Doses Of Dihydrotestosterone Research Articles

Dihydrotestosterone induces reactive oxygen species accumulation and mitochondrial fission leading to apoptosis of granulosa cells

Dihydrotestosterone (DHT), which has significant androgenic activity，is a major player in follicle development and ovary function in females. However, an excess of androgens may result in increased follicular apoptosis with adverse effects on female fertility. This study aimed to explore the mechanism by which DHT induces apoptosis in human ovarian granulosa cells (GCs). The association between DHT and GC apoptosis was explored by the construction of rat models of polycystic ovary syndrome (PCOS). It was found that serum DHT levels were negatively correlated with thickness of the GC layer in PCOS model rats (R2=0.8342, p＜0.0001), compared with control rats, together with significant increases in cofactors (Fis1: p=0.008; MFF: p=0.044). The GC SVOG cell line was used to clarify the mechanism by which DHT influenced GC apoptosis in in vitro experiments. The results confirmed that apoptosis in SVOG cells was positively associated with the DHT dose. The expression of the autophagy-related proteins LC3A/B (p=0.027) and the proapoptotic protein Bax (p=0.0095) were increased, while that of the anti-apoptotic protein Bcl-2 (p=0.0005) was decreased in the high-dose DHT group. ROS levels were significantly increased (p=0.0237) and the mitochondrial membrane potential ΔΨm was decreased (p=0.0194). Moreover, ultrastructural analysis of the mitochondria indicated significant damage. The results of RT-qPCR and western blotting showed that two fission cofactor-Fis1（p=0.034） and MFF (p=0.039) were significantly increased after treatment with high doses of DHT. Even though the overall expression of Drp1 did not change significantly (p=0.5961), that of activated Phosphor-Drp1(Ser616) was significantly increased (p=0.046), while the expression of Phosphor-Drp1 (Ser637) was markedly reduced (p=0.007) following exposure to high concentrations of DHT. All these effects could be reversed by the Drp1 inhibitor Mdivi-1. These findings indicated the impact of DHT on ROS aggregation and mitochondrial fission, resulting in GC apoptosis. An imbalance in Drp1 phosphorylation may be the key link in DHT-induced excessive mitochondrial fission.

Mailuoning oral liquid ameliorates vasculitis in thromboangiitis obliterans rats via inactivating cGAS-STING-IRF3 and TLR4-MAPKs/NF-κB signaling pathways

Ethnopharmacological relevanceMailuoning oral liquid (MLN O), one traditional Chinese patent medicine, has a good therapeutic effect on thromboangiitis obliterans (TAO) in clinical practice. However, the underlying mechanism remains unclear. Aim of the studyThis study aimed to explore the effects and potential mechanisms of MLN O against TAO based on network pharmacology and experimental verification. Materials and methodsNetwork pharmacology was used to identify the intersectional targets and signaling pathways of MLN O and TAO. In vivo, the TAO model was established by injecting sodium laurate and dihydrotestosterone (DHT) into the femoral arteries of Wistar rats. Rats were given the indicated drugs by intragastric administration (i.g.), intravenous injection (i.v.), or subcutaneous injection (s.c.) per day for 21 days since a week before surgery. In vitro, HUVECs, RAW264.7, and THP-1 cells were stimulated by LPS and DHT to simulate the pathological changes of TAO. The anti-inflammatory, anticoagulant, and immunomodulatory effects of MLN O were evaluated by histological observation, blood biochemical indexes detection, H&E staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), qRT-PCR, western blotting and immunofluorescence assays. Furthermore, the vascular ring test was applied to explore the vasodilatory activity of MLN O. ResultsMLN O significantly improved the pathological signs in TAO rats through its excellent anti-inflammatory, anticoagulant, immunomodulatory, and vasodilatory effects. Specifically, MLN O alleviated the gangrene and reduced the thrombosis in TAO rats, meanwhile, suppressed the expressions of inflammatory factors and clotting factors, which is related to the inactivations of cGAS-STING-IRF3 and TLR4-MAPKs/NF-κB signaling pathways. However, the superphysiological dose of DHT deteriorated the pathological development of TAO in vitro and in vivo. Moreover, the results of network pharmacology are consistent with the experimental verification. ConclusionCollectively, this study indicates for the first time that MLN O could alleviate TAO by inhibiting cGAS-STING-IRF3 and TLR4-MAPKs/NF-κB signaling pathways, which sheds light on a novel clinical therapeutic strategy for TAO.

