Solid tumors have an increased reliance on Hsp70/Hsp90 molecular chaperones for proliferation, survival and maintenance of intracellular signaling systems. An underinvestigated component of the chaperone system is the tetratricopeptide repeat (TPR)-containing cochaperone, which coordinates Hsp70/Hsp90 involvement on client proteins as well as having diverse individual actions. A potentially important cochaperone in prostate cancer (PCa) is small glutamine-rich TPR-containing protein alpha (SGTA), which interacts with the androgen receptor (AR) and other critical cancer-related client proteins. In this study, the authors used small interfering RNA coupled with genome-wide expression profiling to investigate the biological significance of SGTA in PCa and its influence on AR signaling. Knockdown of SGTA for 72 hr in PCa C4-2B cells significantly altered expression of >1,900 genes (58% decreased) and reduced cell proliferation (p < 0.05). The regulation of 35% of <em>5α</em>-dihydrotestosterone (<em>DHT</em>) target genes was affected by SGTA knockdown, with gene-specific effects on basal or <em>DHT</em>-induced expression or both. Pathway analysis revealed a role for SGTA in p53, generic PCa and phosphoinositol kinase (PI3K) signaling pathways; the latter evident by a reduction in PI3K subunit p100β levels and decreased phosphorylated Akt. Immunohistochemical analysis of 64 primary advanced PCa samples showed a significant increase in the AR:SGTA ratio in cancerous lesions compared to patient-matched benign prostatic hyperplasia tissue (p < 0.02). This study not only provides insight into the biological actions of SGTA and its effect on genome-wide AR transcriptional activity and other therapeutically targeted intracellular signaling pathways but also provides evidence for PCa-specific alterations in SGTA expression.

The existing drugs for benign prostatic hyperplasia (BPH) are partially effective with undesirable side-effects; hence new agents acting by different mechanism(s) are required as supplements. Modulation of estrogen receptor signaling using selective estrogen receptor modulators (SERMs) offers an alternative approach for BPH management. Using human BPH-derived stromal cells and tissue explants in culture we evaluated two SERMs, DL-2-[4-(2-piperidinoethoxy)phenyl]-3-phenyl-2 H-1-benzopyran (BP) and Ormeloxifene (Orm) in comparison to Tamoxifen (Tam) and 4-hydroxytamoxifen (OHT). BP, OHT and Tam were more effective than Orm in reducing stromal cell proliferation of human BPH. BP was either equipotent or more effective than OHT and Tam in increasing estrogen receptor(ER)-ß, TGFß1, Fas and FasL, and in decreasing ER-α, AR, EGF-R and IGF-I expressions in BPH stromal cells. BP, Tam and Orm (1.0 mg/Kg) reduced rat prostate weights by almost same extent as Finasteride (Fin, 5.0 mg/Kg); however combination treatment (SERM+Fin) was more effective. BP was exceptionally efficient in reducing IGF-1 and cleaving PARP while combination treatments more effectively increased bax:bcl-2 ratio. Fin reduced acinar diameter and prostatic <em>DHT</em> level but increased testosterone, estradiol (E(2)) and E(2)/T+<em>DHT</em> ratio. SERMs, especially BP, reduced epithelial cell height drastically without significantly altering steroid hormone levels and E(2)/T+<em>DHT</em> ratio. Combination treatment reduced both acinar diameter and epithelial cell height with modest increase in E(2), T and E(2)/T+<em>DHT</em>. The study reveals the potential of SERMs per se for BPH management, and more effectively in combination with a <em>5α</em>-reductase inhibitor. BP appears promising for further evaluation as a drug candidate for BPH and prostate cancer.

BACKGROUND

Abacopteris penangiana (Hook.) Ching (AP) is a member of parathelypteris glanduligera and used in folk medicine for the treatment of blood circulation and blood stasis, edema and inflammation as recorded in the ″Chinese Materia Medica″.

OBJECTIVE

The purpose of this study was to investigate the effects of total flavanol glycosides (TFA) from AP and its acid hydrolysate (AHT) on testosterone-induced benign prostatic hyperplasia (BPH) in rats by measuring the levels of inflammatory responses, oxidative stress and prostate cell proliferation.

METHODS

BPH was induced in rats by subcutaneous injection of testosterone after castration. Seventy rats were divided into seven groups. After oral administration of AHT and TFA (100 or 200mg/kg/d) for 4 weeks, the prostate index (PI), <em>5a</em>-reductase (<em>5α</em>-R) and dihydrotestosterone (<em>DHT</em>) were determined. Then the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) were determined. In addition, the relative inflammatory factors, cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8) and interleukin 17 (IL-17) were measured. Finally, the prostatic expression of nuclear transcription factor-κB (NF-κB) and phosphoinositide3-kinase (PI3K)/Akt were determined by immunohistochemistry. The prostatic expression of Bcl-2 was determined by western blot analysis.

