Dihydrotestosterone Treatment Research Articles

Gender-specific role of HDAC11 in kidney ischemia- and reperfusion-induced PAI-1 expression and injury

Male gender and the male hormone testosterone increase susceptibility to kidney ischemia and reperfusion (I/R) injury, which is associated with inflammatory responses. Possible involvement of histone deacetylase (HDAC) in inflammatory responses has been suggested. We investigated the gender-specific role of HDACs in plasminogen activator inhibitor type-1 (PAI-1) expression and I/R injury. PAI-1 inhibition protected the kidney from I/R-induced inflammation and functional loss. Among HDACs, only HDAC11 negatively regulated PAI-1 expression in I/R-subjected kidney gender specifically and lipopolysaccharide (LPS)-stimulated mouse monocytes/macrophages. HDAC11 gene silencing increased PAI-1 expression. Chromatin immunoprecipitation assay confirmed binding of HDAC11 to the promoter region of PAI-1 and then release by I/R insult or LPS treatment. I/R-induced HDAC11 release was inhibited by orchiectomy and reversed by dihydrotestosterone treatment. Release of HDAC11 increased acetylation of histone H3. In conclusion, male gender and male hormones accelerate I/R-induced decreases in expression and binding of HDAC11, resulting in an increase in PAI-1 expression. These data provide important insight into gender dimorphism offering HDAC11 as a novel target for I/R injury.

Androgen-dependent impairment of myogenesis in spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disease caused by expansion of a polyglutamine (polyQ) tract in the androgen receptor (AR). SBMA is triggered by the interaction between polyQ-AR and its natural ligands, testosterone and dihydrotestosterone (DHT). SBMA is characterized by the loss of lower motor neurons and skeletal muscle fasciculations, weakness, and atrophy. To test the hypothesis that the interaction between polyQ-AR and androgens exerts cell-autonomous toxicity in skeletal muscle, we characterized the process of myogenesis and polyQ-AR expression in DHT-treated satellite cells obtained from SBMA patients and age-matched healthy control subjects. Treatment with androgens increased the size and number of myonuclei in myotubes from control subjects, but not from SBMA patients. Myotubes from SBMA patients had a reduced number of nuclei, suggesting impaired myotube fusion and altered contractile structures. The lack of anabolic effects of androgens on myotubes from SBMA patients was not due to defects in myoblast proliferation, differentiation or apoptosis. DHT treatment of myotubes from SBMA patients increased nuclear accumulation of polyQ-AR and decreased the expression of interleukin-4 (IL-4) when compared to myotubes from control subjects. Following DHT treatment, exposure of myotubes from SBMA patients with IL-4 treatment rescued myonuclear number and size to control levels. This supports the hypothesis that androgens alter the fusion process in SBMA myogenesis. In conclusion, these results provide evidence of an androgen-dependent impairment of myogenesis in SBMA that could contribute to disease pathogenesis.

Multifocal VZV vasculopathy with temporal artery infection mimics giant cell arteritis

To address the incidence of varicella-zoster virus (VZV) infection in patients with biopsy-negative giant cell arteritis (GCA), we examined archived biopsy-negative temporal arteries from subjects with clinically suspected GCA for the presence of VZV antigen. Formalin-fixed, paraffin-embedded temporal arteries that were pathologically negative for GCA and normal temporal arteries were analyzed immunohistochemically for VZV and herpes simplex virus-1 (HSV-1) antigen. Five (21%) of 24 temporal arteries from patients who were clinically suspect but biopsy negative for GCA revealed VZV but not HSV-1 by immunohistochemical analysis. Thirteen normal temporal arteries did not contain VZV or HSV-1 antigen. All 5 subjects whose temporal arteries contained VZV antigen presented with clinical and laboratory features of GCA and early visual disturbances. Multifocal VZV vasculopathy can present with the full spectrum of clinical features and laboratory abnormalities characteristically seen in GCA.