Dihydrotestosterone induces arterial stiffening in female mice

BackgroundAndrogens are important sex hormones in both men and women and are supplemented when endogenous levels are low, for gender transitioning, or to increase libido. Androgens also circulate at higher levels in women with polycystic ovarian syndrome, a condition that increases the risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via downregulation of GPER.MethodsThe impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice at 15–16 weeks of age were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n = 8–9/group).ResultsIn cultured vascular smooth muscle cells, GPER mRNA was decreased by DHT (P = 0.001) but was not impacted by dexamethasone or medroxyprogesterone. In contrast, ERα expression in cultured cells was significantly suppressed by all three hormones (P < 0.0001). In control mice or mice treated with a single or double dose of DHT, a dose-dependent increase in body weight was observed (control 22 ± 2 g, single dose 24 ± 2 g, double dose 26 ± 2 g; P = 0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9 ± 0.3 m/s, single dose 4.3 ± 0.8 m/s, double dose 4.8 ± 1.0 m/s). This increase in arterial stiffness occurred independent of changes in blood pressure (P = 0.59). Histological analysis of aortic sections using Masson’s trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P = 0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20 ± 5, single dose 10.5 ± 5.6, double dose 10 ± 4 copies/ng; P = 0.001) and ERα (control 54 ± 2, single dose 24 ± 13, and double dose 23 ± 12 copies/ng; P = 0.003).ConclusionsThese findings indicate that androgen promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important for transgender men, women using testosterone for fitness or reduced libido, as well as patients with polycystic ovarian syndrome.

Alterations in the balance of sex hormones may affect rat prostatic inflammation and fibrosis, and osteopontin might be involved in this process.

This study aimed to investigate the effects of sex hormone imbalance on rat prostatic inflammation and fibrosis and identify the key molecules involved. Castrated Sprague-Dawley (SD) rats were treated with a constant dose of oestradiol (E2) and different doses of dihydrotestosterone (DHT) to achieve different oestrogen/androgen ratios. After 8weeks, serum E2 and DHT concentrations, relative seminal vesicle weights, histopathological changes and inflammation were measured, collagen fiber content and oestrogen receptor (ER) and androgen receptor (AR) expression were detected, mRNA sequencing and bioinformatics analysis were performed to identify differentially expressed genes (DEGs). The severity of inflammation in the rat dorsolateral prostate (DLP) was higher, collagen fibre content and ER expression in the rat DLP and prostatic urethra were increased and AR expression in the rat DLP was decreased in the 1:1 E2/DHT-treated group than that in the 1:10 E2/DHT-treated group. RNA-seq analysis identified 487 DEGs, and striking increases in the expression of mRNAs encoding collagen, collagen synthesis and degradation enzymes, growth factors and binding proteins, cytokines and chemokines, and cell-surface molecules were confirmed in the 1:1 E2/DHT-treated group compared to the 1:10 E2/DHT-treated group. mRNA expression of secreted phosphoprotein 1 (Spp1) and protein expression of osteopontin (OPN, encoded by Spp1) were increased in the 1:1 E2/DHT-treated group compared to the 1:10 E2/DHT-treated group, and Spp1 expression correlated positively with Mmp7, Cxcl6 and Igfn1 expression. The imbalance in the oestrogen/androgen ratio may affect rat prostatic inflammation and fibrosis, and OPN might be involved in this process.

Role of SPTSSB-Regulated de Novo Sphingolipid Synthesis in Prostate Cancer Depends on Androgen Receptor Signaling.

