Gaussia Luciferase. We found that AR/p52-02 markedly inhibited growth of both castration-resistant C4-2 (IC50 ∼6 μM) and parental androgen-dependent LNCaP (IC50 ∼4 μM) human prostate cancer cells under low androgen conditions. Growth inhibition was associated with significantly reduced nuclear p52 levels and DNA binding activity, as well as decreased phosphorylation of AR at serine 81, increased AR ubiquitination, and decreased AR transcriptional activity as indicated by decreased prostate-specific antigen (PSA) mRNA levels in both cell lines. AR/p52-02 also caused a reduction in levels of p21WAF/CIP1, which is a direct AR targeted gene in that its expression correlates with androgen stimulation and mitogenic proliferation in prostate cancer under physiologic levels of androgen, likely by disrupting the AR signaling axis. The reduced level of cyclinD1 reported previously for this compound may be due to the reduction in nuclear presence and activity of p52, which directly regulates cyclinD1 expression, as well as the reduction in p21WAF/CIP1, since p21WAF/CIP1 is reported to stabilize nuclear cyclinD1 in prostate cancer. Overall, the data suggest that specifically inhibiting the interaction of AR with p52 and blocking activity of p52 and pARser81 may be an effective means of reducing castration-resistant prostate cancer cell growth.Accumulating evidence shows that androgen receptor (AR) activation and signaling plays a key role in growth and progression in all stages of prostate cancer, even under low androgen levels or in the absence of androgen in the castration-resistant prostate cancer. Sustained activation of AR under androgen-deprived conditions may be due to its interaction with co-activators, such as p52 NF-κB subunit, and/or an increase in its stability by phosphorylation that delays its degradation. Here we identified a specific inhibitor of AR/p52 interaction, AR/p52-02, via a high throughput screen based on the reconstitution of  About 30% of all prostate cancer patients after first line of therapy succumb to recurrent prostate cancer. Although the recurrent prostate cancer regresses after androgen deprivation therapy (ADT), the majority of these patients return to the clinic with the refractory disease known as castration-resistant (CR) prostate cancer, for which successful therapy remains a challenge. Accumulative evidence suggests that castration-resistant activation of androgen receptor (AR) and development of apoptosis-resistant cells play key roles in the transition of androgen-dependent prostate cancer to CR prostate cancer .ser81) occurring 6 to 8 hours after androgen stimulation plays an important role in AR transcriptional activity  that was kindly provided by Shannon Kenney at the University of Wisconsin-Madison was amplified by PCR and subcloned into pcDNA3.1(+) . This construct was used for fusing the C-terminal portion of  (Gluc2)  at the e (Gluc2) .5) were seeded and 1 day later co-transfected with 3μg each of cmv-Gluc1-AR and cmv-p52-Gluc2 vectors or transfected with cmv-Gluc1-AR and cmv-OFp52-Gluc2 as a negative control using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer-supplied protocol. Two to 3 hours after transfection, cells were washed and refed with DMEM without serum. Cells were collected 48h later and lysed for measurement of Gaussia Luciferase (GL) activity using Biolux Gaussia assay kit (E3300L) from New Engalnd Biolabs  per the manufacturer's protocol.Hep3B cells  based on reconstituted GL bioluminescence. Screening of 2,800 small molecules from a Life Chemicals Library for inhibitors of AR interaction with p52 NF-κB subunit was carried out as described before for AR-JunD interaction , with thFor eliminating false positives, including non-specific inhibitors and toxins, a secondary screen was performed on the hits identified from the initial HTS, using lysate from cells that were co-transfected with vectors containing cmv-Gluc1/cmv-Gluc2 (negative control) or with cmv-smad3-Gluc1/cmv-Gluc2-PKB (positive control) , essentiA commercially available assay, PolarScreen™ Androgen Receptor Competitor Assay kit (Invitrogen), was used to determine if the AR/p52 inhibitors identified by the HTS had anti-androgenic  activity. Fluorescence polarization assay with graded concentrations of the compounds of interest was performed according to the manufacturer's supplied protocol, in comparison to clinical anti-androgen Casodex®.For growth assay, DNA levels of control and treated cell cultures were measured as an indicator of growth . The det® by Life Technologies ™ (AM 1723) was used to synthesize cDNA directly from cells following the instructions provided by the manufacturer as reported before [®Green Supermix (Bio-Rad). Each reaction was normalized by coamplification of 18S rRNA. Triplicates of samples were run on a Bio-Rad CFX-96 real time cycler. The sequences of primers used for amplification of PSA and 18S rRNA cDNA were as follows: PSA: Forward 5′-GACCACCTGCTACGCCTCA and Reverse 5′-GGAGGTCCACACTGAAGTTTC; 18S rRNA: Forward 5′-CGCCGCTAGAGGTGAAATCT and Reverse: 5′-CGAACCTCCGACTTTCGTT.Cells treated with or without 10μM AR/p52-02 in the presence or absence of 2nM R1881 were harvested for qrtpcr analysis to determine PSA mRNA levels after 72h of treatment. For qrtpcr, cells-to-cDNA™ II reverse transcription without RNA isolation kit from Ambiond before . The cDNet. al was used [2, 10 mmol/L KCl, and 0.1% NP40 supplemented with protease inhibitors  and incubated on ice for 30 minutes. Nuclei were precipitated by centrifugation at 3000 g for 10 minutes at 4°C. The supernatants were collected as the cytosolic fraction. After washing once with low-salt buffer, nuclei were lysed in high salt lysis buffer containing; 50 mmol/L Tris-HCl (pH8), 150 mmol/L NaCl, 1% Triton X-100 followed by mechanical disruption at 4°C for 30 minutes. The nuclear lysate was cleared by 10,000 rpm centrifugation at 4°C for 15 minutes. Protein concentration was determined using BCA assay kit . Western blots were performed as described before [For protein analyses, cells were treated for 72h in the presence or absence of 2nM R1881 with or without inhibitor AR/p52-02 at 5μM for LNCaP cells or 10μM for C4-2 cells. Whole cell lysates were prepared using modified RadioImmunoPrecipitation Assay (RIPA) buffer containing a tablet of complete protease inhibitors from Roche  as reported before . For nucwas used . Brieflyd before . BrieflyFor AR ubiquitination studies, prior to immunoblot, total protein was extracted, quantified, and 500μl  was subjected to preclearance using 50 μl Protein A/G PLUS-agarose beads  for 20 minutes at room temperature with rotation. The precleared lysates were incubated with 6 μg AR antibody  for an overnight with constant rotation at 4°C. The antibody-protein complexes were pulled down using 50 μl Portein A/G PLUS-agarose beads in an overnight incubation at 4°C with constant rotation following which the beads were separated by centrifugation at 14,000 × g for 1 minute at 4°C. The beads were further washed three times with PBS after which they were boiled with 4 × LDS sample buffer . The protein was resolved using SDS-PAGE as explained above and the western blots were probed with Ubiquitin antibody for detection of Ubiquitinated-AR.2+/Mg2+-free PBS, 1mM Orthovanadate and 0.012% Saponin). One drop of Image-iTFX signal enhance solution (Invitrogen) was added to each well and incubated for 30 minutes and then washed twice with rinse solution I. The cells were blocked by rinse solution II (1% BSA and 0.3 M Glycine) for an hour at 37°C in a 5% CO2 incubator followed by 2-3 times wash with rinse solution I. Three microliter of mouse monoclonal AR antibody  per well diluted (50x) in rinse solution II was added and incubated for 2 hours at 37°C in a CO2 incubator. The cells were Molecule Screening and Synthesis Facility for assistance rinsed and diluted (100x) secondary antibody Alexa Fluor (488) donkey anti-mouse  was added to each well. Finally, the slides were mounted using DAPI containing mounting solution. Images were taken at 20x magnification with a Nikon TI-U inverted fluorescence microscope for quantitation of AR fluorescence normalized to DAPI for AR nuclear levels.Cells treated with or without inhibitor AR/p52-02 at 5μM for LNCaP cells or 10μM for C4-2 cells in the presence or absence of 2nM R1881 for 72h were prepared for ICC analysis. At the end of treatment, cells were fixed using fixative solution containing 3.6% paraformaldehyde, 0.024% Saponin and 1mM sodium Orthovanadate for 10 minutes at room temperature. The fixation was quenched using 0.1% sodium borohydride in PBS followed by 2 changes of rinse solution I , the binding activity of p52 to its consensus sequence (5′-gggactttcc-3′) with its specific antibody (provided in the kit) was determined in all nuclear fractions of cells from different conditions following the manufacturer's instructions. Briefly, for DNA binding assay, 20μl of nuclear extract containing total nuclear protein of 5μg was added to each well of three wells in a 96-well plate provided in the kit. Four wells were also used for positive control: Raji nuclear extract (provided in the kit) at 20μl/well at different concentrations . Three wells also were used for negative control: nuclear extraction buffer at 20μl/well. Thirty microliters of complete binding buffer were added to each well and the plate was covered using the provided adhesive film. The mixture of nuclear extract and binding buffer was incubated for 1h at room temperature with mild agitation followed by washing x3 with wash buffer. One hundred microliters of diluted NF-κB2/p52 antibody were added to each well and incubated for 1h at room temperature followed by washing the wells x3 with wash buffer. One hundred microliters of diluted HRP-conjugated secondary antibody were added to each well and incubated for another hour at room temperature followed by washing x4 with wash buffer. After the final wash 100μl of Developing Solution was added to each well and incubated for 5 minutes. The reaction was stopped by adding 100 μl Stop Solution. The absorbance was measured at 450nm.t-test with a confidence level of 0.05 was performed for data comparisons and significance determinations.An unpaired two-tailed heteroscedastic Student's


