Nuclear Androgen Receptor Levels Research Articles

An Autophagy-Targeting Chimera Induces Degradation of Androgen Receptor Mutants and AR-v7 in Castration-Resistant Prostate Cancer.

Genetic alterations play a pivotal role in various human diseases, particularly cancer. The androgen receptor (AR) is a crucial transcription factor driving prostate cancer (PCa) progression across all stages. Current AR-targeting therapies utilize competitive AR antagonists or pathway suppressors. However, therapy resistance often emerges due to AR mutations and AR splice variants, such as AR-v7. To overcome this, we developed ATC-324, an AR degrader using the innovative protein degradation technology platform AUTOphagy-TArgeting Chimera (AUTOTAC). ATC-324 was designed to comprise enzalutamide, an AR inhibitor, as a target-binding ligand and YT 6-2, a ligand of the autophagy receptor p62/SQSTM1, as an autophagy-targeting ligand. ATC-324 induces the formation of the AR/p62 complex, leading to autophagy-lysosomal degradation of AR. Importantly, ATC-324 effectively degrades AR mutants frequently detected in PCa and co-degrades AR-v7 as a heterodimer with full-length AR. ATC-324 reduces nuclear AR levels and downregulates the target gene expression of AR and AR-v7, leading to cytotoxicity in AR-positive PCa cells. We also provide evidence of the therapeutic potential of ATC-324 in vivo as well as ex vivo bone organ culture. Moreover, ATC-324 remains potent in enzalutamide-resistant PCa cells. These results demonstrate the potential of the AUTOTAC platform to target previously considered undruggable proteins and overcome certain drug resistance mechanisms.

Saw Palmetto may fight prostate cancer.

Saw Palmetto may fight prostate cancer.

Gene dosage changes in KCTD13 result in penile and testicular anomalies via diminished androgen receptor function.

Despite the high prevalence of hypospadias and cryptorchidism, the genetic basis for these conditions is only beginning to be understood. Using array-comparative-genomic-hybridization (aCGH), potassium-channel-tetramerization-domain-containing-13 (KCTD13) encoded at 16p11.2 was identified as a candidate gene involved in hypospadias, cryptorchidism and other genitourinary (GU) tract anomalies. Copy number variants (CNVs) at 16p11.2 are among the most common syndromic genomic variants identified to date. Many patients with CNVs at this locus exhibit GU and/or neurodevelopmental phenotypes. KCTD13 encodes a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3-ubiquitin-protein-ligase complex (BCR (BTB-CUL3-RBX1) E3-ubiquitin-protein-ligase complex (B-cell receptor (BCR) [BTB (the BTB domain is a conserved motif involved in protein-protein interactions) Cullin3 complex RING protein Rbx1] E3-ubiqutin-protein-ligase complex), which has essential roles in the regulation of cellular cytoskeleton, migration, proliferation, and neurodevelopment; yet its role in GU development is unknown. The prevalence of KCTD13 CNVs in patients with GU anomalies (2.58%) is significantly elevated when compared with patients without GU anomalies or in the general population (0.10%). KCTD13 is robustly expressed in the developing GU tract. Loss of KCTD13 in cell lines results in significantly decreased levels of nuclear androgen receptor (AR), suggesting that loss of KCTD13 affects AR sub-cellular localization. Kctd13 haploinsufficiency and homozygous deletion in mice cause a significant increase in the incidence of cryptorchidism and micropenis. KCTD13-deficient mice exhibit testicular and penile abnormalities together with significantly reduced levels of nuclear AR and SOX9. In conclusion, gene-dosage changes of murine Kctd13 diminish nuclear AR sub-cellular localization, as well as decrease SOX9 expression levels which likely contribute in part to the abnormal GU tract development in Kctd13 mouse models and in patients with CNVs in KCTD13.

Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer.

