Assessment of the impact of mitochondrial genotype upon drug-induced mitochondrial dysfunction in platelets derived from healthy volunteers.

Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10-250 µM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.

Development of Androgen-Antagonistic Coumarinamides with a Unique Aromatic Folded Pharmacophore.

First-generation nonsteroidal androgen receptor (AR) antagonists, such as flutamide (2a) and bicalutamide (3), are effective for most prostate cancer patients, but resistance often appears after several years due to the mutation of AR. Second-generation AR antagonists are effective against some of these castration-resistant prostate cancers, but their structural variety is still limited. In this study, we designed and synthesized 4-methyl-7-(N-alkyl-arylcarboxamido)coumarins as AR antagonist candidates and evaluated their growth-inhibitory activity toward androgen-dependent SC-3 cells. Coumarinamides with a secondary amide bond did not show inhibitory activity, but their N-methylated derivatives exhibited AR-antagonistic activity. Especially, 19b and 31b were more potent than the lead compound 7b, which was comparable to hydroxyflutamide (2b). Conformational analysis showed that the inactive coumarinamides with a secondary amide bond have an extended structure with a trans-amide bond, while the active N-methylated coumarinamides have a folded structure with a cis-amide bond, in which the two aromatic rings are placed face-to-face. Docking study suggested that this folded structure is important for binding to AR. Selected coumarinamide derivatives showed AR-antagonistic activity toward LNCaP cells with T877A AR, and they had weak progesterone receptor (PR)-antagonistic activity. The folded coumarinamide structure appears to be a unique pharmacophore, different from those of conventional AR antagonists.

An Intraprostatic Modified Release Formulation of Antiandrogen 2-Hydroxyflutamide for Localized Prostate Cancer.

PURPOSE: We investigated the tolerability, safety and antitumor effects of a novel intraprostatic depot formulation of antiandrogen 2-hydroxyflutamide (in NanoZolid®) in men with localized prostate cancer. MATERIALS AND METHODS: Two clinical trials, LPC-002 and LPC-003, were performed in a total of 47 men. The formulation was injected transrectally into the prostate under ultrasound guidance. In LPC-002 the effects on prostate specific antigen and prostate volume were measured for 6 months in 24 patients. In LPC-003 antitumor effects were evaluated by histopathology and magnetic resonance imaging including spectroscopy during 6 or 8 weeks in 23 patients. In each study testosterone and 2-hydroxyflutamide in plasma were measured as well as quality of life parameters. RESULTS: In LPC-002 (mean dose 690 mg) a reduction was observed in prostate specific antigen and prostate volume. Average nadir prostate specific antigen and prostate volume were 24.9% and 14.0% below baseline, respectively. When increasing the dose in LPC-003 to 920 and 1,740 mg, average prostate specific antigen decreased 16% and 23% after 6 and 8 weeks, respectively. Magnetic resonance imaging and magnetic resonance spectroscopy showed morphological changes and a global reduction in metabolite concentrations following treatment, indicating an antitumor response. Injections did not result in hormone related side effects. Three serious adverse events were reported and all resolved with oral antibiotic treatment. CONCLUSIONS: Intraprostatic injections of 2-hydroxyflutamide depot formulations showed antitumor effects, and proved to be safe and tolerable. However, for better anticancer effects higher doses and better dose distribution are suggested.

Antiandrogens act as selective androgen receptor modulators at the proteome level in prostate cancer cells.