RESULTS

The results showed that AHT and TFA decreased serum <em>DHT</em> and <em>5α</em>-R activities compared with model group, as well as the PI and histopathological examination findings. In addition, oral treatment of AHT and TFA can significantly increase the activities of SOD, GPx and CAT while the level of MDA was significantly decreased compared with the model group. Moreover, AHT and TFA remarkably decreased the levels of inflammatory cytokines in prostatic tissue. Further investigation demonstrated that AHT and TFA treatment down-regulated the protein expressions of p-Akt, NF-κB and Bcl-2.

CONCLUSIONS

These results suggest that AHT and TFA have anti-BPH properties via anti-inflammatory, antioxidant and anti-proliferative effects. Hence, AP represents a potential herb for the treatment of BPH.

Studies show that treatment of men with <em>5α</em>-reductase inhibitors such as finasteride is effective for the primary prevention of prostate cancer. Although it is known that finasteride treatment suppresses serum levels of dihydrotestosterone (<em>DHT</em>) and its distal metabolite, <em>5α</em>-androstane-3α,17β-diol glucuronide (3α-diol G), and increases serum testosterone (T) levels, little is known about its effect on other precursors and metabolites of <em>DHT</em>, as well as on the relationship of these androgens to prostate specific antigen (PSA), a marker of prostatic intraepithelial neoplasia. The present study provides new data on the effect of finasteride on precursors and metabolites of <em>DHT</em>. Fifty-three men, ages 57-79 years, with elevated PSA levels (>4ng/ml), were randomized to treatment with finasteride (5mg/day) or observation (controls) for 12 months. Blood samples were obtained at baseline, 1, 3, 6 and 12 months for measurement of PSA, androstenedione (A), T, <em>DHT</em>, 3α-diol G, androsterone glucuronide (ADT G) and <em>DHT</em> sulfate (<em>DHT</em> S) in serum by validated, highly specific radioimmunoassays. Statistical analysis was carried out using mixed model ANOVA and t-tests. In the control group, PSA and androgen levels were unchanged throughout the 12 months of treatment. In the finasteride group, PSA, <em>DHT</em>, <em>DHT</em> S, 3α-diol G and ADT G decreased from baseline to 1 month by 23.2%, 78.7%, 71.0%, 75.7% and 43.0%, respectively. The change in PSA decreased further to 46.1% and 55.1% at 3 and 12 months of treatment, respectively, whereas the decrease in androgens observed at 1 month did not change by more than 6.9% for <em>DHT</em>, <em>DHT</em> S and 3α-diol G in the subsequent months of sampling. However, the decline in ADT G was only 22.2% at month 3, and remained essentially at this level after that time. In contrast, T and A increased significantly from baseline, and the increase in A of approximately 34.5% was about 1.9 times the increase in T (approximately 18.3%). The present data suggest that either 3α-diol G or <em>DHT</em> S may serve as a potential diagnostic marker of intraprostatic <em>5α</em>-reductase activity during treatment of patients with <em>5α</em>-reductase inhibitors.

It is well known that the androgen/androgen receptor (AR) pathway is involved in both male and female fertility in mammals. AR knockout female mice are reported to exhibit various abnormalities in follicle development, and a subfertile phenotype. In exogenous gonadotropin-induced superovulation, serum androgen levels were robustly elevated in female mice at the periovulatory stage after human chorionic gonadotropin (hCG) treatment. At this stage, ovarian AR proteins were strongly expressed in cumulus cells. Because these results suggested that the androgen/AR pathway is involved in ovulation, we investigated the expression of ovulation-related genes in the mouse ovary treated with the nonaromatizable androgen, <em>5α</em>-dihydrotestosterone (<em>DHT</em>). <em>DHT</em> treatment induced the expression of the genes for cyclooxyganase-2 (Cox-2 or prostaglandin endoperoxidase synthase 2) and the epidermal growth factor-like factor, amphiregulin (Areg), in the ovary, whereas their hCG-induced expression was suppressed by the AR antagonist flutamide. These genes were also induced by <em>DHT</em> in AR-expressing primary granulosa and granulosa tumor-derived cells. Reporter assays, electrophoretic shift mobility assays and chromatin immunoprecipitation assays demonstrated that androgen response sequence(s) existing upstream of each gene were responsible for androgen responsiveness and were occupied by the AR in periovulatory granulosa cells. Our results suggest that the androgen/AR pathway is involved in the ovulatory process via expression of the Cox-2 and Areg genes in periovulatory granulosa cells.