SummaryAnti-androgens are a common therapy in prostate cancer (PCa) targeting androgen receptor (AR) signaling. However, these therapies fail due to selection of highly aggressive AR-negative cancer cells that have no therapeutic options available. We demonstrate that elevating endogenous ceramide levels with administration of exogenous ceramide nanoliposomes (CNLs) was efficacious in AR-negative cell lines with limited efficacy in AR-positive cells. This effect is mediated through reduced de novo sphingolipid synthesis in AR-positive cells. We show that anti-androgens elevate de novo generation of sphingolipids via SPTSSB, a rate-limiting mediator of sphingolipid generation. Moreover, pharmacological inhibition of AR increases the efficacy of CNL in AR-positive cells through de novo synthesis, while SPTSSB knockdown limited CNL's efficacy in AR-negative cells. Alluding to clinical relevance, SPTSSB is upregulated in patients with advanced PCa after anti-androgens treatment. These findings emphasize the relevance of AR regulation upon sphingolipid metabolism and the potential of CNL as a PCa therapeutic.

Prenatal low-dose methyltestosterone, but not dihydrotestosterone, treatment induces penile formation in female mice and guinea pigs†.

Genital tubercle has bisexual potential before sex differentiation. Females exposed to androgen during sex differentiation show masculinized external genitalia, but the effects of different androgens on tubular urethral and penile formation in females are mostly unknown. In this study, we compared the masculinization effects of commonly used androgens methyltestosterone, dihydrotestosterone, and testosterone on the induction of penile formation in females. Our results suggested that prenatal treatment with low doses of methyltestosterone, but not same doses of dihydrotestosterone or testosterone, could induce penile formation in female mice. The minimum dose of dihydrotestosterone and testosterone for inducing tubular urethral formation in female mice was, respectively, 50 and 20 times higher than that of methyltestosterone. In vivo methyltestosterone treatment induced more nuclear translocation of androgen receptors in genital tubercles of female mice, affected Wnt signaling gene expressions, and then led to similar patterns of cell proliferation and death in developing genital tubercles to those of control males. We further revealed that low-dose methyltestosterone, but not same dose of dihydrotestosterone or testosterone, treatment induced penile formation in female guinea pigs. Exposure of female mouse genital tubercle organ culture to methyltestosterone, dihydrotestosterone, or testosterone could induce nuclear translocation of androgen receptors, suggesting that the differential effect of the three androgens in vivo might be due to the hormonal profile in mother or fetus, rather than the local genital tissue. To understand the differential role of these androgens in masculinization process involved is fundamental to androgen replacement therapy for diseases related to external genital masculinization.

Effect of Dihydrotestosterone on Costimulatory Molecules in a Mouse Model of Graves' Disease

Objective Graves' disease is the most common autoimmune thyroid disease and its prevalence and clinical manifestations are disparate between females and males. Costimulatory molecules play an essential role in regulating autoimmune responses. The objective of this study was to determine if expression of inhibitory molecules was correlated with treatment by dihydrotestosterone (DHT) in an in vivo BALB/c mouse model of experimental autoimmune Graves' disease.Methods Female BALB/c mice were immunized three times with thyroid stimulating hormone receptor A-subunit encoded by adenovirus to establish a Graves' disease model. Three different doses of DHT or a matching placebo were administered by implantation of slow-release pellets a week before the first immunization. Four weeks after the third immunization, the mice were euthanatized, and then the spleen and thymus were removed. Total thyroxine and free thyroxine levels in serum of mice were detected using a radioimmunoassay kit. Real-time polymerase chain reaction was performed to estimate the expression of costimulatory molecules in lymphocytes from the spleen and thymus. Flow cytometry was used to analyze the percentage of CD4+ T cells in splenic lymphocytes. Quantitative data were compared with unpaired t-tests. Correlation between two variables was analyzed using Analysis of Variance.Results Treatment with DHT can dramatically reduce total thyroxine and free thyroxine levels. Higher expression of programmed death-1 was found in the spleen of Graves' disease mice receiving 5 mg of DHT treatment (0.635±0.296 vs. 0.327±0.212; t=2.714, P=0.014), similarly, T-cell immunoglobulin domain and mucin domain 3 (TIM-3) in both the spleen (1.004±0.338 vs. 0.646±0.314; t=2.205, P=0.022) and the thymus (0.263±0.127 vs. 0.120±0.076; t=3.221, P=0.004) also increased after 5 mg of DHT treatment compared with the parallel placebo model mice. Moreover, the percentage of CD4+ T cells declined in the splenic lymphocytes of Graves' disease mice treated with 5 mg of DHT (19.90%±3.985% vs. 24.05%±2.587%; t=2.804, P=0.012). A significant negative association was observed between expression of TIM-3 in the spleen and serum levels of total thyroxine (r=-0.7106, P=0.014) as well as free thyroxine (r=-0.6542, P=0.029).Conclusion This study demonstrates that DHT can ameliorate experimental autoimmune Graves' disease, which may occur by up-regulating expression of programmed death-1 and TIM-3 and inhibiting development of CD4+ T cells.