Nuclear localization of the androgen receptor (AR) is necessary for its activation as a transcription factor. Defining the mechanisms regulating AR nuclear localization in androgen-sensitive cells and how these mechanisms are dysregulated in castration-resistant prostate cancer (CRPC) cells is fundamentally important and clinically relevant. According to the classical model of AR intracellular trafficking, androgens induce AR nuclear import and androgen withdrawal causes AR nuclear export. The present study has led to an updated model that AR could be imported in the absence of androgens, ubiquitinated, and degraded in the nucleus. Androgen withdrawal caused nuclear AR degradation, but not export. In comparison with their parental androgen-sensitive LNCaP prostate cancer cells, castration-resistant C4-2 cells exhibited reduced nuclear AR polyubiquitination and increased nuclear AR level. We previously identified 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) in a high-throughput screen for its inhibition of androgen-independent AR nuclear localization in CRPC cells. The current study shows that CPPI is a competitive AR antagonist capable of enhancing AR interaction with its E3 ligase MDM2 and degradation of AR in the nuclei of CRPC cells. Also, CPPI blocked androgen-independent AR nuclear import. Overall, these findings suggest the feasibility of targeting androgen-independent AR nuclear import and stabilization, two necessary steps leading to AR nuclear localization and activation in CRPC cells, with small molecule inhibitors.

A Novel Small Molecule Targets Androgen Receptor and Its Splice Variants in Castration-Resistant Prostate Cancer.

Reactivation of androgen receptor (AR) appears to be the major mechanism driving the resistance of castration-resistant prostate cancer (CRPC) to second-generation antiandrogens and involves AR overexpression, AR mutation, and/or expression of AR splice variants lacking ligand-binding domain. There is a need for novel small molecules targeting AR, particularly those also targeting AR splice variants such as ARv7. A high-throughput/high-content screen was previously reported that led to the discovery of a novel lead compound, 2-(((3,5-dimethylisoxazol-4-yl)methyl)thio)-1-(4-(2,3-dimethylphenyl)piperazin-1-yl)ethan-1-one (IMTPPE), capable of inhibiting nuclear AR level and activity in CRPC cells, including those resistant to enzalutamide. A novel analogue of IMTPPE, JJ-450, has been investigated with evidence for its direct and specific inhibition of AR transcriptional activity via a pulldown assay and RNA-sequencing analysis, PSA-based luciferase, qPCR, and chromatin immunoprecipitation assays, and xenograft tumor model 22Rv1. JJ-450 blocks AR recruitment to androgen-responsive elements and suppresses AR target gene expression. JJ-450 also inhibits ARv7 transcriptional activity and its target gene expression. Importantly, JJ-450 suppresses the growth of CRPC tumor xenografts, including ARv7-expressing 22Rv1. Collectively, these findings suggest JJ-450 represents a new class of AR antagonists with therapeutic potential for CRPC, including those resistant to enzalutamide.

Androgen Receptor as a Biomarker of Oral Squamous Cell Carcinoma Progression Risk.

Oral squamous cell carcinoma (OSCC) is a cancer with poor prognosis due to therapy resistance, locoregional recurrences, and distant metastases. There is on increased interest in profiling the androgen receptor (AR) in cancer biology. The aim of this study was to compare AR and Ki-67 levels in the neoplastic epithelium and stroma between non-metastatic and metastatic stages of OSCC. Tissue specimens of 101 non-metastatic and 95 metastatic OSCC patients were analyzed by immunohistochemistry. More than 20% of AR-positive cytoplasmic staining of OSCC epithelium was significantly associated with nuclear AR levels in the epithelium and increased AR levels in the stroma. In metastatic OSCC patients, Ki-67 was significantly higher than in non-metastatic OSCC patients. More than 20% of AR-positive cytoplasmic staining in neoplastic OSSC epithelium is a significant predictor of OSCC progression risk.

Overcoming resistance to antiandrogens with a TGF-β RI inhibitor in preclinical mouse model of PCa.