Current therapies for prostate cancer include antiandrogens, inhibitory ligands of the androgen receptor, which repress androgen-stimulated growth. These include the selective androgen receptor modulators cyproterone acetate and hydroxyflutamide and the complete antagonist bicalutamide. Their activity is partly dictated by the presence of androgen receptor mutations, which are commonly detected in patients who relapse while receiving antiandrogens, i.e. in castrate-resistant prostate cancer. To characterize the early proteomic response to these antiandrogens we used the LNCaP prostate cancer cell line, which harbors the androgen receptor mutation most commonly detected in castrate-resistant tumors (T877A), analyzing alterations in the proteome, and comparing these to the effect of these therapeutics upon androgen receptor activity and cell proliferation. The majority are regulated post-transcriptionally, possibly via nongenomic androgen receptor signaling. Differences detected between the exposure groups demonstrate subtle changes in the biological response to each specific ligand, suggesting a spectrum of agonistic and antagonistic effects dependent on the ligand used. Analysis of the crystal structures of the AR in the presence of cyproterone acetate, hydroxyflutamide, and DHT identified important differences in the orientation of key residues located in the AF-2 and BF-3 protein interaction surfaces. This further implies that although there is commonality in the growth responses between androgens and those antiandrogens that stimulate growth in the presence of a mutation, there may also be influential differences in the growth pathways stimulated by the different ligands. This therefore has implications for prostate cancer treatment because tumors may respond differently dependent upon which mutation is present and which ligand is activating growth, also for the design of selective androgen receptor modulators, which aim to elicit differential proteomic responses dependent upon cellular context.

A Molecular Modeling Study of the Hydroxyflutamide Resistance Mechanism Induced by Androgen Receptor Mutations.

Hydroxyflutamide (HF), an active metabolite of the first generation antiandrogen flutamide, was used in clinic to treat prostate cancer targeting androgen receptor (AR). However, a drug resistance problem appears after about one year's treatment. AR T877A is the first mutation that was found to cause a resistance problem. Then W741C_T877A and F876L_T877A mutations were also reported to cause resistance to HF, while W741C and F876L single mutations cannot. In this study, molecular dynamics (MD) simulations combined with the molecular mechanics generalized Born surface area (MM-GBSA) method have been carried out to analyze the interaction mechanism between HF and wild-type (WT)/mutant ARs. The obtained results indicate that AR helix 12 (H12) plays a pivotal role in the resistance of HF. It can affect the coactivator binding site at the activation function 2 domain (AF2, surrounded by H3, H4, and H12). When H12 closes to the AR ligand-binding domain (LBD) like a lid, the coactivator binding site can be formed to promote transcription. However, once H12 is opened to expose LBD, the coactivator binding site will be distorted, leading to invalid transcription. Moreover, per-residue free energy decomposition analyses indicate that N705, T877, and M895 are vital residues in the agonist/antagonist mechanism of HF.

Corepressive function of nuclear receptor coactivator 2 in androgen receptor of prostate cancer cells treated with antiandrogen.

BACKGROUND: Recruitment of cofactors in the interaction of the androgen receptor (AR) and AR ligands plays a critical role in determining androgenic/antiandrogenic effects of the AR ligand on signaling, but the functions of key cofactors, including nuclear receptor coactivator (NCOA), remain poorly understood in prostate cancer cells treated with AR ligands. METHODS: We examined prostate cancer cell lines LNCaP and VCaP expressing mutated and wild-type ARs, respectively, to clarify the significance of NCOAs in the effect of antiandrogens. Hydroxyflutamide showed antagonistic activity against VCaP and an agonistic effect on LNCaP. Bicalutamide served as an antagonist for both. We analyzed mRNA transcription and protein expression of NCOAs in these cells pretreated with dihydrotestosterone and thereafter treated with the mentioned antiandrogens. Transcriptional silencing of candidate NCOAs and AR was performed using small interfering RNA (siRNA). Cell proliferation was evaluated with MTT assay. RESULTS: LNCaP treated with bicalutamide showed an about four-fold increase in the expression of NCOA2 mRNA compared to those pretreated with dihydrotestosterone alone (P <0.01). In VCaP pretreated with dihydrotestosterone, transcriptions of NCOA2 and NCOA7 were slightly increased with bicalutamide (1.96- and 2.42-fold, respectively) and hydroxyflutamide (1.33-fold in both). With Western blotting, the expression of NCOA2 protein also increased in LNCaP cells treated with bicalutamide compared with that in control cells pretreated with dihydrotestosterone alone. Following silencing with siRNA for NCOA2, PSA levels in media with LNCaP receiving bicalutamide were elevated compared with those in non-silencing controls (101.6 ± 4.2 vs. 87.8 ± 1.4 ng/mL, respectively, P =0.0495). In LNCaP cells treated with dihydrotestosterone and bicalutamide, NCOA2-silencing was associated with a higher proliferation activity compared with non-silencing control and AR-silencing. CONCLUSION: NCOA2, which has been thought to be recruited as a coactivator, possibly plays a corepressive role in AR of prostate cancer cells when treated with antiandrogens, suggesting its potential as a therapeutic target.