Androgen deprivation therapy (ADT) is the standard treatment for patients with prostate-specific antigen progression after treatment for localized prostate cancer. An alternative to continuous ADT is intermittent ADT (IADT), which allows recovery of testosterone during off-cycles to stimulate regrowth and differentiation of the regressed prostate tumor. IADT offers patients a reduction in side effects associated with ADT, improved quality of life, and reduced cost with no difference in overall survival. Our previous studies showed that IADT coupled with <em>5α</em>-reductase inhibitor (5ARI), which blocks testosterone conversion to <em>DHT</em> could prolong survival of animals bearing androgen-sensitive prostate tumors when off-cycle duration was fixed. To further investigate this clinically relevant observation, we measured the time course of testosterone-induced regrowth of regressed LuCaP35 and LNCaP xenograft tumors in the presence or absence of a 5ARI. <em>5α</em>-Reductase inhibitors suppressed the initial regrowth of regressed prostate tumors. However, tumors resumed growth and were no longer responsive to <em>5α</em>-reductase inhibition several days after testosterone replacement. This finding was substantiated by bromodeoxyuridine and Ki67 staining of LuCaP35 tumors, which showed inhibition of prostate tumor cell proliferation by 5ARI on day 2, but not day 14, after testosterone replacement. <em>5α</em>-Reductase inhibitors also suppressed testosterone-stimulated proliferation of LNCaP cells precultured in androgen-free media, suggesting that blocking testosterone conversion to <em>DHT</em> can inhibit prostate tumor cell proliferation via an intracrine mechanism. These results suggest that short off-cycle coupled with <em>5α</em>-reductase inhibition could maximize suppression of prostate tumor growth and, thus, improve potential survival benefit achieved in combination with IADT.

Although the vasorelaxing effects of testosterone (T) and various androgen metabolites have been observed in a variety of blood vessels and species, previous studies have not systematically compared the vasorelaxing effects of androgen metabolites in different vascular beds within the same species. Therefore, we studied the vasorelaxing effects of T and its 5-reduced metabolites (<em>5α</em>- and 5β-<em>DHT</em>) on KCl-induced contractions of the canine left coronary artery, femoral artery and saphenous vein, using standard isometric recordings. KCl contractions were inhibited by each androgen in a concentration-dependent manner from 1.8 to 310μM. Vascular sensitivity and efficacy were expressed as inhibitory concentration 50 (IC₅₀) and maximal relaxation (R(max)), respectively. The coronary artery was significantly more sensitive to androgen-induced vasorelaxation than the saphenous vein or femoral artery. These vasorelaxing responses were unaffected by an antiandrogen (Flutamide) or the sulfhydryl reagent, N-ethylmaleimide, suggesting a nongenomic mechanism independent of signaling mediated by the androgen receptor or G proteins. Concentration-response curves were unchanged in endothelium-denuded preparations; thus, the endothelium appears to have no role in androgen-induced vasorelaxation. 5β-<em>DHT</em> was the most potent androgen in both coronary and femoral artery, but all three androgens were equipotent in the saphenous vein. It is concluded that: 1) significant regional differences exist in vasorelaxing effects of androgen metabolites in the canine vasculature; 2) structural differences in these androgens determine their vasorelaxing efficacy; and 3) regional differences in androgen-induced vasorelaxation may account for some of the conflicting findings reported on the vasorelaxing effects of the androgens.

This study was conducted to evaluate the effect of Grateloupia elliptica, a seaweed native to Jeju Island, Korea, on the prevention of hair loss. When immortalized rat vibrissa dermal papilla cells were treated with extract of G. elliptica, the proliferation of dermal papilla cells significantly increased. In addition, the G. elliptica extract significantly inhibited the activity of <em>5α</em>-reductase, which converts testosterone to dihydrotestosterone (<em>DHT</em>), a main cause of androgenetic alopecia. On the other hand, the G. elliptica extract promoted PGE2 production in HaCaT cells in a dose-dependent manner. The G. elliptica extract exhibited particularly high inhibitory effect on LPS-stimulated IL-12, IL-6, and TNF-α production in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells. The G. elliptica extract also showed inhibitory activity against Pityrosporum ovale, a main cause of dandruff. These results suggest that G. elliptica extract has the potential to treat alopecia via the proliferation of dermal papilla, <em>5α</em>-reductase inhibition, increase of PGE2 production, decrease of LPS-stimulated pro-inflammatory cytokines and inhibitory activity against Pityrosporum ovale.

BACKGROUND

<em>5α</em>-Reductase 2 deficiency (5ARD) is a known cause of 46,XY disorders of sex development (DSD). Traditionally, the diagnosis relies on dihydrotestosterone (<em>DHT</em>) measurement, but the results are often equivocal, potentially leading to misdiagnosis. We reviewed alternative approaches for diagnosis of 5ARD.