W741L/C mutation as proof of concept, we demonstrate that this mutant confers an androgenic-like signalling programme and growth promoting phenotype in the presence of bicalutamide. Transcriptomic profiling of ARW741L highlighted key genes markedly up-regulated by the mutant receptor, including TIPARP, RASD1 and SGK1. Importantly, SGK1 expression was found to be highly expressed in the KUCaP xenograft model and a CRPC patient biopsy sample both of which express the bicalutamide-activated receptor mutant. Using an SGK1 inhibitor, ARW741L transcriptional and growth promoting activity was reduced indicating that exploiting functional distinctions between receptor isoforms in our model may provide new and effective therapies for CRPC patients.The persistence of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the unmet clinical need for the development of more effective AR targeting therapies. A key mechanism of therapy-resistance is by selection of AR mutations that convert anti-androgens to agonists enabling the retention of androgenic signalling in CRPC. To improve our understanding of these receptors in advanced disease we developed a physiologically-relevant model to analyse the global functionality of AR mutants in CRPC. Using the bicalutamide-activated AR Prostate cancer (PC) is the leading cause of male cancer deaths in the western world and remains a major challenge to treat effectively , 2. At pAR gene amplification [in vivo [Aberrant AR signalling is a hallmark of CRPC and is driven by numerous mechanisms including fication , somaticfication , 15, expfication  and de-rfication , 18 that[in vivo .W741L/C and ARH874Y/ART877A mutations that utilise the agents as agonists to promote androgenic signalling and tumour cell growth [F876L mutation in patient samples refractory to enzalutamide and ARN-509 therapies has indicated that this is a phenomenon not limited to first-generation anti-androgens [The acquisition of AR mutations during ADT, that either facilitate transcriptional activity of the receptor in the absence of androgens or switch anti-androgens to AR agonists, is a well characterised mechanism of hormone escape and has been reported to occur in upwards of 60% of CRPC patients , 14. Impndrogens –22.T877A mutant, there is a paucity of information on the functional dynamics and global transcriptomics of CRPC-associated AR mutants in a physiological setting that is likely to provide key biomarkers and additional treatment regimens for anti-androgen-resistant malignancies. Moreover, a major consideration for the development of next-generation AR-targeted therapies is whether they will be effective against pre-existing AR mutants in CRPC hence the development of key research tools to facilitate these studies is of high priority. To address this, we have developed a novel RNAi-rescue approach that utilises stable expression of specific AR mutants in LNCaP cells depleted of the endogenous receptor to facilitate more robust analyses of aberrant receptor signalling. Therefore, it is now possible to assess global transcriptional activity and sensitivity of CRPC-associated AR mutants to new receptor-targeting agents in a more relevant cellular context. Using the ARW741L variant as a paradigm, we demonstrate that this mutant activates several endogenous AR-target genes, including PSA and TMPRSS2, and promotes a hyper-proliferative phenotype in the presence of bicalutamide; a phenomenon that can be reversed by depletion of ARW741L. Global transcriptomics identified a sub-set of ARW741L-driven genes that are markedly up-regulated compared to the endogenous receptor, including SGK1, TIPARP and RASD1. Importantly, treatment with an SGK inhibitor down-regulated bicalutamide-driven receptor activity and cell growth, suggesting this could be a novel avenue of treatment for bicalutamide-resistant patients. In all, we have successfully applied a novel AR replacement strategy to physiologically model the ARW741L mutation in disease and highlighted key distinctions in receptor activity that could be therapeutically-exploited for improved CRPC treatment.Modelling the function of CRPC-relevant AR mutants in their native context is challenging with most studies utilising non-PC cell lines, ectopically-expressed variant receptors and luciferase reporter-based transcriptional assays , 23, 24.W741L as a paradigm for this study  to discriminately deplete endogenous ART877A (approximately 90% knockdown) by targeting the 3′-UTR of the AR transcript that is absent in ectopically-expressed ARW741L mRNA  down-regulated expression of this protein , DHT and bicalutamide activated a subtle increase in recruitment of FLAG-ARW741L to these loci  against the original  and a control transduced cell line (LNCaP-LacZ) that does not overexpress ARW741L. Importantly, both Clone 1 and Clone 2 showed comparable growth stimulation in response to DHT and bicalutamide and was distinct from LNCaP-LacZ that only responded to DHT . This threshold was chosen as it fell within fold change cut-offs that have been used in previous publications investigating AR signalling profiles [W741L cells. Importantly, no genes were identified in the LNCaP-LacZ control line that exhibited >1.5 fold up-regulation in the presence of bicalutamide, suggesting that the identified set of bicalutamide-induced genes in the LNCaP-ARW741L cells were specific to the ectopically-expressed receptor . Genes that exhibited > 2 fold increase in expression following bicalutamide treatment in ARW741L-depleted cells were deemed to be bicalutamide-independent. We identified 38 genes matching to the 869 core data set that were subsequently eliminated  were common to both lists  and KLK2, which were found to be the most DHT-stimulated in LNCaP-LacZ cells were also elevated in response to bicalutamide in LNCaP-ARW741L cells and is consistent with LNCaP-ARW741L gene expression data  polymerase), RASD1 (Ras dexamethasone-induced 1) and SGK1 (serum- and glucocorticoid-regulated kinase 1) were exclusively and markedly up-regulated by bicalutamide in the LNCaP-ARW741L cell line compared to the DHT-stimulated control LNCaP-LacZ derivative (data not shown). Robust bicalutamide-mediated up-regulation of TIPARP (57-fold), RASD1 (93-fold) and SGK1 (109.5-fold) was validated by QPCR, and demonstrated to be exclusively mediated by ARW741L as depletion of this mutant by siARW741L completely abrogated gene expression  and RASD1 (2-fold) expression and failed to elevate TIPARP transcript levels while, as expected, PSA was greatly up-regulated (W741L (data not shown), demonstrated modest enhancement of SGK1, TIPARP and RASD1 transcription in response to DHT stimulation with respective 4.5-, 8.6- and 5.9-fold induction, indicating that the robust up-regulation of the three genes in LNCaP-ARW741L cells is not due to the phenomenon of elevated cellular AR levels  and developed key siRNAs to deplete either endogenous or ectopic ART877A and ARW741L, respectively. Using this model, we demonstrated that in the presence of bicalutamide, ARW741L associates with cis-regulatory elements of several AR target genes, including PSA and TMPRSS2, and facilitates their expression. Significantly, these effects were attenuated upon depletion of the ectopic receptor indicating that the bicalutamide-driven functionality of ARW741L previously characterised in AR null cell line studies  experiments demonstrated an inhibitory effect of ART877A on bicalutamide-activated ARW741L when both receptor isoforms were expressed  identified a series of bicalutamide-induced genes; many of which were known AR-target genes including PSA, KLK2 and TMPRSS2. Furthermore, although the number of bicalutamide-activated genes were in excess of those controlled by DHT in our control cells and those reported in Hieronymous et al., [et al., [W741L maintains a common androgenic expression