Androgens promote the acquisition of maturation competence in bovine oocytes.

Recent studies in mice suggest that androgens are important for normal follicle development. However, there have been few reports concerning the action of androgens in the growth of oocytes from large animals. The purpose of this study was to determine the roles of androgens in bovine oocyte growth in vitro. Oocyte-granulosa cell complexes (OGCs) collected from 0.4-0.7 mm early antral follicles were cultured for 14 days with 17ß-estradiol (E2) and a non-aromatizable androgen, dihydrotestosterone (DHT). We also examined the ability of an androgen receptor (AR) inhibitor, hydroxyflutamide, to antagonize the effect of androgens on the oocytes. During growth culture, the OGC structures collapsed in the medium with DHT alone, while in the presence of E2, the OGC structures were maintained. In the medium with both androgens and E2, the mean diameter of oocytes was increased from 95 µm to around 120 µm, larger than those grown with E2 alone (115 µm). Also in the maturation culture, oocytes grown with androgens (A4 or DHT) and E2 showed higher percentages of metaphase II oocytes (63% or 69%, respectively) than those grown with E2 alone (32%). Moreover, these maturation rates were decreased by hydroxyflutamide in a dose-dependent manner. Immunostaining showed that ARs were expressed in oocytes and granulosa cells in early antral follicles, and the nuclei of granulosa cells showed intense AR expression. In conclusion, although E2 supports the OGC structure, additional androgens promote oocyte growth and their acquisition of meiotic competence via AR during in vitro growth culture.

Suppression of androgen receptor enhances the self-renewal of mesenchymal stem cells through elevated expression of EGFR.

Bone marrow derived mesenchymal stem cells (BM-MSCs) have been widely applied in several clinical trials of diseases, such as myocardial infarction, liver cirrhosis, neurodegenerative disease, and osteogenesis imperfecta. Although most studies demonstrated that transplantation of BM-MSCs did exert a temporary relief and short-term therapeutic effects, eventually all symptoms recur, therefore it is essential to improve the therapeutic efficacy of transplantation by either elevating the self-renewal of BM-MSCs or enhancing their survival rate. Herein we demonstrated that the BM-MSCs and adipocyte derived mesenchymal stem cells (ADSCs) isolated from the androgen receptor (AR) knockout mice have higher self-renewal ability than those obtained from the wild-type mice. Knockdown of AR in MSC cell lines exhibited similar results. Mechanistic dissection studies showed that the depletion of AR resulted in activation of Erk and Akt signaling pathways through epidermal growth factor receptor (EGFR) activation or pathway to mediate higher self-renewal of BM-MSCs. Targeting AR signals using ASC-J9® (an AR degradation enhancer), hydroxyflutamide (antagonist of AR), and AR-siRNA all led to enhanced self-renewal of MSCs, suggesting the future possibility of using these anti-AR agents in therapeutic approaches.

Pharmacokinetics of an injectable modified-release 2-hydroxyflutamide formulation in the human prostate gland using a semiphysiologically based biopharmaceutical model.