METHODS

We conducted a retrospective review of the results of urinary steroid profiling (USP) by GC-MS and mutational analysis of SRD5A2 [steroid-5-alpha-reductase, alpha polypeptide 2 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 2)] by PCR and direct DNA sequencing of all 46,XY DSD patients referred to our laboratory with biochemical and/or genetic findings compatible with 5ARD. We also performed a literature review on the laboratory findings of all 5ARD cases reported in the past 10 years.

RESULTS

Of 16 patients diagnosed with 5ARD between January 2003 and July 2012, 15 underwent USP, and all showed characteristically low <em>5α</em>- to 5β-reduced steroid metabolite ratios. Four patients had <em>DHT</em> measured, but 2 did not reach the diagnostic cutoff. In all 12 patients who underwent genetic analysis, 2 mutations of the SRD5A2 gene were detected to confirm the diagnosis. Twenty-four publications involving 149 patients with 5ARD were published in the review period. Fewer than half of these patients had <em>DHT</em> tested. Nearly 95% of them had the diagnosis confirmed genetically.

CONCLUSIONS

5ARD can be confidently diagnosed by USP at 3 months postnatally and confirmed by mutational analysis of SRD5A2. Interpretation of DHT results may be problematic and is not essential in the diagnosis of 5ARD. We propose new diagnostic algorithms for 46,XY DSD.

OBJECTIVE

Androgens play a crucial role in prostate cancer progression, and trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18) F]FACBC) are used for visualization of prostate cancer. We examined the effect of androgen on the expression of amino acid transporters related to anti-[(18)F]FACBC transport and uptake of trans-1-amino-3-fluoro-[1-(14)C]cyclobutanecarboxylic acid (anti-[(14)C]FACBC).

METHODS

Expression of amino acid transporters and uptake of anti-[(14)C]FACBC in androgen receptor (AR)-positive LNCaP and AR-negative DU145 human prostate cancer cells cultured with/without <em>5α</em>-dihydrotestosterone (<em>DHT</em>) and the effect of bicalutamide, an AR antagonist, on <em>DHT</em>-associated changes were investigated.

RESULTS

DHT stimulated the expression of amino acid transporters ASCT2, SNAT5, 4F2 heavy chain, and LAT3 in LNCaP but not in DU145 cells. Anti-[(14)C]FACBC uptake was enhanced, in a DHT-dependent manner, in LNCaP cells only.

CONCLUSIONS

DHT enhanced the expression of ASCT2, the transporter responsible for anti-[(18)F]FACBC uptake, thereby increasing anti-[(14)C]FACBC uptake in AR-positive LNCaP cells. Androgen-mediated induction may contribute to the distinct anti-[(18)F]FACBC accumulation pattern in prostate cancer.

Nuclear receptor subfamily 4 group A member1 (NR4A1), an orphan nuclear receptor, is involved in the transcriptional regulation of thecal cell androgen biosynthesis and paracrine factor insulin-like 3 (INSL3) expression. Androgens are known to play an important regulatory role in ovarian follicle growth. Using a chronically androgenized rat model, a preantral follicle culture model and virus-mediated gene delivery, we examined the role and regulation of NR4A1 in the androgenic control of preantral follicular growth. In the present study, Ki67 staining was increased in preantral follicles on ovarian sections from <em>5α</em>-dihydrotestosterone (<em>DHT</em>)-treated rats. Preantral follicles from <em>DHT</em>-treated rats cultured for 4 d exhibited increased growth and up-regulation of mRNA abundance of G(1)/S-specific cyclin-D2 (Ccnd2) and FSH receptor (Fshr). Similarly, <em>DHT</em> (1 μm) increased preantral follicular growth and Ccnd2 and Fshr mRNA abundance in vitro. The NR4A1 expression was high in theca cells and was down-regulated by <em>DHT</em> in vivo and in vitro. Forced expression of NR4A1 augmented preantral follicular growth, androstenedione production, and Insl3 expression in vitro. Inhibiting the action of androgen (with androgen receptor antagonist flutamide) or INSL3 (with INSL3 receptor antagonist INSL3 B-chain) reduced NR4A1-induced preantral follicular growth. Furthermore, NR4A1 overexpression enhanced <em>DHT</em>-induced preantral follicular growth, a response attenuated by inhibiting INSL3. In conclusion, <em>DHT</em> promotes preantral follicular growth and attenuates thecal NR4A1 expression in vivo and in vitro. Our findings are consistent with the notion that NR4A1 serves as an important point of negative feedback to minimize the excessive preantral follicle growth in hyperandrogenism.