MON-108 Dihydrotestosterone Lowers Cytosolic Sterol Regulatory Element-Binding Protein-1 in Adipose Tissue from Female Mice

: Hyperandrogenemia (HA) and insulin resistance (IR) are hallmarks of polycystic ovary syndrome (PCOS), a common endocrine disorder that affects roughly 1 in 5 women, according to the Rotterdam criteria. These hallmarks are also integral elements of non-alcoholic fatty liver disease (NALFD), a disorder that is common in women with PCOS. In lean female mouse models of dihydrotestosterone (DHT)-induced PCOS, a low dose of DHT promotes IR and hepatic lipogenesis via the androgen receptor (AR), thus resulting in NAFLD (1). However, the molecular mechanism of HA-induced NAFLD has not been determined. We hypothesized that a low dose of DHT would interrupt hepatic lipid metabolism leading to NAFLD. To investigate the role of androgen and AR on a master regulator of lipogenesis, sterol regulatory element-binding protein 1 (SREBP-1), we extracted white adipose tissue (WAT) from lean, female wild-type mice and lean PCOS female mice, aka "DHT mice." Then we analyzed the effect of low-dose DHT on lipogenic protein and gene expression as a control to low-dose DHT's impact on the liver. We accomplished this by performing Western blots and real-time quantitative polymerase chain reaction (qRT-PCR) analysis of the cytosolic lipogenic proteins and gene expression of WAT. A low dose of DHT lowered the active form of SREBP-1 in DHT mice in comparison to the control mice, but there was no significant change in fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), phosphorylated ACC (p-ACC), and the active and inactive forms of SREBP-2. We speculate that the low-dose DHT promotes the translocation of SREBP-1 from the cytosol to the nucleus to influence lipogenic gene expression leading to increased lipogenesis contributing to NAFLD. (1) Andrisse et al., Endocrinology. 2017 Mar 1;158(3):531-544.

Sustained Delivery of Dihydrotestosterone Impact on the Pathophysiology of Isolated Endothelial and Smooth Muscle Cells

Sex steroid hormones appear to be a risk factor for the development of atherosclerosis. Proliferation of vascular smooth muscle cells (SMC) was documented to be a mediator in atherosclerotic plaque formation. The objective of this study was to investigate the effect of sustained delivery of dihydrotestosterone (DHT) on the growth of cultured aortic endothelial cells (EC) and SMC incubated individually, or in combination. The SMC were explanted from the media and intima of the abdominal and thoracic aorta of adult male rabbits, and EC were prepared by trypsinization of the vessel. Cells were seeded and grown undisturbed for up to 6 weeks in culture. A total of four equal (n=4 wells) groups was used in Phase I. Groups 1–3 were plated with EC, SMC, and EC+SMC, respectively. Group IV served as control. Half of the wells (n=4 per group) in each group were exposed to tricalcium phosphates capsules loaded with 40 mg DHT (release profile: 3–5 ng/ml/day) and second half was exposed to sham capsules. Resultes revealed that sustained release of DHT to EC+SMC cultures induced greater cellular proliferation than in control wells, as determined by cell counts after 96 hours of culture. In contrast, the number of cells/well of a single cell type, grown in the presence of DHT, did not differ from the control wells. In the second phase of this investigation, various capsules releasing different concentrations of DHT were used. The release rate of 10 ng/ml/day of DHT regressed the proliferation rate by approximately 40% compared to other lower doses (2.4, or 6 ng/ml/day). Results of this investigation suggest that: (1) sustained delivery of a physiological dose of DHT to combined cultures of EC+SMC significantly increased cellular growth, while the exposure of DHT to EC or SMC grown individually had no effect on the rate of cellular proliferation, and (2) the effect of sustained delivery of DHT on vascular smooth muscle cells was found to be dose dependent.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Low‐Dose Dihydrotestosterone Lowers Lipogenic Master Regulator in Liver and Adipose Tissue from Female Mice