The local distribution of 2-hydroxyflutamide (2-HOF) in prostate tissue after a single intraprostatic injection of a novel parenteral modified-release (MR) formulation in patients with localized prostate cancer was estimated using a semiphysiologically based biopharmaceutical model. Plasma concentration-time profiles for 2-HOF were acquired from a phase II study in 24 patients and the dissolution of the MR formulation was investigated in vitro. Human physiological values and the specific physicochemical properties of 2-HOF were obtained from the literature or calculated via established algorithms. A compartmental modeling approach was adopted for tissue and blood in the prostate gland, where the compartments were modeled as a series of concentric spherical shells contouring the centrally positioned depot formulation. Discrete fluid connections between the blood compartments were described by the representative flow of blood, whereas the mass transport of drug from tissue to tissue and tissue to blood was described by a one-dimensional diffusion approximation. An empirical dissolution approach was adopted for the release of 2-HOF from the formulation. The model adequately described the plasma concentration-time profiles of 2-HOF. Predictive simulations indicated that the local tissue concentration of 2-HOF within a distance of 5 mm from the depot formulation was approximately 40 times higher than that of unbound 2-HOF in plasma. The simulations also indicated that spreading the formulation throughout the prostate gland would expose more of the gland and increase the overall release rate of 2-HOF from the given dose. The increased release rate would initially increase the tissue and plasma concentrations but would also reduce the terminal half-life of 2-HOF in plasma. Finally, an in vitro-in vivo correlation of the release of 2-HOF from the parenteral MR formulation was established. This study shows that intraprostatic 2-HOF concentrations are significantly higher than systemic plasma concentrations and that increased distribution of 2-HOF throughout the gland, using strategic imaging-guided administration, is possible. This novel parenteral MR formulation, thus, facilitates good pharmacological effect while minimizing the risk of side effects.

The impact of antiandrogen 2-hydroxyflutamide on the expression of steroidogenic enzymes in cultured porcine ovarian follicles.

We used our model system for agonism and antagonism of the androgen receptor (AR), in which the porcine ovarian follicles were exposed on the excessive concentration of an AR agonist- testosterone (T) or an AR antagonist- 2-hydroxyflutamide (2-Hf) to: (1) analyze the spatiotemporal expression of ovarian 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 17α-hydroxylase/c17,20-lyase (P450c17) and cytochrome P450 aromatase (P450arom); (2) to determine the contribution of AR-mediated action during steroidogenesis and (3) to establish some correlations between the onset and expression pattern of the investigated proteins. Whole follicles (6-8 mm in diameter) isolated from mature porcine ovaries have been incubated (for 24 h) in an organ culture system in the presence of T (10(-7 )M), 2-Hf (1.7 × 10(-4) M) or both T and 2-hydroxyflutamide (T+2-Hf, at the same concentrations as when added separately). Thereafter, sections obtained from cultured follicles were processed for main steroidogenic enzymes detection by immunohistochemistry. Moreover, expression of their mRNA and protein was determined by real-time PCR and Western blot analysis. Progesterone, androgens and estradiol concentrations in the culture media were measured by radioimmunoassays (RIA). Our results demonstrated that 2-Hf can influence the steroidogenic activity of porcine follicles in vitro through the blockade of AR. It was shown that follicular 2-Hf treatment brought about dramatic decline in the production of the investigated steroids. What is more the addition of 2-Hf separately caused a negative effect on 3ß-HSD and P450c17 mRNA and protein expression by ovarian follicles, while it was without effect on P450arom mRNA level. Quite opposite effect was observed in case of the simultaneous addition of 2-Hf and T. It caused high increase, in both P450arom mRNA and its protein. What was interesting, addition T+2-Hf evoked 3ß-HSD and P450c17 increase on mRNA level, but decreased their protein expression. This was against our expectations but the reason for that finding remains undiscovered, intriguing and worth reporting. These results suggest that alike, steroidogenic enzymes activity and their expression is associated with the presence of androgens and AR in the porcine ovary.

[Application effect of a dual release system of androgen and its antagonist in the repair of full-thickness burn wounds in mice].