174 DIHYDROTESTOSTERONE ENHANCES RENAL CELL CARCINOMA CELL SURVIVAL THROUGH SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION-5 ACTIVATION VIA ANDROGEN RECEPTOR AND/OR GLUCOCORTICOID RECEPTOR

You have accessJournal of UrologyKidney Cancer: Basic Research (I)1 Apr 2013174 DIHYDROTESTOSTERONE ENHANCES RENAL CELL CARCINOMA CELL SURVIVAL THROUGH SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION-5 ACTIVATION VIA ANDROGEN RECEPTOR AND/OR GLUCOCORTICOID RECEPTOR Yunlim Kim, Wansuk Kim, Sejun Park, Myungsun Shim, Dalsan You, Cheryn Song, and Hanjong Ahn Yunlim KimYunlim Kim Seoul, Korea, Republic of More articles by this author , Wansuk KimWansuk Kim Busan, Korea, Republic of More articles by this author , Sejun ParkSejun Park Seoul, Korea, Republic of More articles by this author , Myungsun ShimMyungsun Shim Seoul, Korea, Republic of More articles by this author , Dalsan YouDalsan You Seoul, Korea, Republic of More articles by this author , Cheryn SongCheryn Song Seoul, Korea, Republic of More articles by this author , and Hanjong AhnHanjong Ahn Seoul, Korea, Republic of More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1554AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen receptor (AR) and glucocorticoid receptor (GR) mediate the action of steroid hormones in various tissues, including the kidney. Signal transducer and activator of transcription-5 (STAT5) regulates the expression of genes that mediate cell survival, proliferation, and angiogenesis and is activated in various types of malignancies, including renal cell carcinoma (RCC). Although several important transcriptional targets of STAT5 are known, the effect and molecular mechanism of STAT5 are not well elucidated, especially in RCC cells. Therefore, we examined interaction between STAT5 activation and steroid receptor in RCC. METHODS Using clear cell RCC cell lines Caki-2, A498 and SN12C cell viability assay and Western blot for phosphorylated STAT5 was done after dihydrotestosterone (DHT) treatment at various concentration (0.1, 1, 10nM). Endogenous levels of AR, GR and estrogen receptor (ER) were identified by RT-PCR and Western blot. Using AR antagonist (bicalutamide)/GR antagonist (RU486) and AR/GR-specific siRNA, changes in DHT-induced STAT5 activation were assessed. In human primary RCC cells from patients, cell viability and STAT5 activation after treatment of DHT was assessed by Western blot. RESULTS DHT treatment enhanced STAT5 phosphorylation and promoted cell proliferation up to 34.3% in Caki-2, 25.0% in A498 and 15.7% in SN12C, respectively. Endogenous AR was identified strongly in Caki-2 (193 fold relative to SN12C), weakly in A498 (54 fold relative to SN12C), but not in SN12C. GR was identified strongly in A498, weakly in Caki-2 (0.55 fold relative to A498) and SN12C (0.68 fold relative to A498). Pretreatment of bicalutamide/RU486 abolished DHT-induced cell proliferation and STAT5 activation in these cell lines. Similarly, AR/GR-specific siRNA completely suppressed DHT-induced STAT5 activation. In addition, DHT enhanced cell proliferation and STAT5 activation in 75% of human primary RCC cells expressing AR and/or GR. CONCLUSIONS In RCC cells, DHT activated STAT5 enhancing cell survival and proliferation through STAT5 via AR and/or GR. AR and GR may be new therapeutic target in RCC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e71 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yunlim Kim Seoul, Korea, Republic of More articles by this author Wansuk Kim Busan, Korea, Republic of More articles by this author Sejun Park Seoul, Korea, Republic of More articles by this author Myungsun Shim Seoul, Korea, Republic of More articles by this author Dalsan You Seoul, Korea, Republic of More articles by this author Cheryn Song Seoul, Korea, Republic of More articles by this author Hanjong Ahn Seoul, Korea, Republic of More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effects of vitamin D3 derivative – calcitriol on pharmacological reactivity of aortic rings in a rodent PCOS model