Women with polycystic ovary syndrome (PCOS) are at increased risk of miscarriage, which often accompanies the hyperandrogenism and insulin resistance seen in these patients. However, neither the combinatorial interaction between these two PCOS-related etiological factors nor the mechanisms of their actions in the uterus during pregnancy are well understood. We hypothesized that hyperandrogensim and insulin resistance exert a causative role in miscarriage by inducing defects in uterine function that are accompanied by mitochondrial-mediated oxidative stress, inflammation, and perturbed gene expression. Here, we tested this hypothesis by studying the metabolic, endocrine, and uterine abnormalities in pregnant rats after exposure to daily injection of <em>5α</em>-dihydrotestosterone (<em>DHT</em>; 1.66 mg·kg body wt^-1·day^-1) and/or insulin (6.0 IU/day) from gestational day 7.5 to 13.5. We showed that whereas <em>DHT</em>-exposed and insulin-exposed pregnant rats presented impaired insulin sensitivity, <em>DHT</em> + insulin-exposed pregnant rats exhibited hyperandrogenism and peripheral insulin resistance, which mirrors pregnant PCOS patients. Compared with controls, hyperandrogenism and insulin resistance in the dam were associated with alterations in uterine morphology and aberrant expression of genes responsible for decidualization (<i>Prl8a2</i>, <i>Fxyd2</i>, and <i>Mt1g</i>), placentation (<i>Fcgr3</i> and <i>Tpbpa</i>), angiogenesis (<i>Flt1</i>, <i>Angpt1</i>, <i>Angpt2</i>, <i>Ho1</i>, <i>Ccl2</i>, <i>Ccl5</i>, <i>Cxcl9</i>, and <i>Cxcl10</i>) and insulin signaling (Akt, Gsk3, and Gluts). Moreover, we observed changes in uterine mitochondrial function and homeostasis (i.e., mitochondrial DNA copy number and the expression of genes responsible for mitochondrial fusion, fission, biogenesis, and mitophagy) and suppression of both oxidative and antioxidative defenses (i.e., reactive oxygen species, Nrf2 signaling, and interactive networks of antioxidative stress responses) in response to the hyperandrogenism and insulin resistance. These findings demonstrate that hyperandrogenism and insulin resistance induce mitochondria-mediated damage and a resulting imbalance between oxidative and antioxidative stress responses in the gravid uterus.

The oxidative effect of nicotine was investigated using androgen biomarkers of redox status and wound healing in fibroblasts; using the antioxidant glutathione for confirmation of responses. Cultures of human gingival (HGF) and periosteal fibroblasts (HPF) were incubated with substrates 14C-testosterone/14C-4-androstenedione in the presence or absence of serial concentrations of nicotine (N(100-500)), glutathione (G(1-5)) and their combinations, in medium. At 24 h the medium was solvent extracted for metabolites, separated by TLC and quantified using radioisotope scanning. Nicotine caused significant inhibition in yields of the physiologically active metabolite <em>5α</em>-dihydrotestosterone (<em>DHT</em>) in HGF and HPF, overcome to varying degrees by the anti-oxidant glutathione (n = 6; p<0.01, one way ANOVA); this is suggestive of moderation of an oxidative mechanism induced by nicotine. Down-regulation of <em>5α</em>-reductase activity by nicotine resulting in reduced yields of <em>DHT</em> was overcome by glutathione. Overcoming oxidative stress in a redox environment is applicable to treatment outcome.

Estrogenic and androgenic steroids can be synthesised in the brain and rapidly modulate synaptic transmission and plasticity through direct interaction with membrane receptors for estrogens (ERs) and androgens (ARs). We used whole cell patch clamp recordings in brainstem slices of male rats to explore the influence of ER and AR activation and local synthesis of 17β-estradiol (E2) and <em>5α</em>-dihydrotestosterone (<em>DHT</em>) on the long-term synaptic changes induced in the neurons of the medial vestibular nucleus (MVN). Long-term depression (LTD) and long-term potentiation (LTP) caused by different patterns of high frequency stimulation (HFS) of the primary vestibular afferents were assayed under the blockade of ARs and ERs or in the presence of inhibitors for enzymes synthesizing <em>DHT</em> (<em>5α</em>-reductase) and E2 (P450-aromatase) from testosterone (T). We found that LTD is mediated by interaction of locally produced androgens with ARs and LTP by interaction of locally synthesized E2 with ERs. In fact, the AR block with flutamide prevented LTD while did not affect LTP, and the blockade of ERs with ICI 182,780 abolished LTP without influencing LTD. Moreover, the block of P450-aromatase with letrozole not only prevented the LTP induction, but inverted LTP into LTD. This LTD is likely due to the local activation of androgens, since it was abolished under blockade of ARs. Conversely, LTD was still induced in the presence of finasteride the inhibitor of <em>5α</em>-reductase demonstrating that T is able to activate ARs and induce LTD even when <em>DHT</em> is not synthesized. This study demonstrates a key and opposite role of sex neurosteroids in the long-term synaptic changes of the MVN with a specific role of T-<em>DHT</em> for LTD and of E2 for LTP. Moreover, it suggests that different stimulation patterns can lead to LTD or LTP by specifically activating the enzymes involved in the synthesis of androgenic or estrogenic neurosteroids.