Objective: To explore the optimal ratio of dihydrotestosterone and hydroxyflutamide (hereinafter referred to as DH), construct a dual release system of androgen and its antagonist, and analyze the application effect of this system in the repair of full-thickness burn wounds in mice. Methods: This study was an experimental study. The HaCaT cells were divided into blank group (without drug culture), low baseline group, medium baseline group, and high baseline group according to the random number table (the same grouping method below), and the last three groups of cells were cultured by adding three different ratios of DH. Under a medium ratio, the mass of dihydrotestosterone in the three baseline groups from low to high was 1.4, 2.8, and 4.0 µg, respectively, and the mass of hydroxyflutamide was 1.2, 1.6, and 2.0 µg, respectively. On this basis, under a small ratio, the mass of dihydrotestosterone was reduced by half and the mass of hydroxyflutamide was increased by half; under a large ratio, the mass of dihydrotestosterone was increased by half and the mass of hydroxyflutamide was reduced by half. After culture of 2 days, the cell proliferation level was detected by cell counting kit 8 (n=4). Sixteen 6-8-week-old male BALB/c mice were used to establish a full-thickness burn wound on the back and divided into blank group, small ratio group, medium ratio group, and large ratio group, with 4 mice in each group. On post injury day (PID) 7, normal saline containing different ratios of DH was locally dropped to the wounds of mice in the last three groups of mice (the total mass of DH in the three ratio groups from small to large was 127.5, 165.0, and 202.5 µg, respectively, and the mass ratios of dihydrotestosterone to hydroxyflutamide (hereinafter referred to as drug mass ratio) were 8∶9, 8∶3, and 8∶1, respectively), afterwards, the administration was repeated every 48 hours until PID 27; normal saline was dropped to the wound of mice in blank group at the aforementioned time points. The wound healing status on PID 0 (immediately), 7, 14, 21, and 28 was observed, and the wound healing rates on PID 7, 14, 21, and 28 were calculated (n=4). On PID 28, the wound tissue was taken, which was stained with hematoxylin and eosin for observing re-epithelialization and with Masson for observing collagen fibers, and the proportion of collagen fibers was analyzed (n=3). Twenty 6-8-week-old male BALB/c mice were used to establish a full-thickness burn wound on the back and divided into ordinary scaffold group, small proportion scaffold group, medium proportion scaffold group, and large proportion scaffold group (with 5 mice in each group). On PID 7, the wound was continuously dressed with a polycaprolactone scaffold without drug and a polycaprolactone scaffold containing DH with a drug mass ratio of 1∶3, 1∶1, or 3∶1 (i.e. the dual release system of androgen and its antagonist, with total mass of DH being about 1.7 mg) prepared by using electrospinning technology until the end of the experiment. Histopathological analyses of tissue (n=3) at the same time points as those in the previous animal experiment were performed. On PID 7 and 14, the wound exudates were collected and the relative abundance of bacterial communities was analyzed using 16S ribosomal RNA high-throughput sequencing (n=3). Results: After culture of 2 days, under a small ratio, the proliferation levels of HaCaT cells in low baseline group and high baseline group were significantly higher than the level in blank group (P<0.05). As the time after injury prolonged, the wounds of all four groups of mice continued to shrink. On PID 14, the wound healing rate of mice in large ratio group was 72.5% (61.7%, 75.1%), which was close to 53.3% (49.5%, 64.4%) in blank group (P>0.05); the wound healing rates of mice in small and medium ratio groups were 74.2% (71.0%, 84.2%) and 70.4% (65.1%, 74.4%), respectively, which were significantly higher than the rate in blank group (with both Z values being -2.31, P<0.05). On PID 21, the wound healing rate of mice in small ratio group was significantly higher than that in blank group (Z=-2.31, P<0.05). On PID 28, the wounds of mice in the three ratio groups were completely re-epithelialized and the epidermis was thicker than that in blank group; compared with that in blank group, the collagen fiber content in the wound tissue of mice in the three ratio groups was higher and arranged more orderly, and the proportions of collagen fibers in the wound tissue of mice in small and large ratio groups were significantly increased (P<0.05). On PID 28, the wounds of mice in ordinary scaffold group were partially epithelialized, while the wounds of mice in the three proportion scaffold groups were almost completely epithelialized. Among them, the wounds of mice in small proportion scaffold group had the thickest epidermis. The proportion of collagen fibers in the wound tissue of mice in small proportion scaffold group was significantly increased compared with that in ordinary scaffold group (P<0.05). On PID 7, the bacterial communities with high relative abundance in the wound exudation of mice in the four groups included bacteria of Corynebacterium, Staphylococcus, and Rhodococcus. On PID 14, the bacterial communities with high relative abundance in the wound exudation of mice in the four groups included bacteria of Stenotrophomonas, Rhodococcus, and Staphylococcus, and the number of bacterial species in the wound exudation of mice in the three proportion scaffold groups was more than that in ordinary scaffold group. Conclusions: When the drug mass ratio is relatively small, DH has the effect of promoting the proliferation of HaCaT cells. The ratio of 8∶9 is the optimal mass ratio of dihydrotestosterone to hydroxyflutamide, and DH with this mass ratio can promote re-epithelialization and collagen deposition of full-thickness burn wounds in mice, and promote wound healing. The constructed dual release system of androgen and its antagonist with DH in a 1∶3 drug mass ratio contributes to the re-epithelialization and collagen deposition of the full-thickness burn wounds in mice, and can improve the diversity of wound microbiota.