BackgroundThe aim of this study was to examine the effects of the hyperandrogenic state in dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS), the vascular responses to different vasoactive agents, and the modulatory role of vitamin D3. MethodsAPCOS model was induced by DHT application in 20 femaleWistar rats. Ten of the DHT treated rats simultaneously received calcitriol treatment. After 10 weeks, myographs were used to test the reactivity of isolated thoracic aortic rings to norepinephrine and acetylcholine. Thereafter, the vascular rings were incubated with the NO-synthase blocker (nitro-L-arginine methyl ester) or the cyclooxygenase inhibitor (indomethacin) for 20min, and the effects of norepinephrine and acetylcholine were re-evaluated. ResultsNorepinephrine-induced vasoconstriction was enhanced after DHT treatment, but this effect was attenuated by calcitriol administration. Vasorelaxation of DHT-treated thoracic aortic rings was impaired, but this could be partly reversed by calcitriol application. Impaired NO-dependent vasorelaxation in DHT-treated animals was mostly reversed by concomitant calcitriol administration, but this effect was diminished by prostanoid-dependent vasoconstriction. ConclusionsThese studies show that the enhanced sensitivity to vasoconstrictors and impaired NO-dependent vasorelaxation in hyperandrogenic PCOS rats could be partially reversed by calcitriol treatment.

Enhanced Aggregation of Androgen Receptor in Induced Pluripotent Stem Cell-derived Neurons from Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motor neuron disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. Ligand-dependent nuclear accumulation of mutant AR protein is a critical characteristic of the pathogenesis of SBMA. SBMA has been modeled in AR-overexpressing animals, but precisely how the polyglutamine (polyQ) expansion leads to neurodegeneration is unclear. Induced pluripotent stem cells (iPSCs) are a new technology that can be used to model human diseases, study pathogenic mechanisms, and develop novel drugs. We established SBMA patient-derived iPSCs, investigated their cellular biochemical characteristics, and found that SBMA-iPSCs can differentiate into motor neurons. The CAG repeat numbers in the AR gene of SBMA-iPSCs and also in the atrophin-1 gene of iPSCs derived from another polyQ disease, dentato-rubro-pallido-luysian atrophy (DRPLA), remain unchanged during reprogramming, long term passage, and differentiation, indicating that polyQ disease-associated CAG repeats are stable during maintenance of iPSCs. The level of AR expression is up-regulated by neuronal differentiation and treatment with the AR ligand dihydrotestosterone. Filter retardation assays indicated that aggregation of ARs following dihydrotestosterone treatment in neurons derived from SBMA-iPSCs increases significantly compared with neurological control iPSCs, easily recapitulating the pathological feature of mutant ARs in SBMA-iPSCs. This phenomenon was not observed in iPSCs and fibroblasts, thereby showing the neuron-dominant phenotype of this disease. Furthermore, the HSP90 inhibitor 17-allylaminogeldanamycin sharply decreased the level of aggregated AR in neurons derived from SBMA-iPSCs, indicating a potential for discovery and validation of candidate drugs. We found that SBMA-iPSCs possess disease-specific biochemical features and could thus open new avenues of research into not only SBMA, but also other polyglutamine diseases.

Age-related Impairment in Paracrine Regulation of Angiogenesis by Androgens

Background: Ageing is associated with a marked reduction in capacity for angiogenesis and vascular regeneration. In elderly males, ageing is also associated with a decline in androgen levels. We examined the effects of ageing on androgenmodulation of angiogenesis in young and old human fibroblasts. Methods:Human dermal fibroblasts were isolated from young (30 yrs) and older males (>65 yrs). Cells were incubated with 40nM dihydrotestosterone (DHT), with or without hydroxyflutamide (HF), or a phosphoinositide 3-kinase (PI3-kinase) inhibitor. Fibroblasts and fibroblastconditioned media was used to stimulate HUVECs, and analysed by ELISA and western blotting. Nuclear fractionation and fluorescencemicroscopywere used to study androgen receptor (AR) localisation. Results: Young male fibroblasts treated with DHT doubled HUVEC tubulogenesis and migration through increased VEGF secretion (p< 0.05). Older male fibroblasts were unresponsive to DHT treatment with a lack of VEGF production and could not stimulate enhanced angiogenesis in HUVECs. DHT-induced stimulation of VEGF secretion from young fibroblasts was dependent on AR binding and increased nuclear AR translocation (p< 0.01). Thiswas associatedwith increasedAKTproduction (p< 0.05) and phosphorylation (p< 0.01), all of which was abrogated by PI3-kinase inhibition. Despite comparable level of ARs, fibroblasts from older males treated with DHT displayed reduced AR nuclear translocation, resulting in a lack of AR-mediated VEGF production. Conclusion: Unlike young male fibroblasts, older male cells cannot respond to DHT treatment with increased VEGF production and angiogenic promotion of endothelial cellsdue todefectivenuclear translocationof androgen receptors.