programme. Importantly, a number of robustly up-regulated bicalutamide-dependent genes were identified by micro-array in LNCaP-ARW741L cells that were not greatly enhanced by DHT in the LNCaP-LacZ line, including TIPARP, RASD1 and SGK1. Depletion of ectopic receptor in LNCaP-ARW741L cells attenuated this bicalutamide-driven response indicating that these genes are potentially discriminate targets of ARW741L. This dramatic and selective activity of the ARW741L mutant is intriguing and may be a consequence of subtle allosteric re-organisation of the receptor that permits hyper-activation of the receptor at distinct genomic loci. By repositioning the C-terminal activation function-2 (AF-2) domain of the receptor, the leucine residue may enable more robust interaction with selective co-regulators to drive acetylation and methylation of the AR to enhance inherent transcriptional activity [In keeping with the utility of this model as an important pre-clinical tool to identify distinctions in the functionality of disease-associated AR mutations, we hypothesised that global transcriptomic profiling of AR et al.,  and Nels[et al.,  there waactivity , 36.SGK1 has been identified to be robustly up-regulated by endogenous ART877A in LNCaP cells and the gene product found to positively reinforce androgenic signalling [SGK1 expression in response to 10 nM DHT by quantitative PCR in the respective LNCaP-LacZ and LNCaP-wtAR cell derivatives, it was significantly less than previous reported.In contrast to our data, however, gnalling , 37. TheW741L cells demonstrated marked up-regulation of SGK1 expression in response to bicalutamide, we speculated that they may also be sensitive to SGK1 inhibition. Consistent with the reported phenomenon in parental LNCaP cells [W741L-driven SGK1 expression was down-regulated in response to GSK650395 treatment, suggesting that ectopic receptor function is dependent upon SGK1-mediated kinase activity. Furthermore, proliferation of both LNCaP and LNCaP-ARW741L cells was significantly down-regulated upon SGK1 inhibition; with similar IC50 values for both cell lines in response to GSK650395 . For drug treatments, 10 nM bicalutamide, 1 nM DHT, or increasing doses of enzalutamide, were administered for 96 hours prior to SRB assay.Sulforhodamine B (SRB) assays were performed according to . BrieflyFor additional Methods please refer to accompanying


Androgen receptor (AR) plays a critical role in the development and progression of prostate cancer (PCa). Current clinically used antiandrogens such as flutamide, bicalutamide, and newly approved enzalutamide mainly target the hormone binding pocket (HBP) of AR. However, over time, drug resistance invariably develops and switches these antiandrogens from antagonist to agonist of the AR. Accumulated evidence indicates that AR mutation is an important cause for the drug resistance. This review will give an overview of the mutation based resistance of the current clinically used antiandrogens and the rational drug design to overcome the resistance, provides a promising strategy for the development of the new generation of antiandrogens targeting HBP. Prostate cancer (PCa) is one of the most common cancer and the second leading cause of cancer death in men in the western countries   has been remaining the mainstay for the treatment of advanced PCa since 1940s. , homodimerizes and translocats into the nucleus where it binds ARE sites of DNA, directly regulates targeting genes transcription in the presence of coactivator which binds to AF2, promoting the recruitment of RNA polymerase II, triggering the transcription process  are initially used for the androgen blockade in patients. However, severe drawbacks such as hepatotoxicity, interference with libido and potency, cardiovascular side effects and low efficacy have limited their clinical use. The later developed non-steroidal antiandrogens including the first-generation antiandrogens such as flutamide, bicalutamide, nilutamide, and the second-generation compounds: enzalutamide and ARN509. These non-steroidal antiandrogens which avoid the typical constraints of the steroidal antiandrogens, have been widely used in clinical nowadays. However, after initially effective response, approximately 50% of patients whose cancer started to grow again under the treatment of antiandrogens, the cancer has been observed to regress by simply stopping the antiandrogen, which referred to the Anti-Androgen Withdrawal Response (AAWR)  demonstrates that the H12 adopts the agonistic conformation, which elucidates that the T877A mutation converts the OHF from an antagonist to an agonist Figure  Bohl et. AnotherAs mentioned previously, PCa is continually undergoing AR mutations that switch the antiandrogen from AR antagonist to AR agonist and eventually relapses to lethal CRPC. Thus the rational development of novel antiandrogens based on the AR acquired mutations driven drug resistant seems like ongoing running race and remains a big challenge , which facilitate the discovery and development of novel antiandrogens to combat the drug resistance by using “structure-based” drug design  simulations and proposed a REMD based methodology to predict agonist/antagonist potency of a ligand. According to the simulation results, a novel flutamide derivative called SC333 Figure  was desiin vivo in normal male mice and suppressed AR activity and tumor cell proliferation in human CRPC xenografts  (Huxley et al., 17 (Figure N,N-bis-2-chloroethylaniline was linked to a steroid hormone that targets AR, allowing the conjugate to simultaneously bind to AR and DNA, resulting in the blockade of DNA repair enzymes in PCa cell lines that overexpressing AR, subsequently leading to the disruption of AR-mediated transcription and signaling (Marquis et al., 18 and 19 (Figure Cross coupling and conjugation strategies are wildly applied to develop the modulator with multivalent and heterobifunctional constructs, which exhibit high affinity and specificity to the biomolecular target Lambert,  Figure . To date9 Figure .20 (Figure 21 (Figure 22 (Figure Except for steroid conjugates, there are several representative examples of promising strategies that have been used to target AR with non-steroidal conjugates. Recently, the Oyelere's lab reported a non-steroidal heterobifunctional conjugate 0 Figure  outfitte0 Figure . In simi1 Figure  containi1 Figure . The El-2 Figure  that sel2 Figure .Despite the great improvements h ave been made in the development of antiandrogen circumventing mutation-based resistance, it remains a big challenge. First of all, there is still no antagonistic or apo AR LBD structure available, current LBD actually are all agonistic, which add the hurdle of structure-based antiandrogen design. Up to now, the adopted antagonistic AR LBD model included the homology model based on the antagonistic LBD of other nuclear receptor such as ER, GR and PR, the model with the H12 simply deleted from the agonistic AR LBD, and the model equilibrated via molecular dynamics from the started structure of the agonistic AR LBD. Thus, the antagonistic or apo AR LBD structure is highly in demand to provide more accurate model in structural based drug design of antiandogens. However, as the antagonistic or apo AR LBD protein is formidable to be prepared and crystallized due to its flexibility and tight association with the bacterial chaperonin (Bohl et al., Besides of HBP, several other binding sites on AR like AF2, BF3, the DNA binding site, and AF1 have been attracted attentions in novel antiandrogen development (Haendler and Cleve, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Helicobacter pylori infection and high-salt diet (HSD). Within T cells, Helicobacter pylori (Hp) infection and high-salt dietcan up-regulated SGK1 expression and in turn enhance expression of Lnc-SGK1 through JunB activation. And expression of Lnc-SGK1 can further enhance transcription of SGK1 through cis regulatory mode. Lnc-SGK1 can induce Th2 and Th17 and reduce Th1 differentiation via SGK1/JunB signaling. Serum Lnc-SGK1 expression in combination with H. pylori infection and/or HSD in T cells was associated with poor prognosis of GC patients, and could be an ideal diagnostic index in human GC.Serum and glucocorticoid-inducible kinase (SGK) 1can be triggered in several malignancies. Most research on SGK1has focused on its role in cancer cells, and we sought to investigate its potential upstream non-coding RNA nominated as Lnc-SGK1, and their expression and diagnostic value in T cells in human gastric cancer (GC). Excessive expression of Lnc-SGK1 and SGK1 were observed in T cell either within the tumor or peripheral T cells, and furthermore associated with  A few studies have addressed the role of SGK1 in T cell differentiation. SGK1 upregulates pathogenic IL-23-dependent IL-17-producing CD4lignancy , 16. Recacis-regulatory process. Interestingly, we found a lncRNA (LOC105378009) located in the upstream of SGK1 promoter region within 10kb space, therefore, in the present study, we sought to investigate the