Discovery of Novel Anti-Resistance AR Antagonists Guided by Funnel Metadynamics Simulation.

Androgen receptor (AR) antagonists are widely used for the treatment of prostate cancer (PCa), but their therapeutic efficacy is usually compromised by the rapid emergence of drug resistance. However, the lack of the detailed interaction between AR and its antagonists poses a major obstacle to the design of novel AR antagonists. Here, funnel metadynamics is employed to elucidate the inherent regulation mechanisms of three AR antagonists (hydroxyflutamide, enzalutamide, and darolutamide) on AR. For the first time it is observed that the binding of antagonists significantly disturbed the C-terminus of AR helix-11, thereby disrupting the specific internal hydrophobic contacts of AR-LBD and correspondingly the communication between AR ligand binding pocket (AR-LBP), activation function 2 (AF2), and binding function 3 (BF3). The subsequent bioassays verified the necessity of the hydrophobic contacts for AR function. Furthermore, it is found that darolutamide, a newly approved AR antagonist capable of fighting almost all reported drug resistant AR mutants, can induce antagonistic binding structure. Subsequently, docking-based virtual screening toward the dominant binding conformation of AR for darolutamide is conducted, and three novel AR antagonists with favorable binding affinity and strong capability to combat drug resistance are identified by in vitro bioassays. This work provides a novel rational strategy for the development of anti-resistant AR antagonists.

Androgen receptor signals regulate UDP-glucuronosyltransferases in the urinary bladder: a potential mechanism of androgen-induced bladder carcinogenesis.

UDP-glucuronosyltransferases (UGTs), major phase II drug metabolism enzymes, play an important role in urinary bladder cancer initiation by detoxifying carcinogens. We aimed to determine if androgens regulate UGT expression via the androgen receptor (AR) pathway in the bladder. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to assess UGT1A levels in the normal urothelium SVHUC cell line stably expressed with AR and in bladder tissues from AR knockout (ARKO) and castrated male mice. Immunohistochemistry was also performed in radical cystectomy specimens. Dihydrotestosterone (DHT) treatment in SVHUC-AR reduced mRNA expression of all the UGT1A subtypes (19-75% decrease), and hydroxyflutamide antagonized the DHT effects. In contrast, DHT showed only marginal effects on UGT1A expression in SVHUC-Vector. Of note were higher expression levels of UGT1As in SVHUC-Vector than in SVHUC-AR. In ARKO mice, all the Ugt1a subtypes were up-regulated, compared to wild-type littermates. In wild-type male mice, castration increased the expression of Ugt1a8, Ugt1a9, and Ugt1a10. Additionally, wild-type female mice had higher levels of Ugt1a than wild-type males. Immunohistochemical studies showed strong (3+) UGT1A staining in 11/24 (46%) cancer tissues, which was significantly lower than in corresponding benign tissues [17/18 (94%) cases (P = 0.0009)]. These results suggest that androgen-mediated AR signals promote bladder carcinogenesis by down-regulating the expression of UGTs in the bladder.