288 Background: Epithelial-mesenchymal transition (EMT) is a significant contributor to PCa metastatic progression and therapeutic resistance in patients treated with the androgen receptor (AR) directed therapies. We previously demonstrated that aberrant TGF-β signaling accelerates prostate tumor progression in the TRAMP mouse model of tumorigenesis via selective effects on EMT. Methods: We hypothesize that the combination of the TGF-β receptor inhibitor, galunisertib (G), and enzalutamide (E) will perturb the interactive signaling between TGF-β and AR signaling affecting the phenotypic landscape of EMT. This perturbation may be exploited in our mouse model, towards enhanced anti-tumor efficacy in advanced castration-resistant PCa (CRPC). We treated 2-week old mice for two weeks with the G (75mg/kg) and/or E (30mg/kg) in combination and as single agents. Results: Treatment with G alone or in combination with E resulted in a significant reduction in prostate tumor weight without affecting total body weight. Immunohistochemical (IHC) and Western blot analysis showed that, while treatment with the G alone led to increased apoptosis and decreased cell proliferation, combination of G and E had significantly higher efficacy in inducing apoptosis and inhibiting cell proliferation than either E or G alone. As expected treatment with the G decreased the levels of nuclear Smad4 protein; the combination of G and E further decreased nuclear Smad4 expression. Furthermore the combination of G and E reversed phenotypic EMT to MET (mesenchymal-epithelial-transition), as assessed by the increase in E-cadherin among the prostate tumor cell populations. IHC and Western blot analysis also revealed that the combined treatment of G and E led to a significant decrease in nuclear AR levels compared to E-only-treated or vehicle-control tumors. Conclusions: These results provide significant insights as to the therapeutic impact of G to effectively impair the TGF-β signaling and overcome resistance of PCa patients to E by reversing EMT to potentially sensitize tumors to the antiandrogen effect. This study has major translational relevance; the combination of G and E may lead to synergistic anti-tumor impact in patients with CRPC.

Abstract 1303: In vivo analysis of EGFR family signalling as a bypass mechanism in prostate cancer

Abstract Background: Prostate cancers (PCa) rely on androgenic ligands and the androgen receptor (AR) for their growth and survival, making AR inhibition a predominant therapeutic strategy for these tumors. Some prostate tumors however fail this therapy due to ‘bypass’ mechanisms that emerge as a result of prolonged AR targeting. This in vivo study attempted to assess the expression and activation of the epidermal growth factor receptor (EGFR) family (whose role is well-documented in PCa) in response to androgen deprivation therapy (ADT). Methods: Nude mice were implanted (s.c.) with CWR22 tumors (human-patient-derived, androgen-dependent ‘AD’) and its castration-resistant (‘CR’) subline CWR22-Rv1 (relapsed CWR22). Androgen deprivation (i.e. AR inhibition) was achieved by surgical or ‘sham’ castration of mice. Tumors were analyzed (immunohistochemistry/immunoblot) for EGFR/ErbB2/ErbB3/AR proteins and proliferative/apoptotic markers. Results: Castration caused significant tumor regression in AD but not CR tumors. in vitro viability assays demonstrated that castration (mimicked by using charcoal-stripped serum, ‘css’) did not slow down CR cells to the same degree as it did AD cells. At baseline, intratumoral EGFR protein was unchanged in R22 tumors, ErbB2 levels decreased and ErbB3 protein increased in Rv1 tumors. Castration increased ErbB3 but not EGFR or ErbB2 proteins in CWR22 tumors. Phosphorylated forms of these receptors were generally difficult to detect but there was more phosphorylated ErbB3 protein in Rv1 tumors. Downstream of the EGFR family, there was less phosphorylated Erk but not Akt protein in CWR22-Rv1 tumors. Castration decreased Erk protein in AD tumors but increased it in CR tumors. Immunohistochemical quantification revealed that cytoplasmic EGFR and ErbB3 proteins were elevated in CR tumors but reduced in AD tumors. Castration greatly decreased Ki-67 staining in AD but not in CR tumors while the number of TUNEL-positive nuclei and intensity of PARP staining decreased in castrated CR but not in AD tumors. ErbB3 and AR proteins were significantly correlated with DNA damage and proliferation in CWR22 tumors but only nuclear AR levels and proliferation were significantly correlated in CR tumors. Conclusions: We conclude that androgen deprivation therapy may alter EGFR and ErbB3 protein levels and localization in androgen-dependent and castration-resistant tumors. The EGFR family is typically activated at the cell surface hence their presence and activity there, in response to castration, may initiate signalling pathways encouraging tumor cell proliferation and survival. Citation Format: Maitreyee K. Jathal, Thomas M. Steele, Salma Siddiqui, Benjamin A. Mooso, Leandro S. D’Abronzo, Christiana M. Drake, Paramita M. Ghosh. In vivo analysis of EGFR family signalling as a bypass mechanism in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1303.

The interaction between androgen receptor and PDGF-D in the radiation response of prostate carcinoma.

