 beta-ol-3-one to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol by rat testicular cells in vitro

Conversion of labelled 5 alpha-androstane-17 beta-ol-3-one (DHT) by isolated testicular cells from rats of different ages was examined under saturating substrate conditions in vitro (5--10 micrograms DHT/ml in a 24 h incubation). Two detectable metabolites of DHT were produced by testicular cells in vitro. 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol). Production of these diols during a 24 h period was linear, and the amounts formed were directly related to the cell number. The amount of 3 alpha- and 3 beta-diols formed by testicular cells of rats of different ages increased from Day 10 to Day 25, then declined. Testicular cells from rats 10 to 20 days of age converted DHT mainly to 3 alpha-diol, but thereafter 3 beta-diol was the predominant testicular metabolite of DHT.