Selective androgen receptor modulator, YK11, regulates myogenic differentiation of C2C12 myoblasts by follistatin expression.

The myogenic differentiation of C2C12 myoblast cells is induced by the novel androgen receptor (AR) partial agonist, (17α,20E)-17,20-[(1-methoxyethylidene)bis-(oxy)]-3-oxo-19-norpregna-4,20-diene-21-carboxylic acid methyl ester (YK11), as well as by dihydrotestosterone (DHT). YK11 is a selective androgen receptor modulator (SARM), which activates AR without the N/C interaction. In this study, we further investigated the mechanism by which YK11 induces myogenic differentiation of C2C12 cells. The induction of key myogenic regulatory factors (MRFs), such as myogenic differentiation factor (MyoD), myogenic factor 5 (Myf5) and myogenin, was more significant in the presence of YK11 than in the presence of DHT. YK11 treatment of C2C12 cells, but not DHT, induced the expression of follistatin (Fst), and the YK11-mediated myogenic differentiation was reversed by anti-Fst antibody. These results suggest that the induction of Fst is important for the anabolic effect of YK11.

Fluorescence lifetime imaging microscopy for the monitoring of green fluorescent protein-tagged androgen receptors in living cells.

Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the interaction between androgen receptor (AR) tagging of a green fluorescent protein (GFP) and the ligands in living cells. The fluorescence lifetime of the AR-GFP without ligands was ca. 3.1 ns, which was reduced to ca. 2.5 ns after treatment with agonist 5α-dihydrotestosterone. On the other hand, the fluorescence lifetime of AR-GFP was not changed after treatment with antagonist hydroxyflutamide. The reaction kinetics was simulated in the present study, and the obtained results indicated the possibility of the presence of an intermediate complex during the reaction. FLIM can be used to record the ratio of the AR as it reacts with an agonist, and, therefore, it is useful for acquiring information concerning the interaction between AR and ligands in living cells.

The influence of the antiandrogen 2-hydroxyflutamide on the androgen receptor expression in the porcine ovarian follicles - an in vitro study.

Androgens are one of the most important agents influencing ovarian follicles growth and development. The biological action of androgens is primarily exerted through transcriptional regulation by the androgen receptor (AR), a member of the steroid hormone receptor superfamily. The purpose of this study was to test the role of androgen receptor agonist testosterone (T) or antagonist 2-hydroxyflutamide (2-Hf) and in combination on AR expression in cultured porcine granulosa cells (GC) or whole follicles. Granulosa cells isolated from mature pig follicles were cultured for 48 h. During the last 12 and 24 h of culture, they were incubated in the presence of T (10(-7)  m/ml), 2-Hf (1.7 × 10(-4)  m) or both T and 2-Hf (T + 2-Hf, at the same concentrations as when added separately). To better imitate in vivo conditions, whole follicles (6-8 mm in diameter) isolated from porcine ovaries have been incubated (for 12 and 24 h) in an organ culture system with the addition of the same factors. Thereafter, cells or sections obtained from cultured follicles were processed for AR detection by immunocytochemistry or immunohistochemistry. Moreover, expression of AR mRNA and protein was determined by real-time PCR and Western blot analysis. It was shown that the addition of 2-Hf in the presence of T had a positive effect on AR mRNA and protein expression in porcine GC and ovarian follicles. Moreover, the addition of 2-Hf influenced AR distribution in GC cultures which is seen as change of its localization from nuclear to perinuclear. Our results suggest that androgens acting through AR could be involved in the control of AR expression in porcine GC in vitro and in vivo.

Does 2-hydroxyflutamide inhibit apoptosis in porcine granulosa cells? - An in vitro study.