To determine the functional relationship between androgen receptor (AR) and PDGF D as it relates to the radiation response of PTEN-null prostate cancer (PCa) cells and the effect of enzalutamide on these interactions. Using murine PTEN-null prostate epithelial cell line and human prostate carcinoma LNCaP (PTEN-mutant) models, nuclear and cytosolic AR levels were determined by immunoblot analysis and the transcriptional activity of nuclear AR was assessed by RT-PCR analysis of its target genes with or without irradiation. Cell survival was evaluated by clonogenic assay or sulforhodamine B (SRB) assay upon irradiation in the absence or presence of the AR antagonist enzalutamide. PTEN loss resulted in upregulation of AR expression in a PDGF-D dependent manner and irradiation selectively increased the nuclear AR protein level and its activity in a murine cell model. When the functional significance of AR in cell survival was tested, treatment with enzalutamide resulted in radiosensitization of human LNCaP cells. Similarly to the murine model, PDGF-D overexpression increased the nuclear AR level and its transcriptional activity in LNCaP cells. PDGF-D over-expression was associated with radioresistance and enzalutamide treatment effectively reversed PDGF-D-mediated radioresistance in LNCaP cells. We have demonstrated that AR, a target of the PTEN and PDGF D-downstream signaling program, contributes to radiation resistance in human PCa cells. In addition, this study suggests that anti-androgens such as enzalutamide may serve as radiation sensitizers for the treatment of PCa patients, particularly so in patients with loss of PTEN or overexpression of PDGF-D.

The Interaction Between Androgen Receptor and PDGF-D in the Radiation Response of a Human Prostate Cancer Cell Line

The Interaction Between Androgen Receptor and PDGF-D in the Radiation Response of a Human Prostate Cancer Cell Line

Endostatin: A novel inhibitor of androgen receptor function in prostate cancer

Acquired resistance to androgen receptor (AR)-targeted therapies compels the development of novel treatment strategies for castration-resistant prostate cancer (CRPC). Here, we report a profound effect of endostatin on prostate cancer cells by efficient intracellular trafficking, direct interaction with AR, reduction of nuclear AR level, and down-regulation of AR-target gene transcription. Structural modeling followed by functional analyses further revealed that phenylalanine-rich α1-helix in endostatin-which shares structural similarity with noncanonical nuclear receptor box in AR-antagonizes AR transcriptional activity by occupying the activation function (AF)-2 binding interface for coactivators and N-terminal AR AF-1. Together, our data suggest that endostatin can be recognized as an endogenous AR inhibitor that impairs receptor function through protein-protein interaction. These findings provide new insights into endostatin whose antitumor effect is not limited to inhibiting angiogenesis, but can be translated to suppressing AR-mediated disease progression in CRPC.

Abstract 737: Assessment of cabozantinib activity in diverse prostate cancer xenograft models

Abstract Cabozantinib (cabo), an inhibitor of tyrosine kinases including MET, VEGFR2, RET, KIT, and AXL, is undergoing clinical investigation in patients with castration-resistant prostate cancer (CRPC). A phase II randomized discontinuation trial of cabo showed clinical activity in CRPC patients. However, not all patients responded to cabo, and most of the responding patients eventually experienced disease progression. The objective of our study was to evaluate the efficacy of cabo in a preclinical setting using xenograft models of CRPC with differential phenotypes to obtain insight into which tumor types might be sensitive to cabo, and to investigate mechanisms of action of this agent. We used LuCaP 35CR, a PTEN- ERG+ Rb+ CRPC patient-derived xenograft (PDX) that exhibits amplification of the androgen receptor (AR); LuCaP 96CR, a PTEN+ ERG- Rb+ CRPC PDX with high levels of intra-tumoral androgens; LuCaP 86.2, a PTEN- ERG+ Rb- CRPC PDX that expresses the AR v5-7 variant and is not responsive to endocrine therapy; and LuCaP 93, a PTEN- ERG- Rb- neuroendocrine CRPC PDX. Expression of cabo targets in these models was determined by qPCR. Animals were treated with 30 mg/kg cabo p.o. for six weeks (5d on/2d off), and tumor volume (TV), serum PSA and body weights were monitored. IHC and expression arrays were used to analyze effects of cabo. Cabo was effective in halting tumor growth in all four PDXs. Significant inhibition of TV was evident after one week of cabo treatment compared to control groups. PSA values were also significantly lower after one week of the treatment in all three adenocarcinomas. Furthermore, cabo treatment resulted in significant survival benefits in LuCaP 35CR, LuCaP 96CR and LuCaP 93 without significant decreases in body weight. IHC analysis showed decreased microvessel density in all cabo-treated groups vs control groups, suggesting effects on tumor environment. qPCR analysis showed that all models expressed some cabo targets though at different levels, but we did not observe any associations between magnitude of cabo effects and levels of the its targets in the tumors; inhibition of LuCaP 93, which expresses the highest levels of cabo targets, was similar to that of LuCaP 96CR, which expresses low levels of cabo targets. Our analyses of expression profiles of tumors following cabo treatment showed very little overlap between the models. However, E2F1 signaling, was inhibited by cabo in Rb- PDXs LuCaP 93 and LuCaP 86.2, but not in the Rb+ PDXs LuCaP 35CR and LuCaP 96CR. Also, expression of genes regulated by androgen was increased in the Rb- tumors and decreased (LuCaP 35CR) or was not altered (LuCaP 96CR) in the Rb+ tumors, and the levels of nuclear AR showed a similar pattern. Further investigations are ongoing to obtain more detailed insight into mechanisms of cabo effects. Citation Format: Lisha G. Brown, Holly M. Nguyen, Ilsa M. Coleman, Peter S. Nelson, Robert L. Vessella, Dana T. Aftab, Eva Corey. Assessment of cabozantinib activity in diverse prostate cancer xenograft models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 737. doi:10.1158/1538-7445.AM2014-737

