Androgen receptor (AR) is a ligand-activated transcription factor that plays a pivotal role in the development and progression of many severe diseases such as prostate cancer, muscle atrophy, and osteoporosis. Binding of ligands to AR triggers the conformational changes in AR that may affect the recruitment of coactivators and downstream response of AR signaling pathway. Therefore, AR ligands have great potential to treat these diseases. In this study, we searched for novel AR ligands by performing a docking-based virtual screening (VS) on the basis of the crystal structure of the AR ligand binding domain (LBD) in complex with its agonist. A total of 58 structurally diverse compounds were selected and subjected to LBD affinity assay, with five of them (HBP1-3, HBP1-17, HBP1-38, HBP1-51, and HBP1-58) exhibiting strong binding to AR-LBD. The IC values of HBP1-51 and HBP1-58 are 3.96 µM and 4.92 µM, respectively, which are even lower than that of enzalutamide (Enz, IC = 13.87 µM), a marketed second-generation AR antagonist. Further bioactivity assays suggest that HBP1-51 is an AR agonist, whereas HBP1-58 is an AR antagonist. In addition, molecular dynamics (MD) simulations and principal components analysis (PCA) were carried out to reveal the binding principle of the newly-identified AR ligands toward AR. Our modeling results indicate that the conformational changes of helix 12 induced by the bindings of antagonist and agonist are visibly different. In summary, the current study provides a highly efficient way to discover novel AR ligands, which could serve as the starting point for development of new therapeutics for AR-related diseases.
Androgen Receptor Antagonists ; pharmacology ; Androgens ; metabolism ; pharmacology ; Biological Assay ; Cell Line, Tumor ; Drug Discovery ; methods ; Humans ; Ligands ; Male ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Phenylthiohydantoin ; analogs & derivatives ; pharmacology ; Principal Component Analysis ; Prostatic Neoplasms ; drug therapy ; Protein Binding ; physiology ; Protein Conformation ; drug effects ; Receptors, Androgen ; metabolism

Aged ; Aged, 80 and over ; Androgen Antagonists/pharmacology* ; Androgens/blood* ; Cohort Studies ; Gonadotropin-Releasing Hormone/antagonists & inhibitors* ; Hormones/blood* ; Humans ; Luteinizing Hormone/blood* ; Male ; Middle Aged ; Orchiectomy ; Prostatic Neoplasms/surgery* ; Toremifene/therapeutic use*

Objective: To investigate the effects of down-regulation of PTTG1 expression on the proliferation, invasiveness and apoptosis of androgen-independent human prostate cancer LNCaP-AI cells and their sensitivity to androgen antagonists. METHODS: Human prostate cancer LNCaP-AI cells were transfected with siRNA targeting the PTTG1 gene using the Lipofectamine 2000 transfection reagent. The proliferation, invasiveness and apoptosis of the cells were detected by MTT, Transwell assay and flow cytometry, respectively. The protein expressions of PTTG1, p-Akt, and p-ERK were determined by Western blot and the mRNA expression of PTTG1 measured by agarose gel electrophoresis. RESULTS: The siRNA expression vector markedly down-regulated the expression of PTTG1, which effectively suppressed the proliferation of the LNCaP-AI cells, with the inhibition rates of (19.47 ± 2.12), (24.01 ± 2.13) and (48.02 ± 2.22)% at 24, 48 and 72 hours, respectively, after transfection, with statistically significant differences among the three groups (P <0.05). The number of the cells passing through the polycarbonate film was remarkably decreased at 24, 48 and 72 hours (74.67 ± 9.85, 56.44 ± 8.66 and 37.33 ± 6.14) as compared with the baseline (111.11 ± 13.47) (P <0.01), while the apoptosis rate of the cells was significantly increased at 24, 48 and 72 hours (18.32 ± 0.94), (19.94 ± 1.30) and (21.73 ± 1.88)% in comparison with the baseline (［2.17 ± 0.49］%)， (P <0.05). PTTG1 siRNA combined with androgen antagonist flumatide exhibited even more significant effects in inhibiting the proliferation and promoting the apoptosis of the LNCaP-AI cells than either used alone, and in a flumatide dose-dependent manner. The inhibition and apoptosis rates of the LNCaP-AI cells treated with 50 nmol/L flumatide were (27.13 ± 3.52) and (3.94 ± 0.48)%, and those treated with siRNA + 50 nmol/L flumatide were (67.51 ± 5.13) and (19.93 ± 1.72)%, respectively, both with statistically significant differences between the two groups (P <0.05). The inhibition and apoptosis rates of the cells treated with 100 nmol/L flumatide were (43.72 ± 3.90) and (5.33 ± 0.66)%, and those treated with siRNA + 100 nmol/L flumatide were (73.19 ± 4.78) and (23.43 ± 1.76)%, respectively, both with statistically significant differences between the two groups (P <0.05). CONCLUSIONS The siRNA expression vector can down-regulate the expression of PTTG1, which can inhibit the proliferation and invasiveness of LNCaP-AI cells, promote their apoptosis, and increase their sensibility to androgen antagonists. Suppressing the expression of PTTG1 may enhance the effect of androgen-deprivation therapy on advanced prostate cancer.
Androgen Antagonists ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Humans ; Male ; Neoplasm Invasiveness ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; RNA, Small Interfering ; metabolism ; Securin ; genetics ; metabolism ; Time Factors ; Transfection