role of SGK1 within T cells of patients with gastric cancer (GC), and the association between SGK1 and LOC105378009 as well as the characteristics of GC and their potential prediction value for diagnosis or prognosis.The long non-coding RNAs (lncRNAs) were reported as a biomarker for predicting survival, metastasis, and in the diagnosis of multiple diseases , 19. Thecis-regulatory relationships may exist between lncRNAs and known genes. According to the bioinformatics analyses, LOC105378009 was located upstream of SGK1, with a spacing of about 5 kb  targeting LOC105378009 was designed, we found that the activation affection was significantly reduced comparing to control group Figure , 1E. FroTo investigate the distribution of Lnc-SGK1 and SGK1 in human GC, 245 tumor tissues and adjacent normal tissues were analyzed. GC and normal tissues both expressed SGK1, especially in the parenchymal cells. However, expression of SGK1 was increased significantly in GC compared with both tumor adjacent tissues and normal tissues Figure . It was Next, Expression of both SGK1 and Lnc-SGK1 was determined by real-time PCR. Lnc-SGK1 and SGK1 transcription was significantly higher in GC tumor infiltration cells (TILS) than adjacent normal tissue and peripheral blood mononuclear cells (PBMCs). A significant difference was also found for Lnc-SGK1 and SGK1 expression between the healthy control and GC PBMCs groups Figure , 2C.H. pylori infection  was higher than in those without infection   Figure . Similar) Figure . In geneH. pylori infection and HSD: Hp.Pos+HsDiet: 85 cases; Hp.Pos+Non-HsDiet: 102cases; Hp.Neg+HsDiet: 33 cases; and Hp.Neg+Non-HsDiet: 25 cases. Expression of SGK1 in T cells in each group was compared. Hp.Pos+HsDiet patients had the highest Lnc-SGK1 and SGK1 expression among these four groups. There was no significant difference between Hp.Pos.+Non-HsDiet and Hp.Neg.+HsDiet, which might implied that H. pylori infection and HSD contribute equally to overexpression of SGK1 and Lnc-SGK1. The Hp.Neg.+Non-HsDiet group had the lowest expression of lnc-SGK1and SGK1, while still significantly higher than in the healthy controls  and >0.173 . The overall survival rate was investigated based on follow-up data  between these two groups. Therefore, Lnc-SGK1 expression in serum of GC patients is not an ideal prognostic indicator in GC  in the healthy control group indicated that 0.173 was the threshold from discriminating normal from elevated systemic levels in the serum. The 245 GC patients were divided into two groups accordingly: ≤0.173 , Lnc-SGK1high+Hp (n = 48), Lnc-SGK1high+HSD (n = 45), Lnc-SGK1low+NHp+NHSD (n = 65), Lnc-SGK1low+NHp (n = 12) and Lnc-SGK1low+NHSD (n = 16). We assessed the 5-year survival rate in the four groups with >40 cases. The 5-year survival rate in the Lnc-SGK1high+Hp+HSD group was only 10.27%, which was significantly lower than in the other three groups. There was no significant difference in the 5-year survival rates between the Lnc-SGK1high+Hp and Lnc-SGK1high+HSD groups. The 5-year survival rate in the Lnc-SGK1low+NHp+NHSDgroup was significantly higher than in the other three groups , and decreased using Lnc-SGK1+Hp and Lnc-SGK1+HSD . However, sensitivity and specificity were better than for H. pylori infection and HSD, which are well-known diagnostic indices of GC   of 0.414(95% CI: 0.300–0.528). The diagnostic value was enhanced by combining with either ) Figure . These rH.pylori, which accounts for >60% of cases [H. pylori infection. Salt has been found to increase the growth and action of H. pylori, thus increasing the risk of cancer [H. pylori infection and HSD were related to Lnc-SGK1 and SGK1 expression in T cells of GC.GC is defined as cancer that forms in the tissues lining the stomach. Globally, GC is the fifth leading cause of cancer and the third leading cause of cancer mortality, comprising 7% of cases and 9% of deaths. In 2012 GC occurred in 950,000 people and caused 723,000 deaths . The mosof cases , 27. HSDf cancer , 29. HowH. pylori is specifically adapted to survive in the gastric environment, resulting in the development of gastritis, and recruitment of neutrophils, followed by B and T lymphocytes, macrophages and plasma cells. Consequently, large amounts of reactive oxygen or nitrogen species, involved in epithelial cell damage and carcinogenesis, are generated. However, inflammation is variable, including Th1, Th2, Th17 and regulatory T cell responses [H. pylori-induced chronic atrophic gastritis is characterized by marked infiltration of Th1 cells, However, Th2 and Th17 were more seriously associated with development of GC. Besides the Th1 response, the Th2 response was still observed in human GC, and was related to the development of H. pylori-related cancer. Kido et al. have reported that H. pylori can directly trigger epithelial cells to produce thymic stromal lymphopoietin, and in turn mediate dendritic cell activation, which is involved in the Th2 responses that trigger B-cell activation in H. pylori-induced gastritis. In our study, the Th2 and Th17 response was confirmed to be dominant in H. pylori-positive gastric cancer, which is linked to Lnc-SGK1 and SGK1 was overexpression. In addition, Although T cell Lnc-SGK1 expression was related to tumor size and metastasis, Lnc-SGK1 alone was not sufficient to determine the prognosis of GC. However, Lnc-SGK1 expression in T cells could be a good prognostic indicator in GC when combined with other factors: either H. pylori infection or HSD.esponses –32. Alth+H. pylori strains [H. pylori infection and HSD can serve as an ideal diagnostic indicator in GC. Expression of Lnc-SGK1 was associated with Th2 and Th17 response in GC patients with H. pylori infection and/or HSD. Previous studies have suggested the mechanism of the effect of SGK1 on T cell differentiation. Heikamp et al. (2004)have shown that expression of SGK1 in T cells promotes Th2 differentiation. SGK1 is indispensible in upregulating pathogenic IL-23-dependent Th17 cells [HSD can increase the carcinogenesis induced by enhanced gastritis and CagA expression. HSD causes severe gastritis, high gastric pH, increased parietal cell loss, increased gastric expression of IL-1β, and decreased gastric expression of hepcidin and hydrogen potassium ATPase , compared to individuals on a regular diet . These r strains . In the 17 cells .H. pylori infection and HSD can serve as ideal diagnostic indicator in GC.In summary, we investigated the expression of Lnc-SGK1 in human GC, and showed that Lnc-SGK1 in combination with A total of 245 paired GC and adjacent tissues were obtained at the time of surgical resection between January 2009 and December 2015 at the People's Hospital of Kunshan City, China. Samples were collected in accordance with the principles expressed in the Declaration of Helsinki. Written documented informed consent for gene expression analyses of all tissues was obtained from all patients prior to surgery or endoscopy examination. The study and consent procedures were approved by the Ethics Committee of the First People's Hospital of Kunshan City. Judgment of high-salt diet (HSD) intake of GC patients was obtained by questionnaire. In brief, GC patients who whether belong to HSD population was based on Nutrition and Health Survey of Chinese in 2002 published by Chinese ministry of Healthy, people who intake salt over 10g was classified into HSD patients. The patients whether infected with Hp can be determined by Carbon 13 breath test. The detailed clinical characteristics of the 245 GC patients are listed in Table Fresh GC tissues were washed twice in RPMI 1640. Fatty, connective and necrotic tissue was removed. Tissues were minced into 1–2-mm pieces in RPMI 1640, transferred into 15- or 50-ml conical tubes, and incubated with triple enzyme digestion medium containing DNase (30 U/ml), hyaluronidase (0.1 mg/ml), and collagenase (1 mg/ml) for 2 hours at room temperature with gentle shaking. Tissues were resuspended in 10 ml RPMI 1640 and filtered through a 70-μm cell strainer (BD Pharmingen). Tissues trapped by the strainer wereplaced into individual wells containing 1 ml T-cell growth medium in a 24-well plate for isolation by T cell isolation kit (Miltenyi Biotec).PBMCs from GC patients and normal controls were isolated by using Ficoll Paque Plus (GE), according to the manufacturer's instructions, and further isolated by T cell isolation kit (Miltenyi Biotec).Reverse transcription reactions were performed using the SuperScript First-Strand Synthesis System (Invitrogen), and the RNA templates were treated with DNase to avoid genomic DNA contamination. To determine the relative level of cDNA in the reverse transcribed samples, real-time PCR analyses were performed using an Applied Biosystems 7300 Detection System. The primer sequences used for detecting SGK1 were (5′-3′) forward primer:AGGATGGGTCTGAACGACTTT and reverse primerGCCCTTTCCGATCACTTTCAAG for human SGK1, and the amplification length was 208 bp. Primer for Lnc-SGK1: Forward primer AGAGGACGCAGGAGATTGGA; Reverse primer CAAGGCTGAAGCATCTCCGTA. The