An Article on the Predictive Value of on Treatment Platelet Reactivity in Major Vascular Surgery Patients

Background: Ageing is associated with a marked reduction in capacity for angiogenesis and vascular regeneration. In elderly males, ageing is also associated with a decline in androgen levels. We examined the effects of ageing on androgenmodulation of angiogenesis in young and old human fibroblasts. Methods:Human dermal fibroblasts were isolated from young (30 yrs) and older males (>65 yrs). Cells were incubated with 40nM dihydrotestosterone (DHT), with or without hydroxyflutamide (HF), or a phosphoinositide 3-kinase (PI3-kinase) inhibitor. Fibroblasts and fibroblastconditioned media was used to stimulate HUVECs, and analysed by ELISA and western blotting. Nuclear fractionation and fluorescencemicroscopywere used to study androgen receptor (AR) localisation. Results: Young male fibroblasts treated with DHT doubled HUVEC tubulogenesis and migration through increased VEGF secretion (p< 0.05). Older male fibroblasts were unresponsive to DHT treatment with a lack of VEGF production and could not stimulate enhanced angiogenesis in HUVECs. DHT-induced stimulation of VEGF secretion from young fibroblasts was dependent on AR binding and increased nuclear AR translocation (p< 0.01). Thiswas associatedwith increasedAKTproduction (p< 0.05) and phosphorylation (p< 0.01), all of which was abrogated by PI3-kinase inhibition. Despite comparable level of ARs, fibroblasts from older males treated with DHT displayed reduced AR nuclear translocation, resulting in a lack of AR-mediated VEGF production. Conclusion: Unlike young male fibroblasts, older male cells cannot respond to DHT treatment with increased VEGF production and angiogenic promotion of endothelial cellsdue todefectivenuclear translocationof androgen receptors.

Altered insulin-induced relaxation of aortic rings in a dihydrotestosterone-induced rodent model of polycystic ovary syndrome

To clarify the effects of dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS) on the insulin-dependent vasodilatation of the thoracic aorta and the role of vitamin D in a rat model. Controlled experimental animal study. Laboratory. Thirty adolescent female Wistar rats. The PCOS model was induced by 10 weeks of DHT treatment (83 μg/d). One-half of the DHT-treated animals also received vitamin D (120 ng/kg/wk). The aortic rings of the control, DHT, and DHT plus vitamin D-treated animals were isolated. The insulin-dependent vasodilation of the isolated aortic rings was compared in Krebs-Ringer solution and under blockade of nitric oxide (NO) synthase or cyclooxygenase. The insulin-dependent vasorelaxation decreased in both DHT-treated groups independently from the vitamin D treatment; NO-dependent and -independent relaxations were both impaired. In response to prostanoid, vasoconstriction was increased after DHT treatment. The NO-independent relaxation was partially improved by vitamin D treatment, which was neutralized by increased prostanoid-dependent vasoconstriction. Previously, we found that vitamin D treatment prevented systemic insulin resistance; however, in this study, we did not detect any influence on the vascular insulin resistance of the aorta that was induced by DHT treatment. Consequently, controlling insulin resistance with vitamin D alone did not resolve the aortic endothelial dysfunction caused by the hyperandrogenic state.

Arteriolar biomechanics in a rat polycystic ovary syndrome model — Effects of parallel vitamin D3 treatment

To clarify the effects of dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS) on arteriolar biomechanics in a rat model and the possible modulatory role of vitamin D3. The PCOS model was induced in female Wistar rats by ten-weeks DHT treatment. Arteriolar biomechanics was tested in arterioles by pressure arteriography in control as well as DHT- and DHT with vitamin D3-treated animals in contracted and passive conditions. Increased wall stress and distensibility as well as increased vascular lumen were detected after DHT treatment. Concomitant vitamin D3 treatment lowered the mechanical load of the arterioles and restored the vascular diameter. The hyperandrogenic state resulted in more rigid, less flexible arteriolar walls with increased vascular lumen compared with controls. DHT treatment caused eutrophic remodelling of gracilis arteriole. These prehypertensive alterations caused by chronic DHT treatment were mostly reversed by concomitant vitamin D3 administration.