Hyperandrogenemia (HA) and insulin resistance (IR) are hallmarks of polycystic ovary syndrome (PCOS), a common endocrine disorder that affects 1 in 10 women. These hallmarks are also integral elements of non‐alcoholic liver disease (NALFD), a disorder that is common in women with PCOS. In lean female mouse models of dihydrotestosterone (DHT)‐induced PCOS, a low dose of (DHT) promotes IR and hepatic lipogenesis via the androgen receptor (AR), thus resulting in NAFLD. However, the molecular mechanism of HA‐induced NAFLD has not been determined. We hypothesized that a low dose of DHT would interrupt hepatic lipid metabolism leading to NAFLD. In this project, we aimed to investigate how androgen and androgen receptor (AR) impact a key player of lipogenesis, SREBP1. We extracted liver tissue, white adipose tissue (WAT), and skeletal muscle from lean, female wild type mice and mice. Next, we analyzed the effect of low dose dihydrotestosterone (DHT) on lipogenic protein and gene expression. We performed Western blot and real‐time quantitative polymerase chain reaction (qRT‐PCR) analysis of lipogenic intermediates on homogenates of energy storage tissues (liver, WAT, and skeletal muscle). Low‐dose DHT lowered the protein expression of the active form of cytosolic SREBP1 in the liver and WAT. Additionally, low‐dose DHT lowered the protein expression of the inactive SREBP1 in the liver. DHT did not alter the levels of the active and inactive forms of SREBP2 in the liver and WAT. Interestingly, the active form of SREBP2 was lowered in skeletal muscle. Further, phospho acetyl co‐A carboxylase (p‐ACC) protein levels were unaltered in liver and WAT. Fatty acid synthase (FAS) levels were unchanged in WAT and skeletal muscle. Taken together, our findings suggest that hepatic and white adipose cytosolic SREBP1 is being decreased due to its translocation to the nucleus, where it regulates lipogenic protein levels.Support or Funding InformationThank you to Howard University College of Medicine, Johns Hopkins Medicine, and Georgetown University Medical Center (GUMC) for supporting this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dihydrotestosterone enhances growth and infectivity of LeishmaniaMexicana.

A strong sex-associated susceptibility towards Leishmania has been reported in males, yet little is known on the effect of hormones in Leishmania physiopathogenicity. Due to the enhanced susceptibility of males to Leishmaniamexicana infections, we were interested in analysing the effect exerted by the main androgen produced in males (DHT) on L. mexicana promastigotes. Thus, the aim of this study was to assess the regulation exerted by dihydrotestosterone (DHT) on L. mexicana replication, infectivity, survival and development of tissue lesions. Experiments included growth curves of L. mexicana promastigotes incubated with different doses of DHT, their infection rate, intracellular survival and lesion development in BALB/c mice. Our data show that DHT significantly enhances parasite replication, infection rate and survival in bone marrow-derived macrophages (BMMФ). Promastigotes in the presence of DHT produced significantly larger lesions in BALB/c earlobes. These results suggest that DHT probably plays a critical role during L. mexicana infections, and the higher susceptibility of males possibly relates to benefits gained by the parasite from host-derived hormones. Our data shed new light on the physiopathology of Leishmania infections and are the first attempt to understand the direct interaction between Leishmania and androgens, particularly DHT. Understanding this trans-regulation process employed by parasites to exploit host molecules sheds new light on L. mexicana physiopathogenesis and opens a possible field for studies on drug development.