Antiproliferative, antiandrogenic and cytotoxic effects of novel caffeic acid derivatives in LNCaP human androgen-dependent prostate cancer cells

Caffeic acid and its naturally occurring derivative caffeic acid phenethyl ester (CAPE) have antiproliferative and cytotoxic properties in a variety of cancer cell lines without displaying significant toxicity toward healthy cells, and are considered to be potential anticancer agents. However, little is known about their effects on prostate cancer cells. We synthesized and evaluated the effects of caffeic acid, CAPE (2) and 18 synthetic derivatives on cell viability and androgen-dependent cell proliferation, subcellular localisation and expression of androgen receptor (AR) and secretion of prostate-specific antigen (PSA) in LNCaP human hormone-dependent prostate cancer cells. Several synthetic derivatives of CAPE were strong, concentration-dependent cytotoxic agents in LNCaP cells with IC50 values in the 6.8–26.6μM range, potencies that were up to five-fold greater than that of CAPE (33.7±4.0μM). A number of caffeic acid derivatives were inhibitors of androgen-stimulated LNCaP cell proliferation with concomitant inhibition of DHT-stimulated PSA secretion. Compound 24 was the most cytotoxic and antiproliferative caffeic acid derivative (IC50 values of 6.8±0.3 and 2.4±0.8μM, respectively) inhibiting DHT-stimulated cell proliferation and PSA secretion statistically significantly at concentrations as low as 0.3μM. Exposure to DHT increased cytoplasmic and nuclear AR levels and co-treatment with increasing concentrations of compound 24 or CAPE (2), notably, further increased these levels. In conclusion, a number of synthetic derivatives of caffeic acid are potent inhibitors of androgen-dependent prostate cancer cell proliferation and viability, acting, at least in part, via an antiandrogenic mechanism that involves increased nuclear accumulation of (presumably inactive) AR.

The role of 20‐HETE in the regulation of androgen receptor nuclear translocation

Androgen has been shown to increase the synthesis of 20‐Hydroxyeicosatetraenoic acid (20‐HETE), a cytochrome P450 (CYP4A)‐arachidonate metabolite which in turn mediates cardiovascular effects of androgen including vascular dysfunction and hypertension. The mechanism underlying these interactions are unclear. We used LNCaP prostate cancer cell line the growth of which is androgen‐dependent. LNCaP cells produced high levels of 20‐HETE (290± 59 pg/mg) while in normal prostate epithelial cells 20‐HETE was undetectable. LNCaP cell growth was inhibited by HET0016, a selective CYP4A inhibitor, suggesting that 20‐HETE mediates the growth effect of androgen. Since androgen receptor (AR) nuclear translocation is critical for AR‐mediated growth and survival of prostate cancer, we examined whether 20‐HETE is involved in AR nuclear translocation. Treatment of LNCaP cells with 20‐HETE (100nM; 30min) showed a 2.5±0.48‐fold increase in nuclear AR levels. Since HSP90 is believed to tether AR in the cytoplasm towards a high affinity ligand binding conformation, we examined the effect of 20‐HETE on the HSP90‐AR association. 20‐HETE induced 2‐fold increase in cytoplasmic HSP90 association with AR. These results suggest that 20‐HETE, by increasing the association of HSP90 to AR, maintains AR in a high affinity ligand binding conformation and thereby increasing AR activation and translocation.