Epidermal growth factor induces bladder cancer cell proliferation through activation of the androgen receptor

Androgen receptor (AR) signals have been suggested to contribute to bladder tumorigenesis and cancer progression. Activation of epidermal growth factor receptor (EGFR) also leads to stimulation of bladder tumor growth. However, crosstalk between AR and EGFR pathways in bladder cancer remains uncharacterized. We have recently shown that androgens activate the EGFR pathway in bladder cancer cells. The purpose of this study was to investigate the effects of EGF on AR activity in bladder cancer. EGF increased AR transcriptional activity by 1.2-, 1.9- and 2.0-fold in UMUC3, 5637-AR and J82-AR cell lines, respectively, over mock treatment and a specific EGFR inhibitor, PD168393, antagonized the EGF effect. Combined treatment of EGF and dihydrotestosterone (DHT) further induced AR transactivation while an AR antagonist, hydroxyflutamide (HF), abolished the effect of not only DHT but also EGF. In growth assays, EGF alone/DHT alone/EGF+DHT increased cell numbers by 16/12/19%, 6/14/18% and 30/12/38% in UMUC3-control-shRNA, 5637-AR and J82-AR, respectively, whereas the effects of EGF were marginal or less significant in UMUC3-AR-shRNA (8%) or AR-negative 5637-V (<1%) and J82-V (17%) cells. HF treatment at least partially counteracted the EGF effect on the growth of AR-positive cells. Western blotting demonstrated that EGF, especially in the presence of DHT, upregulated the expression of the p160 coactivator TIF2 and HF again blocked this stimulation. Co-immunoprecipitation revealed the association between AR and estrogen receptor (ER)-β or Src in UMUC3 cells and stronger associations with EGF treatment, implying the involvement of the AR/ER/Src complex in EGF-increased AR transactivation and cell growth. Current results, thus, suggest that EGF promotes bladder cancer cell proliferation via modulation of AR signals. Taken together with our previous findings, crosstalk between EGFR and AR pathways can play an important role in the progression of bladder cancer.

Androgen Initiates Sertoli Cell Tight Junction Formation in the Hypogonadal (hpg) Mouse1

Sertoli cell tight junctions (TJs) form at puberty as a major component of the blood-testis barrier (BTB), which is essential for spermatogenesis. This study characterized the hormonal induction of functional Sertoli cell TJ formation in vivo using the gonadotropin-deficient hypogonadal (hpg) mouse that displays prepubertal spermatogenic arrest. Androgen actions were determined in hpg mice treated for 2 or 10 days with dihydrotestosterone (DHT). Follicle-stimulating hormone (FSH) actions were studied in hpg mice expressing transgenic human FSH (hpg+tgFSH) with or without DHT treatment. TJ formation was examined by mRNA expression and immunolocalization of TJ proteins claudin-3 and claudin-11, and barrier functionality was examined by biotin tracer permeability. Immunolocalization of claudin-3 and claudin-11 was extensive at wild-type (wt) Sertoli cell TJs, which functionally excluded permeability tracer. In contrast, seminiferous tubules of hpg testes lacked claudin-3, but claudin-11 protein was present in adluminal regions of Sertoli cells. Biotin tracer permeated throughout these tubules, demonstrating dysfunctional TJs. In hpg+tgFSH testes, claudin-3 was generally absent, but claudin-11 had redistributed basally toward the TJs, where function was variable. In hpg testes, DHT treatment stimulated the redistribution of claudin-11 protein toward the basal region of Sertoli cells by Day 2, increased Cldn3 and Cldn11 mRNA expression, then induced the formation of functional TJs containing both proteins by Day 10. In hpg+tgFSH testes, TJ protein redistribution was accelerated and functional TJs formed by Day 2 of DHT treatment. We conclude that androgen stimulates initial Sertoli cell TJ formation and function in mice, whereas FSH activity is insufficient alone, but augments androgen-induced TJ function.