The major androgen within the prostate is dihydrotestosterone (<em>DHT</em>). <em>DHT</em> and <em>5α</em>-reductase are highly associated with prostate cancer. It has been hypothesised that inhibition of <em>5α</em>-reductase activity might reduce the risk of prostate cancer development, slow tumour progression and even treat the existing disease. The basis for endocrine treatment of prostate cancer is to deprive the cancer cells of androgens. Every type of endocrine treatment carries adverse events which influence quality of life in different ways. <em>5α</em>-Reductase inhibitors (5-ARI) reduce risk of being diagnosed with prostate cancer but they do not eliminate it. By suppressing PSA from BPH and indolent prostate cancers 5-ARI enhances the ability of a rising PSA to define a group of men at increased risk of clinically significant prostate cancer. Also fewer high-grade cancers are missed because biopsy is more accurate in smaller prostates. Androgen deprivation is an effective treatment for patients with advanced prostate cancer. However, it is not curative, and creates a spectrum of unwanted effects that influence quality of life. Castration remains the frontline treatment for metastatic prostate cancer, where orchiectomy, oestrogen agonists, GnRH agonists and antagonists produce equivalent clinical responses. MAB is not significantly more effective than single agent GnRH agonist or orchiectomy. Nonsteroidal antiandrogen monotherapy is as effective as castration in treatment of locally advanced prostate cancer offering quality of life benefits. Neoadjuvant endocrine treatment has its place mainly in the external beam radiotherapy setting. Increasing data suggest IAD is as effective as continuous ADT. The decision regarding the type of androgen deprivation should be made individually after informing the patient of all available treatment options, including watchful waiting, and on the basis of potential benefits and adverse effects. There are new promising secondary or tertiary forms of endocrine therapies under evaluation, like CTP17A1 inhibitors and more potent antiandrogens including MDV3100, which give new hope for patients developing castration resistant prostate cancer.

Clinical evidence has demonstrated that the accumulation of <em>5α</em>-dihydrotestosterone (<em>DHT</em>) in dermal papilla cells (DPCs) is implicated in androgenetic alopecia. Whether this accumulation in <em>DHT</em> may have direct cellular effects leading to androgenetic alopecia remains to be elucidated. The present study aimed to determine whether <em>DHT</em> affects cell growth, cell cycle arrest, cell death, senescence and the induction of reactive oxygen species (ROS), and whether these effects are mediated by microRNA (miRNA)-dependent mechanisms. The cell viability and cell cycle were determined, levels of ROS were examined and senescence-associated β-galactosidase assays were performed in normal human DPCs (nHDPCs). Furthermore, miRNA expression profiling was performed using an miRNA microarray to determine whether changes in the expression levels of miRNA were associated with the cellular effects of <em>DHT</em>. The results revealed that <em>DHT</em> decreased cell growth by inducing cell death and G2 cell cycle arrest, and by increasing the production of ROS and senescence in the nHDPCs. In addition, 55 miRNAs were upregulated and 6 miRNAs were downregulated in the <em>DHT</em>-treated nHDPCs. Bioinformatic analysis demonstrated that the putative target genes of these upregulated and downregulated miRNAs were involved in cell growth, cell cycle arrest, cell death, senescence and the production of ROS. Specifically, the target genes of five highly upregulated and downregulated miRNAs were identified and were associated with the aforementioned effects of <em>DHT</em>. These results demonstrated that the expression of miRNA was altered in the <em>DHT</em>-treated nHDPCs and suggest the potential mechanisms of <em>DHT</em>-induced cell growth repression, cell cycle arrest, cell death, senescence and induction of ROS.

A simple spectrophotometric method for the assay of steroid <em>5α</em>-reductase (<em>5α</em>-SR) was developed in which <em>5α</em>-dihydrotestosterone (<em>5α</em>-<em>DHT</em>) and <em>5α</em>-androstane-3α,17β-diol (<em>5α</em>-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of <em>5α</em>-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that <em>5α</em>-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate >> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of <em>5α</em>-<em>DHT</em> and <em>5α</em>-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of <em>5α</em>-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of <em>5α</em>-SR.

OBJECTIVE

The effects on scalp and serum dihydrotestosterone (<em>DHT</em>) of different doses of a novel topical solution of 0.25% finasteride (P-3074), a type 2 <em>5α</em>-reductase, were investigated in men with androgenetic alopecia.