Inhibition of Glycogen Synthase Kinase-3β Counteracts Ligand-Independent Activity of the Androgen Receptor in Castration Resistant Prostate Cancer

In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from in vitro experiments that in castration-resistant prostate cancer (CRPC) cells the AR is able to translocate into the nucleus in a ligand-independent manner. The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.

Inhibitory mechanisms of the transcriptional activity of androgen receptor by resveratrol: Implication of DNA binding and acetylation of the receptor

Androgen receptor (AR) is a ligand-dependent transcription factor and plays a key role in the development of prostate cancer. Resveratrol, a polyphenolic compound, inhibits AR function and reduces the level of prostate-specific antigen (PSA), a notable target gene of AR. Here, we investigated the mechanisms by which resveratrol inhibits AR function. Although the protein levels of AR were decreased by resveratrol treatment for 24 h, the decrease could not fully account for the suppression of AR function. The total and the nuclear AR levels were not affected after incubation with 10 μM resveratrol for 3 h, whereas resveratrol inhibited the binding of AR to the enhancer region of PSA and decreased the acetylation of AR even at this early phase. Inhibition of transcription by resveratrol was weaker in the AR acetylation site mutant than in the wild-type. In later phase (24 h) after incubation with resveratrol, the ligand-induced nuclear accumulation of AR was markedly decreased by resveratrol. These data show that resveratrol inhibits DNA binding of AR, presumably by decreasing its level of acetylation and suggest that acetylation of AR is involved in its accumulation in the nucleus.

Studies on molecular mechanisms of growth inhibitory effects of thymoquinone against prostate cancer cells: role of reactive oxygen species

Thymoquinone (TQ), an active ingredient of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against a variety of experimental tumors. However, the precise mechanism of action of TQ is not known. We investigated the mechanism of action of TQ in androgen receptor (AR)-independent (C4-2B) and AR naïve (PC-3) prostate cancer cells, as models of aggressive prostate cancers. Exposure (24-48 h) to TQ (25-150 micromol/L) inhibited the growth of both C4-2B and PC-3 cells, with IC(50) values of approximately 50 and 80 micromol/L, respectively. Within one hour, TQ increased reactive oxygen species (ROS) levels (3-fold) and decreased glutathione (GSH) levels (60%) in both cell types. Pretreatment with N-acetylcysteine (NAC) inhibited both TQ-induced ROS generation and growth inhibition. TQ did not increase the activity of caspases and the caspase inhibitor, z-VAD-FMK did not decrease TQ-induced apoptosis. Furthermore, although TQ treatment resulted in the activation of Jun kinase (JNK), pretreatment with the JNK inhibitor, SP600125, did not protect cells from TQ. However, TQ significantly up-regulated the expressions of growth arrest and DNA damage inducible gene (GADD45alpha) and apoptosis-inducing factor-1 and down-regulated the expressions of several Bc12-related proteins, such as BAG-1, Bcl2, Bcl2A1, Bcl2L1 and BID. In C4-2B cells, TQ dose dependently inhibited both total and nuclear AR levels (4-5 fold) and AR-directed transcriptional activity (10-12 fold). Interestingly, this suppressive effect on AR was not prevented by NAC, which clearly suggested that TQ-induced cytotoxicity is not due to changes in AR regulation. These data suggest that TQ-induced cell death is primarily due to increased ROS generation and decreased GSH levels, and is independent of AR activity.

Nuclear and cytosol androgen receptor in androgen dependent dermatoses in female patients.

Determination of cytosol and nuclear androgen receptor (AR) was performed in females with androgen dependent dermatoses. 34 patients with acne, hirsutism and androgenetic alopecia were punch biopsied on day 21 of the menstrual cycle under local anesthesia within correspondent predilection sites. The tissue was snap frozen in liquid nitrogen and stored until receptor assays were performed. Overall evaluation of both, cytosol and/or nuclear AR was positive in 76% of all cases. In 86% of (10 out of 14) females with androgenetic alopecia, in 80% (8 out of 10) of patients with acne and 60% (6 out of 10) of hirsute females cytosol and/or nuclear AR were positive. No significant differences of cytosol androgen receptor levels became evident between the dermatoses. In contrast, nuclear androgen receptor levels showed a trend towards distinct differences with highest levels in hirsutism, followed by androgenetic alopecia and acne. Androgen stimulability thus seems to be superior in the first and minor in the latter. Comparison with previous studies on cytosol androgen receptor in androgen dependent dermatoses shows significantly higher number of positive results by additive nuclear receptor determination. Additional nuclear androgen receptor assay, thus gives a more complete picture of local hormone action.