Hepatic Protein Expression Networks Associated with Masculinization in the Female Fathead Minnow (Pimephales promelas)

Endocrine disruptors that act via the androgen receptor (AR) are less well studied than environmental estrogens, and there is evidence that treatment with AR agonists can result in masculinization of female fish. In this study, female fathead minnows (FHM) were exposed to the model nonaromatizable androgen 5-alpha dihydrotestosterone (DHT) (100 μg/L), the ureic-based herbicide linuron (LIN) (100 μg/L), and a mixture of DHT and LIN (100 μg/L each) to better characterize androgen action in females. LIN was used because of reports that this chemical has an antiandrogenic mode of action in fish. After 21d, DHT and LIN treatments resulted in a significant depression of plasma vitellogenin (Vtg) and DHT and DHT+LIN increased the prevalence of nuptial tubercles in female FHMs indicating masculinization. Using iTRAQ and an LTQ Orbitrap Velos, ∼2000 proteins were identified in the FHM liver and the number of proteins quantified after exposures was >1200. Proteins that significantly and consistently changed in abundance across biological replicates included prostaglandin E synthase 3, programmed cell death 4a, glutathione S transferases, canopy, selenoprotein U, and ribosomal proteins. Subnetwork enrichment analysis identified that interferon and epidermal growth factor signaling were regulated by DHT and LIN, suggesting that these signaling pathways are correlated to depressed plasma vitellogenin. These data provide novel insight into hepatic protein networks that are associated with the process of masculinization in teleosts.

Pharmacological reactivity of resistance vessels in a rat PCOS model – vascular effects of parallel vitamin D3 treatment

The aim of this study was to clarify the effects of dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS) on pharmacological reactivity of a resistance vessel in a rat model and the possible modulatory role of 1,25-(OH)2-cholecalciferol (vitamin D3). The PCOS model was induced in adolescent female Wistar rats by a 10-week DHT treatment. Norepinephrine induced contractility and acetylcholine relaxation were tested in arterioles by pressure arteriography in control as well as DHT- and DHT plus vitamin D3-treated (DHT+D3) animals. Decreased vasoconstriction and dilatation were detected after DHT treatment. Concomitant vitamin D3 treatment increased the contractile response and resulted in more relaxed vessels. Endothelial dilation tested with acetylcholine was lower after DHT treatment, this effect was not depend on vitamin D3 supplementation. In conclusion, hyperandrogenic state resulted in reduced endothelium- and smooth muscle-dependent vasorelaxation and constriction with a complete loss of nitric oxide (NO)-dependent relaxation compared with controls. These alterations caused by chronic DHT treatment were partially reversed by concomitant vitamin D3 administration.

Dihydrotestosterone ameliorates degeneration in muscle, axons and motoneurons and improves motor function in amyotrophic lateral sclerosis model mice.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by a progressive loss of motoneurons. The clinical symptoms include skeletal muscle weakness and atrophy, which impairs motor performance and eventually leads to respiratory failure. We tested whether dihydrotestosterone (DHT), which has both anabolic effects on muscle and neuroprotective effects on axons and motoneurons, can ameliorate clinical symptoms in ALS. A silastic tube containing DHT crystals was implanted subcutaneously in SOD1-G93A mice at early symptomatic age when decreases in body weight and grip-strength were observed as compared to wild-type mice. DHT-treated SOD1-G93A mice demonstrated ameliorated muscle atrophy and increased body weight, which was associated with stronger grip-strength. DHT treatment increased the expression of insulin-like growth factor-1 in muscle, which can exert myotrophic as well as neurotrophic effects through retrograde transport. DHT treatment attenuated neuromuscular junction denervation, and axonal and motoneuron loss. DHT-treated SOD1-G93A mice demonstrated improvement in motor behavior as assessed by rota-rod and gait analyses, and an increased lifespan. Application of DHT is a relatively simple and non-invasive procedure, which may be translated into therapy to improve the quality of life for ALS patients.