In mammalian ovaries, the majority of follicles are lost before ovulation by atresia. This degenerative process is initiated or caused by granulosa cell apoptosis. To reveal the androgen-dependent mechanism of selective follicular atresia, the culture model system for agonism and antagonism of the androgen receptor has been established. We examined the influence of an androgen receptor antagonist, 2-hydroxyflutamide (2-Hf), on the incidence of apoptosis in cultured porcine granulosa cells. They were incubated (6 and 12-h) in the presence of testosterone (T, 10⁻7M), 2-Hf (1.7×10⁻4 M) or both T and 2-Hf (T+2-Hf), and then analyzed by flow cytometry with fluorescein labelled annexin V. To better imitate in vivo conditions, the intact porcine follicles (6-8 mm in diameter) have been incubated in an organ culture system with the addition of the same factors. Sections obtained from follicles fixed after culture were stained with hematoxylin and eosin, and the presence of apoptosis-related DNA strand breaks was evaluated by the TUNEL method. Estradiol and progesterone concentrations in the culture media were measured by radioimmunoassays. The addition of T or 2-Hf to the culture media caused an increase in the number of apoptotic granulosa cells, while treatment with T+2-Hf decreased it in both in vitro and organotypic models. Follicles cultured with the addition of T or 2-Hf exhibited morphological changes indicating follicular atresia. Granulosal estradiol secretion was considerably stimulated by T+2-Hf. The highest increase in follicular estradiol secretion was observed after the anti-androgen addition. In both granulosal and follicular cultures, the production of progesterone declined in the presence of T or 2-Hf but increased after their simultaneous addition. In conclusion, androgen receptor antagonist 2-Hf attenuates induction of granulosa cell apoptosis in the presence of a high T level. The nature of this protective mechanism as yet is unknown and requires further research.

Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 µg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p < 0.05) in flutamide-treated boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

Systematic structure-function analysis of androgen receptor Leu701 mutants explains the properties of the prostate cancer mutant L701H.

One mechanism of prostate tumors for escape from androgen ablation therapies is mutation of the androgen receptor (AR). We investigated the unique properties of the AR L701H mutant, which is strongly stimulated by cortisol, by a systematic structure-function analysis. Most amino acid substitutions at position 701 did not affect AR activation by 5alpha-dihydrotestosterone. Further analysis of the AR Leu(701) variants showed that AR L701M and AR L701Q, like AR L701H, had changed ligand responsiveness. AR L701M was strongly activated by progesterone but not by cortisol, whereas the opposite was observed for AR L701Q and AR L701H. Next, we analyzed a panel of structurally related steroids to study which of the OH groups at positions 11beta, 17alpha, and 21, which discriminate cortisol from progesterone, underlie the differential responses to both hormones. The results showed that the 17alpha-OH group was essential for activation of AR L701H and AR L701Q, whereas its absence was important for activation of AR L701M. Modeling indicated a conserved H-bonding network involving the steroidal 17alpha-OH group, His(701) or Gln(701), and the backbone of Ser(778). This network is absent in Leu(701) and in other mutants. A hydrophobic leucine or methionine at position 701 is unfavorable for the 17alpha-OH group. Our results indicate that the specific amino acid residue at position 701, its interaction with the backbone of Ser(778), and the steroidal 17alpha-hydroxyl group of the ligand are all important for the distinct transcriptional responses to progesterone and cortisol of AR mutants, including the prostate cancer mutant L701H.

Production of human metabolites of the anti-cancer drug flutamide via biotransformation in Cunninghamella species.

Fungi belonging to the genus Cunninghamella have enzymes similar to those employed by mammals for the detoxification of xenobiotics, thus they are useful as models of mammalian drug metabolism, and as a source for drug metabolites. We report the transformation of the anti-cancer drug flutamide in Cunninghamella sp. The most predominant phase I metabolites present in the plasma of humans, 2-hydroxyflutamide and 4-nitro-3-(trifluoromethyl)aniline, were also produced in Cunninghamella cultures. Other phase I and phase II metabolites were also detected using a combination of HPLC, GC-MS and (19)F-NMR.