METHODS

Two randomized, parallel-group studies were conducted. Study I: 18 men received 1 mL (2.275 mg) P-3074, applied to the scalp once a day (o.d.) or twice a day (b.i.d), or 1 mg oral tablet o.d. for 1 week. Study II: 32 men received P-3074 at the dose of 100 (0.2275 mg), 200 (0.455 mg), 300 (0.6285 mg), or 400 (0.91 mg) μL or the vehicle o.d. for 1 week. Scalp and serum DHT and serum testosterone were evaluated at baseline and treatment end.

RESULTS

Change from baseline in scalp DHT was -70% for P-3074 o.d. and approx. -50% for P-3074 b.i.d. and the tablet. Serum DHT decreased by 60 - 70%. The doses of 100 and 200 μL P-3074 resulted in a -47/-52% scalp DHT reduction, similar to the 300 and 400 μL doses (i.e., -37/-54%). A -5.6% inhibition was observed for the vehicle. Serum DHT was reduced by only -24/-26% with 100 and 200 μL P-3074 and by -44/-48% with 300 and 400 μL P-3074. No relevant changes occurred for serum testosterone.

CONCLUSIONS

The novel finasteride 0.25% solution applied o.d. at the doses of 100 and 200 μL results in an appropriate inhibition of scalp DHT potentially minimizing the untoward sexual side-effects linked to a systemic DHT reduction.

Traditional literature and textbooks generally describe that estradiol (E2) and dihydrotestosterone (<em>DHT</em>) are synthesized from the aromatization and <em>5α</em>-reduction of testosterone (T), respectively, following a pathway in which T is an essential intermediate (Tpath). This pathway implies that the steps of aromatization and <em>5α</em>-reduction follow the reaction of the androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) that catalyzes the conversion of 4-androstenedione (4-dione) into T, and that estrogenic 17β-HSDs are not required. Contrary to this belief, the cloning of many estrogen-specific 17β-HSDs and the observation of higher affinity of aromatase and <em>5α</em>-reductase for 4-dione than T are strongly in favor of biosynthetic pathways in which the steps catalyzed by aromatase and <em>5α</em>-reductase precede that catalyzed by 17β-HSDs. Such pathways do not require T as an intermediate, as demonstrated by experiments using [14C]-labeled DHEA and 4-dione as substrates and incubation with SZ95 sebaceous gland, DU-145 prostate cancer and JEG-3 choriocarcinoma cell lines cultured in the presence of inhibitors of <em>5α</em>-reductase and aromatase. A review of early literature about patients with testicular 17β-HSD deficiency and of steroid metabolism appears to confirm the physiological functionality of the E2 and <em>DHT</em> biosynthetic pathway not requiring T as intermediate (noTpath).

Regucalcin (RGN) is a calcium (Ca(2)(+))-binding protein which regulates intracellular Ca(2)(+) homeostasis by modulating the activity of enzymes regulating Ca(2)(+) concentration and enhancing Ca(2)(+)-pumping activity. Several studies have described the pivotal role of proper Ca(2)(+) homeostasis regulation to spermatogenesis and male fertility. Recently, RGN was identified as a sex steroid-regulated gene in prostate and breast; however, a possible role of RGN in spermatogenesis has not been examined. In this study, the expression and localization of RGN in rat and human testis, and other rat reproductive tissues was analyzed. Moreover, we studied whether RGN protein was present in seminiferous tubule fluid (STF). Finally, we examined the effect of <em>5α</em>-dihydrotestosterone (<em>DHT</em>) on the expression of Rgn mRNA in rat seminiferous tubules (SeT) cultured ex vivo. The results presented in this study show that RGN is expressed in Leydig and Sertoli cells, as well as in all types of germ cells of both rat and human testis. RGN is also expressed in rat prostate, epididymis, and seminal vesicles. Moreover, RGN protein is present in rat STF. The results also demonstrate that Rgn expression is age dependent in rat testis, and is upregulated by the non-aromatizable androgen <em>DHT</em> in rat SeT cultured ex vivo. Taken together, these findings indicate that Rgn is a novel androgen-target gene in rat testis and that it may have a role in male reproductive function, particularly in the control of spermatogenesis.

BACKGROUND

D-004, a lipid extract of the fruit of the Cuban royal palm (Roystonea regia), has been found to reduce prostatic hyperplasia (PH) induced with testosterone (T), but not PH induced with dihydrotestosterone (<em>DHT</em>), in rodents, suggesting the inhibition of prostate <em>5α</em>-reductase activity.

OBJECTIVE

The aims of this study were to assess whether D-004 inhibits prostate <em>5α</em>-reductase activity in vitro and to examine the effects of D-004 on enzyme kinetics.