Abstract 201: DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling

Abstract The prostate cancer antigen 3 (DD3/PCA3) is a non-coding RNA (ncRNA) specifically expressed in prostate tissues and overexpressed in prostate cancer (PCa) tumors. Although widely applied as a diagnostic marker for PCa, to date nothing has described about its role in PCa biology. We used herein small interfering RNA (siRNA) in order to knockdown DD3 mRNA message as an approach to elucidate DD3 functional roles in PCa cells. LNCaP cell line was been used herein as an in vitro model for DD3 functional assays. siRNA sequences were specifically designed for DD3 exon 4 mRNA sequences (siDD3), as well an scrambled siRNA (siScr), as negative control. LNCaP cells were transiently transfected with siDD3 or siScr and DD3 expression was analysed by real time PCR (qRT-PCR) using DD3 specific oligonucleotides. LNCaP cells transfected with siDD3 demonstrated a marked decrease in cell proliferation and viability, as compared to siScr transfected cells. Further, LNCaP cells in which DD3 was knocked-down presented a significant increase in proportion of cells in SubG0/G1 phase of cell cycle and presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly uregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide. Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation of AR target genes. Notably, siDD3/LNCaP transfected cells significantly inhibited the expression of tested AR responsive genes. Besides, DD3 knockdown was able to counteract DHT stimulatory effects over AR target gene expression. Despite negatively modulating the transcription of AR target genes, DD3 knockdown did not alter Akt and ERK phosphorylation, suggesting that DD3 is mainly controlling the expression of signaling pathways downstream to AR activation. In summary, our findings indicate that DD3 is a ncRNA whose expression is AR regulated and is involved on the control of PCa cell survival and proliferation, in part by modulating the AR signaling pathway and its target genes. These [[Unsupported Character - &amp;#64257;]]ndings correspond to the first description of DD3 roles on PCa cells and could provide new insights into understanding prostate carcinogenesis, besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches aiming to inhibit PCa growth by negatively modulating AR pro-survival signal and their target genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 201. doi:1538-7445.AM2012-201

Dihydrotestosterone suppresses oxidative stress and improves vascular function following induction of inflammation in the cerebral circulation

During pathophysiological events (i.e. stroke) enzymatic actions of cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase result in reactive oxygen species (ROS) generation that could potentially alter vascular function. Since dihydrotestosterone (DHT) augments COX‐2 in the absence of inflammation and attenuates COX‐2 in the presence of inflammation in rat and human vascular smooth muscle (VSM), we hypothesized that DHT would attenuate ROS and thus improve vascular function following an inflammatory insult. Oxidative stress was assessed in primary human brain VSM cells stimulated with cytokine in the absence or presence of DHT (18 h; 10 nM) using ROS‐indicator dyes (H2DCFDA and DAF‐FM). Myogenic tone was measured in middle cerebral arteries (MCA) isolated from castrated male rats treated with DHT (2 wk; 25 mg pellet/21 days) followed by endotoxin (6 h; 0.5 mg/ml i.p.). In VSM cells, DHT attenuated cytokine‐induced ROS production. In MCA, chronic DHT treatment increased pressure‐induced tone. However, in the presence of endotoxin, DHT's ability to increase tone was blunted without altering the myogenic response to step pressure increases. These data suggest that DHT may provide protection against inflammation‐induced injury in the cerebrovasculature in part by decreasing ROS (e.g. peroxynitrite and hydrogen peroxide) and thus improving vascular function. Sarver Heart Center &amp; AHA (RG &amp; KO)

Flutamide treatment induces anxiolytic-like behavior in adult castrated rats

BackgroundIt has previously been speculated that the androgen receptor antagonist flutamide produces behavioral effects that are not mediated by androgen receptors. These earlier studies were performed in intact rodents and thus, flutamide may have interfered with endogenous testosterone produced by the testes. The main objective of the present study was to examine whether flutamide induces anxiolytic-like behavior in castrated rats. MethodsMale Wistar rats (8–9 weeks old) were castrated and thereafter, in the same operation, the rats received silastic capsules subcutaneously (sc) that were filled with dihydrotestosterone (DHT) or were left empty. Three weeks later, rats were sc administered flutamide 50mg/kg/day or vehicle for seven days. Four hours after the last injection, anxiolytic-like behavior was studied in a modified Vogel’s drinking conflict model. In a separate experiment, shock threshold and drinking motivation were estimated. ResultsFlutamide induced anxiolytic-like behavior in castrated rats irrespective of administration of DHT. Treatment with DHT alone did not induce a significant behavioral effect. Shock threshold and drinking motivation were not affected by flutamide and/or DHT treatment. ConclusionsThis study demonstrates that flutamide induces anxiolytic-like behavior in a modified Vogel’s conflict model in castrated rats, which indicates that flutamide has anxiolytic-like properties that are not dependent on testes-produced testosterone.