amplification length was 198 bp. The primer sequences used for detecting GAPDH were (5′-3′) forward primer:TGTGGGCATCAATGGATTTGG and reverse primerACACCATGTATTCCGGGTCAAT for human GAPDH, and the amplification length was 116 bp, which served as an internal control. The primers were synthesized by Genscript. Real-time PCR reactions were performed in accordance with the instructions of the SYBR Premix Ex Taq kit (Takara). Data were normalized tothe GAPDH levels in the samples.All the tissues were removed and fixed in 4% paraformaldehyde overnight at 4°C, processed, and sectioned at 5-μm thickness. The sectioned slides were stained immunohistochemically for SGK1 (purchased from Abcam) following routine protocols.For intracellular cytokine staining, cells were stimulated at 37° C for 5 hours with a Leukocyte Activation Cocktail (BD Pharmingen). Cells were then stained with surface markers, fixed, and permeabilized with IntraPre Reagent (Beckman Coulter), and finally stained with intracellular markers. Data were acquired on FACSVantage SE and analyzed with CellQuest software. Fluorochrome-conjugated monoclonal antibodies against SGK1, CD3, CD11b, INF-γ, IL-4 and IL-17A were purchased from BD Pharmingen.In this system, the BoxB RNA stem loop is fused to LOC105378009, LUNAR1 was used as a positive control . The plahttp://www.idtdna.com) using the chimeric 25-mer setting. The top 3 ASOs generated by the design tool were ordered and tested for knockdown efficiency for further investigation. Sequence for the control group in ASO assay was taken from Thomas Trimarchi, et al [Antisense oligonucleotides (ASOs) were designed using the IDT Antisense Design Tool , according to the manufacturer's instructions. Data were normalized by the activity of Renilla luciferase.T cell line Jurkat were culture in medium with IL-2 supplied. The Lnc-SGK1 promoter (either wild type or mutant type) luciferase reporter vector (pGL4 packaged) and mock pGL4 vector was added to 3×10ttest or Mann–Whitney U test, as appropriate. Risk score analysis was performed to investigate the effectiveness of the three-factors signature for tumor occurrence. Frequency tables and receiver operating characteristic (ROC) curves were then used to evaluate the diagnostic effects of the factors. All statistical analyses were performed using SPSS version 13.0, and two-tailed t tests were applied to all data unless otherwise specified, with P < 0.05 considered statistically significant.The results are expressed as mean±SD. Comparisons between two groups were performed using Student's The authors declare no competing financial interests.


The androgen receptor (AR) surface-directed antagonist MJC13 inhibits AR function and proliferation of prostate cancer (PC) cells. These effects are related to arrest of an AR/chaperone complex in the cytoplasm. Here, we compared MJC13 and classic AR antagonists such as flutamide and bicalutamide. Microarray analysis and confirmatory qRT-PCR reveals that MJC13 and flutamide inhibit dihydrotestosterone (DHT)-dependent genes in LNCaP PC cells. Both compounds are equally effective on a genome wide basis and as effective as second generation AR antagonists  at selected genes. MJC13 inhibits AR binding to the prostate specific antigen (PSA) promoter more strongly than flutamide, consistent with different mechanisms of action. Examination of efficacy of MJC13 in conditions that reflect aspects castrate resistant prostate cancer (CRPC) reveals that it inhibits flutamide activation of an AR mutant (ART877A) that emerges during flutamide withdrawal syndrome, but displays greatly restricted gene-specific activity in 22Rv1 cells that express a constitutively active truncated AR and is inactive against glucocorticoid receptor (GR), which can co-opt androgen-dependent signaling networks in CRPC. Importantly, MJC13 inhibits AR interactions with SRC2 and β-catenin in the nucleus and, unlike flutamide, strongly inhibits amplification of AR activity obtained with transfected SRC2 and β-catenin. MJC13 also inhibits DHT and β-catenin-enhanced cell division in LNCaP cells. Thus, a surface-directed antagonist can block AR activity in some conditions in which a classic antagonist fails and may display utility in particular forms of CRPC. Androgen receptor (AR) mediates growth promoting effects of androgens in prostate cancer (PC) and is a major target for therapeutics –4. Androin vitro and AR activity in cultured cells. X-ray screening revealed that useful ligands clustered in a novel AR surface, BF-3, which comprises a shallow cleft formed between helices (H) 1 and 9 and the H3–H4 loop [Effective blockade of AR action in CRPC should inhibit disease recurrence and extend patient survival  and seve–H4 loop . BF-3 is–H4 loop . Further–H4 loop . In addi–H4 loop . Althoug–H4 loop ,26. ThusThe AR inhibitor MJC13 blocks FKBP52-stimulated AR activity in yeast and cell culture but does not interact with FKBP52 and was unable to displace androgens from the AR pocket . InsteadMammalian expression vectors for human AR, SRC-2, MMTV-luc, human β-catenin, Gal4-AR LBD and Gal4-tk-luc were described previously ,27,28. AHEK293T cells (purchased from ATCC) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing phenol red and supplemented with 10% fetal bovine serum. LNCaP cells (purchased from the ATCC) and 22RV1 cells  were maintained in RPMI 1640 containing phenol red and supplemented with L-glutamine and 10% fetal bovine serum. For luciferase assays, cells were switched to medium with commercial charcoal stripped serum (Sigma) one day prior to transfection and were maintained in this medium after hormone induction. Cells were transfected using Fugene HD Transfection reagent (Roche) and luciferase assays performed as described [LNCaP cells were treated +/- DHT, +/- Flutamide, and +/- MJC-13 at indicated concentrations for 24h. Microarray hybridizations were performed as described . Arrays 5'-GCCTGGATCTGAGAGAGATATCATC-3'; 5’-ACACCTTTTTTTTTCTGGATTGTTG-3’), which amplify regions spanning the PSA androgen response element (ARE). IgG was used as an immunoprecipitation control.LNCaP cells were treated +/- 1nM DHT, +/- 1μM Flutamide, or +/- 30μM MJC-13 for 16h. ChIP was performed by SimpleChip Enzymatic Chromatin IP Kit  according to manufacturer’s instructions. 10% (v/v) of the supernatant was saved as ‘input’ chromatin prior to immunoprecipitation. Anti-AR antibody (Santa Cruz Biotechnology) was used for immunoprecipitation. Immunoprecipitated DNA and input-sheared DNA were subjected to PCR using primer pair . Cells were treated +/- hormones and antagonists as mentioned above and described in individual Figure Legends. Proteins were subjected to Western blot analysis with anti-AR (Santa Cruz Biotechnology), anti-SRC-2 (Bethyl Laboratories), anti-β-catenin , and anti-GAPDH  and detected with an ECL kit (Amersham Pharmacia).4cells/well. Cells were infected with adenovirus expressing β-catenin (Ad-β-catenin) or null adenovirus (Ad-Null) at multiplicity of infection = 50, maintained in normal culture media with steroid depleted serum and treated with 1nM DHT, 1μM Flutamide, 1μM bicalutamide or 30μM MJC13 for 24 h. After 24 h, XTT solution was added to each well and cells were incubated for 4 h at 37°C, the absorbance was measured using an ELISA reader at a wavelength of 450 nm.Adenovirus that expresses mouse wild type β-catenin was purchased from Applied Biological Materials. Cell proliferation was assessed by XTT Cell Viability Kit  according to manufacturer’s instructions. LNCaP cells were plated in 96-well plates at 10All results are means ± SD. Statistical analysis was performed using GraphPad Prism software . Comparisons of groups were performed using ANOVA followed by a post hoc multiple comparisons test. P ≤ 0.05 was considered statistically significant. All experiments were performed at least three times. Throughout the manuscript, the following convention was used to denote levels of statistical significance ***,P < 0.001; **,P < 0.01; *,P < 0.05.We first compared effects of increasing doses of flutamide and MJC13 on androgen (DHT)-dependent reporter genes in the presence of 1nM DHT in prostate cancer and non-prostate cell lines. Representative results with a minimal ARE responsive reporter gene are shown in We next determined effects of both AR antagonists on the endogenous androgen regulated gene program using microarray analysis in LNCaP cells . AnalysiGiven strong similarities between actions of flutamide and MJC13 on DHT response in LNCaP, we verified that the ligands act in different ways. ChIP analysis of AR binding to the PSA ARE region  revealedSince we observed inhibitory effects of AR antagonists on basal ARE-dependent reporter gene activity in LNCaP cells , we applLNCaP cells express an AR mutant (T877A) that has been reported to display partial agonist activity in the presence of flutamide . The ARTWe next compared activities of both compounds in 22RV1 cells, which express wild type AR and a truncated AR variant with constitutive activity . We obseDespite the fact that very high doses of