Effects of dihydrotestosterone on synaptic plasticity of the hippocampus in mild cognitive impairment male SAMP8 mice.

The current study focused on how dihydrotestosterone (DHT) regulates synaptic plasticity in the hippocampus of mild cognitive impairment male senescence-accelerated mouse prone 8 (SAMP8) mice. Five-month-old SAMP8 mice were divided into the control, castrated and castrated-DHT groups, in which the mice were castrated and treated with physiological doses of DHT for a period of 2 months. To determine the regulatory mechanisms of DHT in the cognitive capacity, the effects of DHT on the morphology of the synapse and the expression of synaptic marker proteins in the hippocampus were investigated using immunohistochemistry, qPCR and western blot analysis. The results showed that the expression of cAMP-response element binding protein (CREB), postsynaptic density protein 95 (PSD95), synaptophysin (SYN) and developmentally regulated brain protein (Drebrin) was reduced in the castrated group compared to the control group. However, DHT promoted the expression of CREB, PSD95, SYN and Drebrin in the hippocampus of the castrated-DHT group. Thus, androgen depletion impaired the synaptic plasticity in the hippocampus of SAMP8 and accelerated the development of Alzheimer's disease (AD)-like neuropathology, suggesting that a similar mechanism may underlie the increased risk for AD in men with low testosterone. In addition, DHT regulated synaptic plasticity in the hippocampus of mild cognitive impairment (MCI) SAMP8 mice and delayed the progression of disease to Alzheimer's dementia. In conclusion, androgen-based hormone therapy is a potentially useful strategy for preventing the progression of MCI in aging men. Androgens enhance synaptic markers (SYN, PSD95, and Drebrin), activate CREB, modulate the fundamental biology of synaptic structure, and lead to the structural changes of plasticity in the hippocampus, all of which result in improved cognitive function.

MiR-141 and miR-375 induction and release are different from PSA mRNA and PCA3 upon androgen stimulation of LNCaP cells

Recent studies have demonstrated the differential expression of miR-141 and miR-375 in circulation of patients with advanced/metastatic prostate cancer (PCa). The aim of this study was to investigate the regulation of miR-141 and miR-375 by androgens and their release into the incubation medium in relation to prostate-specific antigen (PSA) mRNA and prostate cancer antigen 3 (PCA3). Plasma levels of these molecules were measured in a small cohort of patients with localized PCa. As an in vitro cell model we used androgen-sensitive LNCaP cells exposed to an androgen ablation of 48 h, and then treated with dihydrotestosterone (DHT) for 24 h. Expression of the four RNA molecules was measured by quantitative polymerase chain reaction (qPCR). miR-141 and miR-375 were induced in a dose-dependent manner where the median stimulation reached only 1.5-fold at maximum. The effect of DHT on PSA mRNA (up to 30-fold) and PCA3 (up to 195-fold) was much more evident. With regard to the release into the incubation medium, similar results were obtained with the exception of PCA3. At the highest DHT dose (100 nM), median miR-141 and miR-375 release was increased 1.7- and 1.4-fold (P=0.07), respectively. DHT treatment led to a significant release of PSA mRNA (up to 12-fold) into the medium while PCA3 could not be amplified from the incubation medium. In plasma only PCA3 differed significantly between localized PCa patients and healthy subjects. In conclusion, our study provides evidence that miR-141 and miR-375 are increasingly released into incubation medium from androgen-stimulated cells. However, the extent of their induction was weaker than PSA mRNA or PCA3, suggesting differential regulation by androgens.

Abstract LB-322: Synergistic effects of FOXO3 and AR to increase tolerogenicity of tumor associated dendritic cells.