METHODS

This experimental study was conducted at the Pharmacology Department, Center of Natural Products, National Center for Scientific Research, Havana, Cuba. Soluble rat prostate preparations were used as the source of <em>5α</em>-reductase, and ((3)H)-<em>DHT</em> production was measured to determine prostate <em>5α</em>-reductase activity. Cell-free rat prostate homogenates were pre-incubated with carboxymethyl cellulose 2% alone (control tubes) or D-004 (0.24-125 μg/mL) suspended in the vehicle (treated tubes) for 10 minutes prior to adding the labeled substrate ((3)H)-T Once the reaction was stopped, sterols were extracted with chloroform and aliquots were applied on silica gel plates developed in benzene-acetone (4:1, v/v). Areas containing <em>DHT</em> were scraped and radioactivity was counted. The median inhibitory concentration (IC50) was determined by measuring the conversion of T to <em>DHT</em> The apparent Michaelis-Menten constant (Km) and Vmax values before and after adding D-004 were determined in kinetic studies using labeled T (0.5-25 μmol/L).

RESULTS

Compared with controls, D-004 significantly and dose-dependently inhibited the enzymatic reaction at doses of 1.95 to 125.0 μg/mL) (all, P < 0.05). The IC50 of D-004 required to inhibit 5a-reductase activity was 2.25 μg/mL. Enzyme inhibition was noncompetitive, since D-004 lowered the Vmax from 15.3 to 10.0 nmol DHT/min · mg(-1) protein, while the Km (4.54 μmol/L) was almost unaffected.

CONCLUSIONS

D-004 dose-dependently and noncompetitively inhibited in vitro <em>5α</em>-reductase activity in soluble fractions of rat prostate. Although the extent of the maximal inhibition was high and the value of IC50 was low, the relevance of such inhibition requires further study in vivo.

The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ((20)LxxLL(24) and (92)LxxLL(96)), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of <em>5α</em>-dihydrotestosterone (<em>DHT</em>), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant (20)LxxLL(24) motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant (92)LxxLL(96) motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact (92)LxxLL(96) motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.

OBJECTIVE

Controversy surrounds the gender basis of progression in chronic kidney disease. Unfortunately, most experimental studies addressing this question do not distinguish between direct effects of estrogen and indirect activation of estrogen receptors through conversion of testosterone to 17β-estradiol by aromatase. We examined the pathogenesis of renal fibrosis in female aromatase knockout (ArKO) mice, which lack circulating and stored estrogens, while having normal levels of testosterone.

METHODS

ArKO mice and their wild-type (ArWT) counterparts were subjected to unilateral ureteric obstruction (UUO), with kidney tissue collected at day(D) 0, 3 and 9 post-UUO. Effects of <em>5α</em>-dihydrotestosterone (<em>DHT</em>) administration on each genotype were also studied. Tissue was assessed biochemically and histochemically for fibrosis. Western blot analysis was used to measure α-smooth muscle actin (α-SMA) expression and TGF-β1 signalling. Matrix metalloproteinase-2 (MMP-2) activity was measured by zymography.

RESULTS

UUO increased collagen content over time (p<0.05 (D3) and p<0.01 (D9) vs day 0), with no difference between genotypes in qualitative (collagen IV staining) and quantitative (hydroxyproline concentration) analyses. Systemic administration of non-aromatizable DHT increased collagen content after 3days of UUO in both genotypes. This was not paralleled by any change in α-SMA (myofibroblast burden) or TGF-β1 signalling but was commensurate with DHT reducing MMP2 activity in both genotypes (p<0.05 vs genotype controls).

CONCLUSIONS

Physiological concentrations of estrogens do not protect the injured kidney from fibrosis progression. Androgens rather than estrogens are the relevant factor involved in regulating disease-related renal scarring in this model.

We have previously reported that androstenedione induces abnormalities of follicle development and oocyte maturation in the mouse ovary. In granulosa cells of the ovarian follicle, androstenedione is aromatized to 17β-estradiol (E2). To determine whether the androgen or estrogen acts directly on the follicle to induce the above-mentioned abnormalities, we compared the effects of a non-aromatizable androgen, <em>5α</em>-dihydrotestosterone (<em>DHT</em>), with those of E2 on murine follicular development and oocyte maturation in a single follicle culture system. The high dose (10(-6) M) of <em>DHT</em> prompted normal follicular development, and there was no effect on oocyte meiotic maturation after stimulation with human chorionic gonadotropin (hCG) and epidermal growth factor (EGF). In contrast, culture with the high dose (10(-6) M) of E2 delayed follicular growth and also suppressed proliferation of granulosa cells and antrum formation. Furthermore, culture with E2 delayed or inhibited oocyte meiotic maturation, such as chromosome alignment on the metaphase plate and extrusion of the first polar body, after addition of hCG and EGF. In conclusion, these findings demonstrate that E2, but not <em>DHT</em>, induces abnormalities of follicular development, which leads to delay or inhibition of oocyte meiotic maturation.