MJC13 have previously been shown to inhibit GR in a yeast system , we founWe next compared abilities of flutamide and MJC13 to inhibit AR/coactivator interactions. We performed co-immunoprecipitations from LNCaP cells with anti-AR antibody in the presence and absence of DHT and antagonists. SRC2 associated with AR in the presence of DHT . While fSince SRC2 overexpression and β-catenin activation may occur in CRPC, we determined whether overexpression of either AR coactivator influences flutamide or MJC13 sensitivity. To do this, we performed transient transfections with relatively low amounts of AR expression vectors to highlight coactivator effects, as previously described . As expeFinally, we examined effects of MJC13 on androgen-dependent LNCaP cell growth +/- β-catenin . As was We showed here that two different classes of AR antagonists display similar effects on androgen responsive genes in LNCaP cells in the presence of DHT. MJC13, which acts at the AR surface, inhibits DHT response at standard ARE-dependent reporters and androgen responsive genes and, further, inhibitory effects are indistinguishable from the classic AR antagonist flutamide. There were gene-specific variations in blockade of DHT response. For example, flutamide and MJC13 only inhibit DHT response at the PSA gene by about 60% but block the highly induced ORM1 and ORM2 genes by close to 100%. We also observed one case in which MJC13 was moderately more effective than flutamide (TRPM8). Notably, however, we did not detect any DHT-responsive genes that were unaffected by either of the two antagonists. Two lines of evidence presented in this paper confirm previous suggestions that MJC13 and flutamide work in different ways. Flutamide did not display significant effects on DHT-dependent AR binding to the PSA ARE whereas MJC13 almost completely inhibited AR binding. Further, both compounds blocked AR interactions with SRC2, but MJC13 was uniquely able to inhibit AR interactions with β-catenin in the presence of DHT. Thus, optimal doses of flutamide and MJC13 result in effective and specific inhibition of AR activity and effects of flutamide and MJC13 are almost indistinguishable at the level of highly DHT induced genes.Our results differ from a previous comparison of a classic thyroid hormone (TH) receptor (TR) antagonist (NH-3) and a compound that blocks TR activity via covalent attachment to TR AF-2 (SJ-AK) . Here, bThe two AR antagonists exhibited no major effects on gene expression in the absence of DHT. Microarray analysis revealed few statistically significant changes in gene expression in the absence of DHT and qRT-PCR analysis confirmed this impression for flutamide and also showed that MJC13 could inhibit basal activity of selected genes, such as PSA and TMPRSS2 see  and otheWhile not the main focus of this paper, our results also reveal complex effects of flutamide in LNCaP. As mentioned, LNCaP cells express an ART877A mutant that has confers the capacity to respond to flutamide as a partial agonist , 35. We Reasons that we do not detect flutamide-dependent induction of endogenous genes in the presence of the ART877A mutant in LNCaP are not clear but our results are not outside of the norm. Some groups have detected weak flutamide-dependent induction of selected genes, but not others, in conditions in which high levels of DHT induction are obtained see, for example, . Other gThe fact that flutamide and MJC13 work in different ways raises the exciting possibility that surface-directed antagonists could block AR action in several conditions in which classical antagonists are ineffective. Although we were not able to detect flutamide partial agonist effects on gene expression in LNCaP, transfection assays revealed that MJC13 inhibits flutamide action through ART877A mutant . This maPerhaps more strikingly, MJC13 remains effective in the presence of overexpressed coactivators and, notably, β-catenin. While flutamide blocks AR interactions with SRC2 in co-immunoprecipitations, SRC2 overexpression overcomes flutamide blockade. This effect is probably related to the inability of small ligands such as flutamide to induce a stable inactive AF-2 conformation, thus permitting overexpressed AR cofactors to overcome this inhibitory effect . Flutamiin vitro (not shown). Our accompanying paper indicates that β-catenin cooperates with FKBP52 to stimulate AR and we therefore suggest that this effect may be related to preferential inhibition of DHT-dependent changes in the AR/FKBP52 complex and nuclear translocation rather than direct effects on AR/β-catenin binding [We do not know how MJC13 blocks AR/β-catenin interactions but emphasize that we do not think that MJC13 directly blocks AR/β-catenin interactions in the presence of DHT because we have been unable to demonstrate effects of MJC13 upon DHT-AR/β-catenin interactions in pulldowns  binding . In eithS1 FigResults of transfection analysis showing basal activity of the ARE-Luc reporter in LNCaP Cells +/- Flutamide and MJC13. Transfections employed 200ng of ARE-luc and 100ng of β-gal.(TIF)Click here for additional data file.S2 Fig(A-F) PGC-1, ORM1, ORM2, WDR76, KLK2, TRPM8. Note that MJC13 displays more potent inhibitory effects at the latter two genes.qPCR analysis of LNCaP cells extracts treated +/- DHT, +/- Flutamide or MJC13. The data are representative of at least three independent experiments. All values represent the mean ± SD of triplicate samples. (TIF)Click here for additional data file.S3 FigInformation from microarray analysis, presented as in (TIF)Click here for additional data file.S4 Fig qPCR analysis of 22RV1 cells extracts treated +/- DHT (1nM), +/- Flutamide (1μM), or +/- MJC13 (30μM) for 24 hours as in (TIF)Click here for additional data file.


Testosterone and DHT treatment increased tissue non-specific alkaline phosphatase (Alpl) mRNA expression. Testosterone-induced calcification was blunted in VSMC-specific AR-ablated (SM-ARKO) VSMCs compared to WT. Consistent with these data, SM-ARKO VSMCs showed a reduction in Osterix mRNA expression. However, intriguingly, a counter-intuitive increase in Alpl was observed. These novel data demonstrate that androgens play a role in inducing vascular calcification through the AR. Androgen signalling may represent a novel potential therapeutic target for clinical intervention.Vascular calcification powerfully predicts mortality and morbidity from cardiovascular disease. Men have a greater risk of cardiovascular disease, compared to women of a similar age. These gender disparities suggest an influence of sex hormones. Testosterone is the primary and most well-recognised androgen in men. Therefore, we addressed the hypothesis that exogenous androgen treatment induces vascular calcification. Immunohistochemical analysis revealed expression of androgen receptor (AR) in the calcified media of human femoral artery tissue and calcified human valves. Furthermore,  Being male is associated with an increased risk of cardiovascular disease23457Testosterone is the primary androgen in men. The biological actions of testosterone, and particularly of nonaromatizable dihydrotestosterone (DHT), are predominantly mediated by the androgen receptor (AR)131512i) accelerates this phenotypic trans-differentiation, evident in the loss of characteristic smooth muscle markers and the development of osteoblastic features, such as the expression of tissue-nonspecific alkaline phosphatase (TNAP), sodium-dependent phosphate co-transporter (PiT-1), osteocalcin and osteopontin, and osteocyte markers including sclerostin and podoplanin (Pdpn/E11)25i), matrix Gla protein (MGP) and fetuin A27Vascular calcification is a life-threatening complication of cardiovascular disease, affecting tissues including arteries, heart valves and cardiac muscle19202124in vitro studies have suggested an inhibitory effect of testosterone on vascular calcificationin vitro calcification studies in C57BL/6 and SM-ARKO mice to address the hypothesis that testosterone induces vascular calcification through stimulation of AR.The prevalence of vascular calcification is sexually dimorphic, showing a relative increase in men compared to women, which suggests possible hormonal influenceTo investigate the role of the AR in vascular calcification, a critical and advanced phenotype of atherosclerosis, localisation studies were undertaken. Calcification of human femoral artery and aortic valve tissues was confirmed by alizarin red and von kossa staining  I-II. ThIsolated VSMCs were positively stained with alpha smooth muscle actin (SMA) antibody , red. ARi levels, VSMCs were cultured in growth medium containing high Pi (3 mM) as previously described33i medium for up to 9 days. A minimum concentration of 10 nM testosterone treatment significantly increased calcium deposition of VSMCs at 9 days, as evaluated by HCl leaching   . No chanobserved .Esr2 mRNA expression in SM-ARKO mice versus WT controls  mice. Protein expression studies confirmed AR ablation in the VSMCs . Furthernaltered . Consistnaltered , 100 nM naltered . ConsistApoptosis plays