Abstract The immune system has the ability to recognize and destroy cancerous cells long before they grow, multiply and form tumors. However, once tumors develop they severely suppress anti-tumor immune responses. The underlying causes of immune suppression are complex and not completely understood. Recent studies indicate that tumor-associated dendritic cells (TADC) within the tumor microenvironment (TME) are key regulators of cytotoxic T-cell (CTL)function and may serve as a critical target for immunotherapy. The TADC-T cell interaction results in T-cell tolerance that is regulated by DC expression of the transcription factor Forkhead Box O3 (FOXO3). FOXO3 is a transcriptional regulator of a variety of processes associated with cell cycle progression as well as regulation of nuclear receptors, including the androgen receptor (AR). Androgen stimulation of tumor cells has been shown to promote tumor growth and androgen deprivation therapy is the standard of care to treat aggressively growing prostate cancers. However, less is known regarding the impact of androgen signaling on immune cell function in the TME. In this study we show a potential combined effect of AR signaling and FOXO3 to promote tolerogenic functions in TADC. Our preliminary data, using the TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP), a murine model of spontaneous prostate cancer, showed that prostates in FOXO3−/− TRAMP mice grew at half the rate compared to prostate tumors of the wild type TRAMP mice, in spite of equivalent expression of SV40 TAg. Additionally, TADC that infiltrated the FOXO3−/- tumors had minimal AR expression, were capable of stimulating T cell proliferation, and most importantly, did not promote CTL tolerance. In contrast, DC purified from wild type TRAMP had increased expression of AR and FOXO3 and were highly tolerogenic. However, upon silencing FOXO3 via siRNA, tolerogenicity subsided and AR expression was down regulated. To test the effect of androgen signaling on DC function, normal prostate dendritic cells were treated with increasing doses of dihydrotestosterone (DHT). It was observed, that FOXO3 expression and tolerogenicity increased with increased concentrations of DHT and AR stimulation. Furthermore, TRAMP mice had a significant increase, over that of wild type mice, in testosterone levels in the seminal vesicle fluid (measured by ELISA) and prostate tissue (measured by RT-PCR). Surgical castration of TRAMP mice abrogated FOXO3 and AR expression and up-regulated co-stimulatory molecules and pro-inflammatory cytokines such as IL-12 in dendritic cells. These data suggest a synergistic effect between FOXO3 and AR to promote DC mediated immune suppression in the prostate TME and may be critical targets to enhance therapies in the treatment of prostate cancer. Citation Format: Matthew Thompson, Stephanie Watkins, Arthur A. Hurwitz. Synergistic effects of FOXO3 and AR to increase tolerogenicity of tumor associated dendritic cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-322. doi:10.1158/1538-7445.AM2013-LB-322

Microinjection of Dihydrotestosterone as a 5α-Reduced Metabolite of Testosterone into CA1 Region of Hippocampus Could Improve Spatial Learning in the Adult Male Rats.

CA1 region of hippocampus has an important role in learning and memory. Previous reports have shown that androgens like testosterone and its metabolites are present in high concentration in CA1 region of hippocampus. Androgen receptors have also high density in this region. Therefore, it is suggested that neurohormones in CA1 have an important role in learning and memory. It is likely that testosterone exerts its effect via its metabolites, especially dihydrotestosterone (DHT), a 5α-reduced androgen. In this research, we conducted an experiment to assess the path of testosterone›s effectiveness on spatial learning and memory. Adult male rats were randomly divided into 4 groups and, bilaterally, cannulated into CA1 region of hippocampus. One week after the surgery, animals received DMSO 0.5 μL as a control group and different doses of dihydrotestosterone (DHT) (0.25, 0.5 and 1 µg/0.5 μL/side) 25-30 min before the training in spatial version of Morris Water Maze task. Training session contained two blocks which animals had to learn the position of hidden platform in 4 trials. On the test session (next day), rats performed a one-trial probe test and then a visible platform one. The results showed that escape latency and traveled distance were decreased significantly in DHT-treated (0.5 µg/0.5 μL/side) rats. This finding suggested that DHT may have improved the effect on acquisition of spatial learning and memory.