ALTERATIONS IN ANDROGEN RECEPTORS AND ANDROGEN RECEPTOR mRNA LEVELS IN PROSTATE GLANDS OF VITAMIN E DIFICIENT RATS WITH AND WITHOUT CASTRATION AND ANDROGEN ADMINISTRATION

Vitamin E has been reported to be essential for germ cell development and maturation, gonadotropin secretion, steroid hormone synthesis and metabolism, and reproduction and fertility. The present study was designed to study if vitamin E deficiency changes prostate androgen receptor (AR) and AR mRNA levels. Male King-Holtzman rats were fed with vitamin E deficient diet and control rats were fed with Purina rat chow for 3 months. Two weeks prior to autopsy, other groups of rats were castrated and administered with testosterone. Prostate tissues were obtained for determination of nuclear AR levels and quantitation of mRNA encoding androgen receptors. Total cellular RNA was prepared by guanidine thiocyanate method and determined by Northern and slot blot analyses. Prostate androgen receptor levels in vitamin E deficient rats were significantly lower than in control animals. Androgen receptor mRNA levels were decreased in vitamin E deficient rats when compared to those of normal controls. However, prostate AR and AR mRNA levels were increased following castration, whereas AR and AR mRNA levels were decreased in castrated rats with androgen administration. These findings indicate that vitamin E deficiency causes a reduction of AR levels in rats with the decline of circulating androgen levels and a decreased transcription of AR mRNA which leads to a defective translation of AR protein, suggesting an important role of dietary vitamin E on prostate AR levels, AR mRNA expression, and reproduction and fertility.

Androgen Receptor Levels of Oral and Genital Ulcers and Skin Pathergy Test in Patients with Behçet’s Disease

Background: Hormonal factors have long been proposed to play a role in Behçet’s disease (BD). Male sex, systemic onset, HLA-B51 positivity and a younger age of onset in BD are associated with severer disease, and the disease generally runs a milder course in women. Vascular involvement is more common, and the skin pathergy test (SPT) is more strongly positive in men. BD rarely develops before puberty or after the age of 50 years. Clinical manifestations of the disease, with the exception of eye symptoms, tend to improve with time. Therefore, BD may be androgen driven to some degree. Objectives: We aimed to investigate androgen receptor (AR) levels of oral ulcers (OU), genital ulcers (GU) and SPT areas and compared them with those of adjacent normal-appearing skin/mucosa from patients with BD. Methods: Thirty-eight patients with BD (16 female, 22 male; mean ± SD age, 36.45 ± 10.2 years), diagnosed according to the criteria of the International Study Group for Behçet’s Disease, were included in the study with blind histological examination. Biopsies from OU of 10 patients, GU of 11 patients, SPT areas of 17 patients and adjacent (approximately 2 cm distant) normal-appearing skin/mucosa in patients with BD were performed. Nuclear AR levels were studied by an immunohistochemical technique, using monoclonal antibodies. The percentage of positively staining cells was recorded as the AR index (ARI). In addition, the prevalence and the positivity rate of SPT has also been evaluated. Results: ARI values in the lesional and control (non-lesional adjacent) skin/mucosa were found to be 14.5 versus 18% for OU, 28.7 versus 25.5% for GU and 36.3 versus 21.8% (p = 0.068) for SPT areas. The positive SPT areas in male patients showed a higher ARI than those of female patients (43.36 and 23.33%; p = 0.078). The ARI values of SPT areas in male patients but not in female patients were found to be significantly higher as compared with non-lesional skin (21.63%; p = 0.039). The SPT positivity was also more common in male patients compared with female patients (86.4% and 62.5%), although the difference was not significant (p = 0.88). SPT have been found to be more strongly positive among the males (4.63 ± 3.3) compared with female patients (3.18 ± 1.9), and the difference was statistically significant (p = 0.022). Conclusions: Our findings indicate that androgens seem to play a role both in the formation and increased positivity of the SPT areas in male patients with BD.