To study the regulation of androgen receptor (AR) expression in human prostate cancer LNCaP cells by triptolide (TP) and the possible mechanism, by using qRT-PCR and Western blot, the AR mRNA and protein levels in TP treated LNCaP cells were detected, and the AR protein level in TP and NF-κB inhibitor treated LNCaP cells was also detected; a series of pGL3-AR promoter reporter gene vectors were built using restriction-free cloning method, and the vectors were employed to investigate the effects of TP on the transcriptional activity of AR promoter in LNCaP cells; the upstream proteins which may play regulatory roles were detected using western blot assay. After treated LNCaP cells with TP for 48 h, AR mRNA and protein expressions decreased with increasing TP concentration. The expression of AR target gene PART1 and prostate specific antigen (PSA) was also downregulated by TP treatment; a series of pGL3-AR promoter reporter vectors were constructed and validated by sequencing and luciferase activity; the results of dual luciferase reporter assay showed that TP downregulated AR at the transcriptional level; PI3K/AKT/NF-κB pathway which is associated with AR promoter activity was drowregulated by TP. In conclusion, our results demonstrated that the transcriptional activity of AR in LNCAP cells was downregulated by TP, and PI3K/AKT/NF-κB pathway may be involved in the regulation mechanism.
Cell Line, Tumor ; Diterpenes ; pharmacology ; Down-Regulation ; Epoxy Compounds ; pharmacology ; Genetic Vectors ; Humans ; Male ; NF-kappa B ; antagonists & inhibitors ; Phenanthrenes ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Promoter Regions, Genetic ; Prostate-Specific Antigen ; metabolism ; Prostatic Neoplasms ; metabolism ; RNA, Messenger ; Receptors, Androgen ; metabolism ; Signal Transduction ; Transcriptional Activation

Prostate cancer generally relapses into castration resistant prostate cancer (CRPC) after androgen deprivation therapy, which may be associated with androgen metabolism, particularly de novo androgen synthesis apart from the amplification and mutation of androgen receptor and the activation of its signaling pathways. This article focuses on the advances in the studies of the changes in androgen metabolism and de novo androgen synthesis in CRPC as well as their possible mechanisms and clinical significance.
Androgen Antagonists ; pharmacology ; Androgens ; biosynthesis ; metabolism ; Humans ; Male ; Orchiectomy ; Prostate ; metabolism ; Prostatic Neoplasms, Castration-Resistant ; metabolism

Adolescent ; Amenorrhea ; diagnosis ; etiology ; Androgen Antagonists ; pharmacology ; Androgens ; blood ; Contraceptive Agents ; pharmacology ; Female ; Humans ; Hyperandrogenism ; complications ; Hypoglycemic Agents ; therapeutic use ; Insulin Resistance ; Luteinizing Hormone ; blood ; Menstruation Disturbances ; diagnosis ; etiology ; Obesity ; complications ; Ovary ; diagnostic imaging ; pathology ; Polycystic Ovary Syndrome ; diagnosis ; etiology ; therapy ; Ultrasonography ; Young Adult

AIMTo determine whether testosterone is involved in morphine withdrawal syndrome (WS).