Dihydrotestosterone differentially modulates the cortisol response of the hypothalamic–pituitary–adrenal axis in male and female rhesus macaques, and restores circadian secretion of cortisol in females

Here we used a within-subject design to evaluate hypothalamic–pituitary–adrenal (HPA) activity following replacement of low and high physiological levels of testosterone (T) to adult, gonadally-suppressed, male rhesus macaques, and replacement with sex-specific low and high physiological doses of dihydrotestosterone (DHT) in the same adult males as well as in adult, gonadally-suppressed, female rhesus macaques. As indexes of HPA axis activation following T and DHT replacement, serum levels of cortisol (CORT) were measured before and following dexamethasone (DEX) inhibition, and corticotrophin-releasing factor (CRF) induced activation. Female monkeys were assessed for differences in response associated with dominant (DOM) and subordinate (SUB) social status. Data show that the high physiological dose of DHT significantly decreased basal CORT in both male and female monkeys irrespective of social status, but reduced CRF-stimulated CORT only in males. SUB female monkeys showed a trend towards increased CRF-stimulated CORT release under high-dose DHT replacement compared to DOM females or males given the same treatment, indicating that androgens likely have no influence on reducing HPA activation under chronic psychosocial stress in females. The normal circadian rhythm of CORT release was absent in placebo-replaced SUB and DOM females and was restored with low-dose DHT replacement. These results indicate that DHT significantly reduces CRF-stimulated CORT release only in male monkeys, and plays a role in maintaining circadian changes in CORT release in female monkeys.

Androgens exert sexually dimorphic effects on angiogenesis: novel insight into the relationship between androgens and cardiovascular disease

Androgens exert sexually dimorphic effects on angiogenesis: novel insight into the relationship between androgens and cardiovascular disease

A role for dihydrotestosterone treatment in older men?

A role for dihydrotestosterone treatment in older men?

Androgen receptor signaling induced by supraphysiological doses of dihydrotestosterone in human peripheral blood lymphocytes

Anabolic androgenic steroids, a class of steroid hormones related to testosterone, are natural ligands of androgen receptor (AR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. AR binds specific DNA elements, known as androgen-response elements. Testosterone, the main male sexual hormone, binds AR directly and indirectly, through conversion into dihydrotestosterone (DHT), its more active metabolite. Anabolic androgenic steroids are frequently detected in the urine of doped athletes; their consumption is also growing among sport amateurs and adolescents. The effects of androgens can differ depending on the target cells and/or tissues. To gain insight into transcription activation mechanisms of AR, we investigated AR protein signaling in human peripheral blood lymphocytes treated with supraphysiological doses of DHT. We performed a comparative proteomic analysis and we identified about 30 differentially expressed proteins. At least five species contained a consensus androgen-response elements sequence in the promoter region of related coding genes. The analysis also revealed that high doses of DHT activate the drug detoxification process, could stimulate an increase in cell motility and exert a prosurvival effect rather than an apoptotic one.

Role of Salt-Induced Kinase 1 in Androgen Neuroprotection against Cerebral Ischemia

Androgens within physiological ranges protect castrated male mice from cerebral ischemic injury. Yet, underlying mechanisms are unclear. Here, we report that, after middle cerebral artery occlusion (MCAO), salt-induced kinase 1 (SIK1) was induced by a potent androgen-dihydrotestosterone (DHT) at protective doses. To investigate whether SIK1 contributes to DHT neuroprotection after cerebral ischemia, we constructed lentivirus-expressing small interference RNA (siRNA) against SIK1. The SIK1 knockdown by siRNA exacerbated oxygen-glucose deprivation (OGD)-induced cell death in primary cortical neurons, suggesting that SIK1 is an endogenous neuroprotective gene against cerebral ischemia. Furthermore, lentivirus-mediated SIK1 knockdown increased both cortical and striatal infarct sizes in castrated mice treated with a protective dose of DHT. Earlier studies show that SIK1 inhibits histone deacetylase (HDAC) activities by acting as a class IIa HDAC kinase. We observed that SIK1 knockdown decreased histone H3 acetylation in primary neurons. The SIK1 siRNA also exacerbated OGD-induced neuronal death in the presence of trichostatin A (TSA), an HDAC inhibitor, and decreased histone H3 acetylation at 4 hours reoxygenation in TSA-treated neurons. Finally, we showed that DHT at protective doses prevented ischemia-induced histone deacetylation after MCAO. Our finding suggests that SIK1 contributes to neuroprotection by androgens within physiological ranges by inhibiting histone deacetylation.