Abstract A1: DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling

Abstract The prostate cancer antigen 3 (DD3/PCA3) is a noncoding RNA (ncRNA) specifically expressed in prostate tissues and overexpressed in prostate cancer (PCa) tumors. Although widely applied as a diagnostic marker for PCa, to date nothing has described about its role in PCa biology. We used herein small interfering RNA (siRNA) in order to knockdown DD3 mRNA message as an approach to elucidate DD3 functional roles in PCa cells. LNCaP cell line has been used herein as an in vitro model for DD3 functional assays. siRNA sequences were specifically designed for DD3 exon 4 mRNA sequences (siDD3), as well an scrambled siRNA (siScr), as negative control. LNCaP cells were transiently transfected with siDD3 or siScr and DD3 expression was analyzed by real time PCR (qRT-PCR) using DD3 specific oligonucleotides. LNCaP cells transfected with siDD3 demonstrated a marked decrease in cell proliferation and viability, as compared to siScr transfected cells. This growth inhibition was specific for DD3 expressing cells. Further, LNCaP cells in which DD3 was knocked-down presented a significant increase in proportion of cells in SubG0/G1 phase of cell cycle and presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly uregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide. Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation of AR target genes. Notably, siDD3/LNCaP transfected cells significantly inhibited the expression of tested AR responsive genes. Besides, DD3 knockdown was able to counteract DHT stimulatory effects over AR target gene expression. Despite negatively modulating the transcription of AR target genes, DD3 knockdown did not alter Akt and ERK phosphorylation, suggesting that DD3 is mainly controlling the expression of signaling pathways downstream to AR activation. Besides, DD3 mRNA expression was mainly detected in nuclei and ribosome cell compartments, indicating that in this cell niches DD3 ncRNA exert its main functional roles. In summary, our findings indicate that DD3 is a ncRNA whose expression is AR regulated and is involved on the control of PCa cell survival and proliferation, in part by modulating the AR signaling pathway and its target genes. These .ndings correspond to the first description of DD3 roles on PCa cells and could provide new insights into understanding prostate carcinogenesis, besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches aiming to inhibit PCa growth by negatively modulating AR prosurvival signal and their target genes. Citation Format: Luciana Bueno Ferreira, Antonio Palumbo, Kivviduarte de Mello, Felipe Leite, Cinthya A. Sternberg, Mauricio Caetano, Adriana Freitas Neves, Luiz Ricardo Goulart, Etel Rodrigues Pereira Gimba. DD3/PCA3 noncoding RNA regulates prostate cancer cell survival and modulates AR signaling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A1.

Combined inhibition of aromatase activity and dihydrotestosterone supplementation attenuates renal injury in male streptozotocin (STZ)-induced diabetic rats.

Our previous studies showed that streptozotocin (STZ)-induced diabetic male rats have increased estradiol and decreased testosterone levels that correlate with renal injury (Xu Q, Wells CC, Garman GH, Asico L, Escano CS, Maric C. Hypertension 51: 1218-1224, 2008). We further showed that either supplementing dihydrotestosterone (DHT) or inhibiting estradiol biosynthesis in these diabetic rats was only partially renoprotective (Manigrasso MB, Sawyer RT, Marbury DC, Flynn ER, Maric C. Am J Physiol Renal Physiol 301: F634-F640, 2011; Xu Q, Prabhu A, Xu S, Manigrassso MB, Maric C. Am J Physiol 297: F307-F315, 2009). The aim of this study was to test the hypothesis that the combined therapy of DHT supplementation and inhibition of estradiol synthesis would afford better renoprotection than either treatment alone. The study was performed in 12-wk-old male nondiabetic (ND), STZ-induced diabetic (D), and STZ-induced diabetic rats that received the combined therapy of 0.75 mg/day of DHT along with 0.15 mg · kg(-1) · day(-1) of an aromatase inhibitor, anastrozole (Dta), for 12 wk. Treatment with the combined therapy resulted in attenuation of albuminuria by 84%, glomerulosclerosis by 55%, and tubulointerstitial fibrosis by 62%. In addition, the combined treatment decreased the density of renal cortical CD68-positive cells by 70% and decreased protein expression of transforming growth factor-β protein expression by 60%, collagen type IV by 65%, TNF-α by 55%, and IL-6 by 60%. We conclude that the combined treatment of DHT and blocking aromatase activity in diabetic male STZ-induced diabetic rats provides superior treatment than either treatment alone in the prevention of diabetic renal disease.