an important role in vascular calcification, we therefore examined the effect of testosterone treatment on VSMC apoptosis. Alamar blue assay showed no effect of either testosterone or DHT on cell viability . DAPI stWhilst testosterone deficiency has been linked with cardiovascular disease in men7in vitro investigations revealed that exogenous testosterone and DHT treatment both had striking effects on the induction of VSMC calcification. A concomitant increase in expression of Alpl, a recognised regulator of osteoblastic differentiation and matrix mineralisation of VSMCs was observed. Indeed, there is a substantial body of evidence linking TNAP elevation with vascular calcification. TNAP upregulation has been observed in calcified coronary atherosclerotic plaques37in vitro study has reported inhibitory effects of androgens on VSMC calcificationOur in vitro studies demonstrated that AR is up-regulated in VSMCs by androgen treatment. This is in agreement with reports of increased local AR expression in the innominate artery in response to testosterone and DHT-induced calcification in the ApoE mouse modelAndrogens act mainly through transcriptional control of target genes mediated by AR14Osterix expression was observed in SM-ARKO VSMCs, suggesting androgen receptor may regulate osteogenic differentiation of VSMCs. Unexpectedly, altered expression of the calcification regulator Mgp and Alpl was also noted in SM-ARKO cells. Further studies are required to investigate their roles in AR-mediated calcification.The utilisation of a novel vascular selective ARKO mouse model was central to this investigation. The employment of a cell-specific mouse line with AR deleted from VSMCsThis study reveals AR expression in diseased vascular tissue and highlights a potential link between pharmacological androgen use and vascular calcification. We note that this is achieved in a small sample, and these data need to be extended with further samples in future investigations. However, the clear demonstration that androgens directly act through the AR to mediate pro-calcificatory effects in VSMCs may have important health ramifications for patients with elevated AR expression in their vascular tissues and for the use of ART. These data also have strong implications for the role of androgens in aortic valve calcification, which independently predicts mortality in aortic stenosis45in situ thrombosis of typical atherosclerotic femoro-popliteal peripheral arterial disease. He was an ex-smoker with no history of any endocrine disorder (including diabetes) and had an entirely normal masculine phenotype. He was on no hormonal treatment but was receiving standard therapy for atherosclerosis, namely aspirin and a HMG-CoA reductase inhibitor (statin). He had normal renal function and so the aetiology of the calcification in his femoral artery was associated with atherosclerosis. Serum testosterone levels were not measured. Human valve tissues were obtained with appropriate ethical approval from patients undergoing valve replacement surgery (Ethics No: 13/ES/0126). The control valve was from a 79 year old woman with aortic incompetence and generalised aortic dilatation with ascending aorta involvement. There was minor to moderate left ventricular impairment. The calcified valve was from a 67 year old woman with severe aortic stenosis detected by echocardiography. The aortic valve was removed at the time of aortic valve replacement operation, and carefully preserved the integrity of the valve architecture. An informed consent was given for the use of the human tissue and this study was performed in conformation with the declaration of Helsinki.Arterial samples were obtained with appropriate ethical approval from a patient undergoing amputation for critical limb ischaemia (Ethics Number: 13/ES/0126). Research ethics committee approval  and the written and informed consent of all participants were obtained. The sample was from an amputated leg from a man who had unsalvagable critical limb ischaemia secondary to The AR antibody  was from Santa Cruz Biotechnology . Testosterone and DHT were obtained from Sigma . The SMA antibody  was from sigma. DAPI, Alexa Fluor@488 donkey-anti goat antibody  and Alexa Fluor@594 goat-anti mouse antibody  were from Invitrogen .Mice with selective ablation of AR from VSMCs (SM-ARKO) were generated and genotyped as previously described−1 trypsin (Invitrogen) for 10 min in order to remove any remaining adventitia and endothelium. After a further overnight incubation at 37 °C in a humidified atmosphere of 95% air/5% CO2 in α-MEM medium (Invitrogen) supplemented with 10% Fetal Bovine Serum (Invitrogen) and 1% gentamicin (Invitrogen), tissues were digested with 425 U/ml collagenase type II  for 5 hrs. Medial cells were released and cell suspensions were centrifuged at 2000 g for 5 min. The cell pellet was washed and resuspended in the above mentioned culture medium. Isolated VSMCs were cultured with growth medium for two passages in T25 tissue culture flasks  coated with 0.25 μg/cm2 laminin (Sigma) to promote maintenance of the contractile differentiation stateMice were euthanized by cervical dislocation. Primary murine VSMCs were isolated as previously described25334/cm2 in multi-well plates. Calcification was induced as previously described2533i)  (Sigma), to reach a final concentration of 3 mM Pi. VSMCs were incubated for up to 9 days in 95% air/ 5% CO2 and the medium was changed every third/fourth day. Testosterone (1–100 nM) or DHT (1–100 nM) were added at day 0, according to previous publications14Primary VSMCs were seeded in growth medium at a density of 1.5 × 10Calcium deposition was quantified as previously described3448RNA was extracted using RNeasy total RNA , according to the manufacturer’s instructions. RNA was reverse transcribed and specific cDNAs were semi-quantified by end point PCR or quantified by real-time PCR using the SYBR green detection method as previously reported33342. Optical density absorbance was analysed at wavelengths of 570 nm and 620 nm using a spectrophotometer .VSMCs were grown on 96-well plates at a density of 5,000 cells/ well. Alamar blue assay was performed as previously describedApoptotic VSMCs were determined by manually counting pyknotic nuclei after staining with DAPI (Invitrogen) as previously describedo C. After washing, cells were incubated with Alexa Fluor@488 donkey-anti goat antibody and Alexa Fluor@594 goat-anti mouse antibody (Invitrogen) for 1 hr in the dark. Glass coverslips were mounted onto slides with Prolong®Gold Anti-Fade Reagent contained DAPI (Invitrogen). Fluorescence signal was detected under a Leica fluorescence microscope .VSMCs were seeded on glass coverslips in 12-well plates at a density of 50, 000 cells/well. Following confluence, VSMCs were serum-restricted for 24 hrs and treated with contro or 100 nM testosterone for 48 hrs. Cells were fixed with 4% paraformaldehyde and washed with PBS. The fixed cells were permeabilised with 0.3% Triton-X 100 (Sigma) and incubated with anti-AR antibody (Santa Cruz Biotechnology) and anti-SMA (Sigma) overnight at 4VSMCs were treated with 100 nM testosterone for 48 hrs. Cells were lysed in PhosphoSafe extraction buffer  containing “Complete” protease inhibitor cocktail  according to manufacturer’s instructions. Western blotting was performed as previously described2332oC with 2 μg IgG/ml anti-AR antibody (Santa Cruz Biotechnology). The sections were then washed in PBS, incubated with diluted biotinylated secondary antibody (1:200 dilution) for 30 min. After washing in PBS for 5 min, the sections were incubated for 30 min with VECTASTAIN® ABC Reagent (Vector Labs). The sections were then incubated with DAB substrate reagent  until the desired stain intensity developed. The sections were finally dehydrated, counterstained with haematoxylin and eosin and mounted in DePeX. Control sections were incubated with non-immune goat IgG (2 μg IgG/ml) in place of the primary AR antibody.Tissues were fixed in 10% neutral buffered formalin (NBF) for 24 hrs before being dehydrated and embedded in paraffin wax and sectioning at 4 μm using standard procedures. Sections were de-waxed in xylene and stained with Von Kossa and alizarin red (Sigma) to visualise phosphate and calcium deposition, respectively. Immunohistochemistry was performed using the VECTASTAIN ABC Kit (Goat IgG)  according to manufacturer’s instructions. Sections were de-waxed in xylene and de-masked with citric acid based antigen unmasking solution (Vector Labs). Endogenous peroxidase and non-specific antibody binding were blocked before overnight incubation at 4 General Linear Model analysis and the Students t-test were used to assess the data. All data are expressed as the mean + /− S.E.M. Statistical analysis was performed using Minitab 16. P < 0.05 was considered to be significant.How to cite this article: Zhu, D. et al. Ablation of the androgen receptor from vascular smooth muscle cells demonstrates a role for testosterone in vascular calcification. Sci. Rep.6, 24807; doi: 10.1038/srep24807 (2016).