METHODSIn order to induce dependency, rats were treated with subcutaneous injection of morphine (days 1-2, 5 mg/kg; days 3-5, 7.5 mg/kg; days 6-8, 10 mg/kg), and after the last dose of morphine (day 8) WS was induced by intraperitoneal injection of naloxone (1 mg/kg). Wet dog shake (WDS), abdomen writhing (AW), and jumps (J) were recorded as indicators of WS.

RESULTSThe severity of WDS, AW, and J in male rats was greater than that in females. Accordingly, in 4-week castrated and flutamide-treated (10 mg/kg/day for 8 days, i.p.) male rats, WDS, AW, and J were significantly decreased compared to male control rats. Testosterone replacement therapy (10 mg/kg/day for 8 days, i.m.) in 4-week castrated rats restored the severity of WDS, AW, and J behaviors to the level of non-castrated male rats, whereas testosterone potentiated the WDS behavior in non-castrated male rats.

CONCLUSIONIt can be concluded that testosterone might be effectively involved in morphine WS.

Androgen Antagonists ; pharmacology ; Androgens ; pharmacology ; physiology ; Animals ; Behavior, Animal ; Female ; Flutamide ; pharmacology ; Male ; Morphine ; pharmacology ; Morphine Dependence ; physiopathology ; Naloxone ; pharmacology ; Narcotic Antagonists ; pharmacology ; Narcotics ; pharmacology ; Orchiectomy ; Rats ; Rats, Wistar ; Severity of Illness Index ; Substance Withdrawal Syndrome ; physiopathology ; Testosterone ; pharmacology ; physiology

BACKGROUNDThe failure of hormone treatment for advanced prostate cancer might be related to aberrant activation of the androgen receptor. We have shown that (125)I labeled triple-helix forming oligonucleotide (TFO) against the androgen receptor gene inhibits androgen receptor expression and cell proliferation of LNCaP prostate cancer cells in vitro. This study aimed at exploring the effects of the (125)I-TFO on prostate tumor growth in vivo using a nude mouse xenograft model.

METHODSTFO was labeled with (125)I by the iodogen method. Thirty-two nude mice bearing LNCaP xenograft tumors were randomized into 4 groups and were intratumorally injected with (125)I-TFO, unlabeled TFO, Na(125)I and normal saline. Tumor size was measured weekly. The tumor growth inhibition rate (RI) was calculated by measurement of tumor weight. The expression of the androgen receptor gene was performed by RT-PCR and immunohistochemical study. The prostate specific antigen (PSA) serum levels were measured by enzyme linked immunosorbent assay. The tumor cell apoptosis index (AI) was detected by TUNEL assay.

RESULTSTumor measurements showed that tumor development was significantly inhibited by either (125)I-TFO or TFO, with tumor RIs of 50.79% and 32.80% respectively. (125)I-TFO caused greater inhibition of androgen receptor expression and higher AIs in tumor tissue than TFO. Both the tumor weight and the PSA serum levels in (125)I-TFO treated mice ((0.93 +/- 0.15) g and (17.43 +/- 1.85) ng/ml, respectively) were significantly lower than those ((1.27 +/- 0.21) g and (28.25 +/- 3.41) ng/ml, respectively) in TFO treated mice (all P < 0.05). Na(125)I did not significantly affect tumor growth and androgen receptor expression in tumor tissue.

CONCLUSIONSThe (125)I-TFO can effectively inhibit androgen receptor expression and tumor growth of human prostate cancer xenografts in vivo. The inhibitory efficacy of (125)I-TFO is more potent than that of TFO, providing a reference for future studies of antigen radiotherapy.

Androgen Receptor Antagonists ; Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunohistochemistry ; Iodine Radioisotopes ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Oligonucleotides ; chemistry ; pharmacology ; therapeutic use ; Prostate-Specific Antigen ; blood ; Prostatic Neoplasms ; drug therapy ; metabolism ; pathology ; Receptors, Androgen ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays ; methods

OBJECTIVETo investigate the antiandrogenic activities of cypermethrin and beta-cypermethrin in vitro and in vivo.