Reduced cardiac β-adrenoceptor (β-AR) expression and cardiovascular dysfunction occur in models of hyperglycemia and hypoinsulinemia. Cardiac β-AR expression in type-2 diabetes models of hyperglycemia and hyperinsulinemia, remain less clear. This study investigates cardiac β-AR expression in type-2 diabetic Zucker diabetic fatty (ZDF) rats.Ex vivo biodistribution experiments with [3H]CGP12177 were performed in Zucker lean (ZL) and ZDF rats at 10 and 16 weeks of age as diabetes develops. Blood glucose, body mass, and diet consumption were measured. Western blotting of β-AR subtypes was completed in parallel. Echocardiography was performed at 10 and 16 weeks to assess systolic and diastolic function. Fasted plasma insulin, free fatty acids (FFA), leptin and fed-state insulin were also measured.3H]CGP12177 was normal in hyperglycemic ZDF (17±4.1mM) compared to ZL, but reduced 16-25% at 16 weeks of age as diabetes and hyperglycemia (22±2.4mM) progressed. Reduced β-AR expression not apparent at 10 weeks also developed by 16 weeks of age in ZDF brown adipose tissue. In the heart, Western blotting at 10 weeks indicated normal β1-AR (98±9%), reduced β2-AR (76±10%), and elevated β3-AR (108±6). At 16 weeks, β1-AR expression became reduced (69±16%), β2-AR expression decreased further (68±14%), and β3-AR remained elevated, similar to 10 weeks (112±9%). While HR was reduced at 10 and 16 weeks in ZDF rats, no significant changes were observed in diastolic or systolic function.At 10 weeks, myocardial [3H]CGP12177 retention and β1- and β2-AR expression are inversely correlated with the progression of type-2 diabetes.Cardiac β-AR are reduced over 6 weeks of sustained hyperglycemia in type-2 diabetic ZDF rats. This indicates cardiac [ Type-2 diabetes mellitus (DM) and associated complications are increasing globally . Type-2 11C]hydroxyephedrine and positron emission tomorgraphy (PET) have identified a positive correlation between blood glucose, plasma NE and subsequent reductions in cardiac NET in hyperglycemic rats CGP12177 PET studies in humans have shown a decrease in cardiac β-AR in vivo in HF [3H]CGP12177 binding has been observed in the hearts of hyperglycemic STZ rats and corresponded with a reduction in β1-AR Western blotting [ex vivo biodistribution studies using [3H]CGP12177 and Western blotting. This downregulation is expected to develop with the deterioration of left ventricular function measured by echocardiography.4-(3-tert-Butylamino-2-Hydroxypropoxy)-Benzimidazol-2-One CPG12177) is a non-selective β-AR antagonist ,18. Due 177 is a blotting ,15. Echoblotting , but the3H]CGP12177 (specific activity 41.6 Ci/mmol) was purchased from Perkin Elmer Health Sciences . Antibodies against rat β1-AR (Ab3546) were purchased from AbCam . Antibodies against β2-AR (SC-570), β3-AR (SC-1473), and GAPDH (SC-32233) were purchased from Santa Cruz Biotechnologies . Secondary horseradish peroxidase conjugated IgG antibodies goat anti-rabbit (SC-2004), donkey anti-goat (SC-2020), and donkey anti-mouse (SC-2314) were also purchased from Santa Cruz Biotechnology.CGP12177 biodistribution studies providing non-tritiated samples for Western blotting and fed-state measurements. A trunk blood sample was collected during terminal biodistribution from fasted animals. Echocardiography was performed at 10 and 16 weeks to assess systolic and diastolic function.Fed state blood glucose and body mass were monitored over the duration of the study. Diet consumptions was measured and calculated at the terminal endpoints of 10 and 16 weeks of age. Rats from each group were excluded from the [Ex vivo biodistribution experiments were performed as described elsewhere [3H]CGP12177 in 200μL of saline and decapitated 30 min post-tracer injection. Trunk blood was collected and used to isolate plasma. Hearts, interscapular brown adipose tissue (BAT), and quadricep skeletal muscle were rapidly excised. Hearts were dissected into left and right atria, left and right ventricular free walls, and intraventricular septum. Samples (100mg) were processed for liquid scintillation counting as described elsewhere [2O2 were added to solubilized tissue in quaternary ammonium hydroxide  followed by 10ml liquid scintillation fluid  with glacial acetic acid (99+%) then counted using a Packard Tri-Carb liquid scintillation analyzer model 2100TR . Total uptake was expressed as [(cpm recovered/g tissue) / (cpm injected/g body mass)].lsewhere . Brieflylsewhere . BrieflyPlasma measures were performed in trunk plasma from fasted and/or fed animals. Insulin was measured in fed and fasted animals using the Rat High-Range Insulin ELISA kit  . Free faInsulin resistance was estimated using the homeostatic model assessment of insulin resistance (HOMA-IR), as defined by the equation: HOMA-IR = [(fasting glucose (mmol/L) x fasting insulin (μIU/ml)] /22.5 .ex-vivo biodistribution experiments, and to observed changes in β-AR subtype expression, Western blotting for cardiac β1-AR, β2-AR and β3-AR was performed at 10 and 16 weeks on whole heart homogenates. Hearts were rapidly removed, frozen in liquid nitrogen, hand-powdered and total protein lysate was extracted. Protein determination was performed by bicinchoninic acid assay. Protein was separated on 8% sodium dodecyl sulfate-polyacrylamide reducing gels and transfered to an Immobilin-P polyvinylidene fluoride membrane  CGP12177 was similar in ZL and ZDF animals across myocardial regions. No differences in [3H]CGP12177 binding were evident in BAT or skeletal muscle, while small but significant increases were observed in ZDF blood and plasma (3H]CGP12177 uptake in all myocardial regions of about 16–25% relative to ZL. Uptake was also 67% lower in ZDF BAT. Consistent with 10 weeks, no changes were observed in skeletal muscle uptake and small but significant increases were observed in ZDF blood (At 10 weeks, uptake of CGP12177 retention at 16 weeks of age but not at 10 weeks suggests β-AR expression in ZDF rats is inversely correlated with the progression type-2 DM. Reduced myocardial β-AR expression appears to be associated with reduced binding in BAT, which also exhibits high β-AR expression CGP12177 binding was not evident until 16 weeks as β1-AR immunoblotting declined, indicating [3H]CGP12177 binding in the heart depended predominantly on β1-AR expression. The redistribution of β2-AR from deep transverse tubules in the healthy heart to the cell surface following myocardial infarction suggests the observed decrease in β2-AR may reflect a loss of receptors expressed in the transverse tubules rather than the cell surface [2-AR expression observed by Western blotting did not appear to have a large impact on [3H]CGP12177 binding at the cell surface. The greater dependence of [3H]CGP12177 binding on cardiac β1-AR is also supported by the greater relative expression of β1-AR in the healthy rat heart of about 62:30:8 compared to β2- and β3-AR CGP12177 uptake early after the induction of hyperglycemia, but binding of [3H]CGP12177 to cardiac β-AR becomes reduced by up to 50% after 6 to 8 weeks of sustained hyperglycemia HHHHhjhsdjfbsdlvhbasekjfvbadsrjvkbasrejfbw[3H]CGP12177 binding in STZ compared to ZDF rats indicates that cardiac β-AR are inversely proportional to the magnitude of hyperglycemia, and their reduction may be further exacerbated by the development of hypoinsulinemia in diabetic animals.Reduced cardiac CGP12177 PET may also be useful to identify patients at risk of cardiac events and help to define a therapeutic window for intervention before overt diastolic or systolic dysfunction develop.A slower HR is commonly observed in ZDF rats ,41,42, b3H]CGP12177 to cardiac β-AR is reduced between 10 and 16 weeks of age as type-2 DM progresses in ZDF rats. Western blotting indicates that while cardiac β1- and β2-AR are reduced, β3-AR expression is increased. ZDF rats have a slower HR as early as 10 weeks of age, with no significant changes in %EF or diastolic filling by 16 weeks. While tritium biodistribution studies underscore the potential of CGP12177 as an imaging agent, further preclinical imaging studies using a [11C] labeled derivative are warranted.In conclusion, binding of [