METHODSTranscriptional activation assay based on MDA-kb2 cell was used to determine the antiandrogenic effect of cypermethrin and beta-cypermethrin in vitro. The cells were treated by 10(-8), 10(-7), 10(-6) and 10(-5) mol/L of cypermethrin and beta-cypermethrin with 1.0 nmol/L DHT at the same time. The effects of antagonism towards the androgenic receptor were studied. In in vivo assays, Hershberger assay was used to determine the antiandrogenic activities of cypermethrin and beta-cypermethrin. Six-week-old castrated male SD rats were administered by cypermethrin (7, 21 and 63 mg/kg) and beta-cypermethrin (6, 18 and 54 mg/kg). After 7-day treatments, all rats were euthanized and androgen-responsive tissues were excised and weighed respectively.

RESULTSThe in vitro experiments showed that 10(-6) and 10(-5) mol/L cypermethrin could inhibit significantly the antagonism activity towards the androgenic receptor of DHT. In in vivo tests, the weight of seminal vesicle, ventral prostate, dorsolateral prostate and preputial glands in the 63 mg/kg cypermethrin [(52.8 +/- 7.1), (42.4 +/- 8.9), (36.6 +/- 4.5) and (43.4 +/- 11.1) mg] decreased significantly compared with those in the control group. In 21 mg/kg cypermethrin treated group only the weights of ventral prostate and dorsolateral prostate decreased significantly, and in 7 mg/kg cypermethrin only the weight of dorsolateral prostate decreased (P < 0.05). For beta-cypermethrin, any antiandrogen effect in in vivo and in vitro experiments was not found in all the groups.

CONCLUSIONCypermethrin is a moderate antiandrogen that elicits antiandrogenic effects at least partly by antagonizing AR and beta-cypermethrin is not an antiandrogen in our experiments.

Androgen Antagonists ; pharmacology ; Animals ; Cells, Cultured ; Male ; Organ Size ; Prostate ; drug effects ; Pyrethrins ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Androgen ; drug effects ; Seminal Vesicles ; drug effects

INTRODUCTIONThe advent of prostate specific antigen (PSA) has resulted in an increased incidence of early detection of prostate cancer recurrence. Patients treated with androgen deprivation therapy (ADT) become hormone-resistant after 18 to 24 months. In patients with biochemical failure, where there is a rise in PSA but no objective evidence of metastases, or in whom there are small volume metastases but who are asymptomatic, there is no standard of care after ADT. Ketoconazole, an antimycotic which affects the synthesis of androgens and other steroids, has shown direct cytotoxic effects in prostate cancer cell lines in in-vitro studies. This study describes our experience with ketoconazole treatment for hormone refractory prostate cancer (HRPC).

MATERIALS AND METHODSA retrospective study of HRPC patients given ketoconazole at the National Cancer Centre and The Cancer Institute from 2004 to 2005 was performed. All eligible patients had histologically proven adenocarcinoma of the prostate and a rising PSA level despite ADT with orchidectomy or luteinising hormone-releasing hormone (LHRH) agonist therapy. All patients received 200 mg of ketoconazole thrice daily. Response was defined as a decline in PSA of at least 50% from the pre-treatment level and confirmed by a second PSA value 4 or more weeks later. The endpoints evaluated were the presence and duration of a response and the toxicity profile of the treatment.

RESULTSA total of 32 patients with HRPC were treated with ketoconazole. Twelve (38%) of the 32 patients had a greater than 50% decrease in their PSA values. The median duration of response was 6.75 months. The median time to reach PSA nadir was 3.5 months. Five patients continue to exhibit progression-free response at the time of writing. Ketoconazole was generally well tolerated. Eighteen (56%) patients recorded mild toxicities related to ketoconazole. There were no grade 3 or 4 toxicities.

CONCLUSIONSLow-dose ketoconazole bridges the gap in the continuum of treatment for patients who have failed ADT and in whom cytotoxic chemotherapy would have a significant impact on the quality of life. Its good toxicity profile, low cost and ease of administration makes it a viable option for this group of patients.

Adenocarcinoma ; Aged ; Aged, 80 and over ; Androgen Antagonists ; administration & dosage ; therapeutic use ; Androgens ; biosynthesis ; Humans ; Ketoconazole ; administration & dosage ; pharmacology ; Male ; Middle Aged ; Prostate-Specific Antigen ; Prostatic Neoplasms ; drug therapy ; Retrospective Studies ; Singapore