[Expression of macrophage inflammatory protein-1alpha in EPS and its significance].

OBJECTIVE: To detect the mRNA and protein expressions of MIP-1alpha in EPS and determine their significance in the sub-typing of chronic prostatitis. METHODS: We collected samples of expressed prostatic secretion (EPS) from 50 cases of chronic prostatitis, including 16 cases of chronic bacterial prostatitis (CBP), 23 cases of chronic nonbacterial prostatitis/chronic pelvic pain syndrome (CPPS) (11 CPPS IIIA, 12 CPPS IIIB), and 11 cases of type-IV asymptomatic inflammatory prostatitis (AIP). Another 15 healthy volunteers were included as normal controls. The mRNA and protein levels of MIP-1alpha in EPS were determined by RT-PCR and ELISA, respectively, followed by statistical analysis with SPSS 15.0. RESULTS: The mRNA expression of MIP-1alpha was markedly higher in the CPPS IIIA and CPPS IIIB groups than in the others (P<0.05). The protein level of MIP-1alpha was (1174.3 +/- 89.2) pg/ml in CPPS IIIA and (842.3 +/- 76.2) pg/ml in CPPS IIIB, significantly higher than (198.0 +/- 37.8) pg/ml in the control, (347.0 +/- 61.6) pg/ml in CBP and (292.0 +/- 56.4) pg/ml in type-IV AIP (P<0.05). CONCLUSION: Determination of mRNA and protein levels of MIP-1alpha in EPS may help the sub-typing and diagnosis of chronic prostatitis.

[Expression and regulation of androgen receptor in the androgen-independent conversion of prostate cancer cells].

OBJECTIVE: To investigate the expression and regulation of androgen receptor (AR) in prostate cancer cells from androgen dependent to androgen independent. METHODS: LNCaP cells were cultured in charcoal-stripped serum for 6 months to establish androgen-independent celline (LNCaP-AI). Proliferation of LNCaP-AI was assayed by cell viability. Expression of AR mRNA and protein was analyzed by RT-PCR and Western blot. Wnt signaling pathway inhibitor IWR-1 and proteasome inhibitor lactacystin were used to investigate effects of Wnt and proteasome pathway on AR expression in LNCaP-AI. RESULTS: LNCaP-AI exhibit enhanced proliferation and up-regulated PSA expression compared with LNCaP. During androgen deprivation, AR mRNA was up-regulated in a short early stage and then declined to a stable level in LNCaP-AI compared with LNCaP, but AR protein kept in downward trend. The mRNA and protein expression of AR was decreased by IWR-1 treatment. AR protein but not mRNA was increased by lactacystin treatment. CONCLUSION: The androgen independent prostate cancer cell line was established by androgen deprivation, in which the protein expression of AR was dramatically decreased. mRNA and protein expression of AR in LNCaP-AI was related to Wnt signaling pathway and proteasome pathway. Increased Wnt signaling or decreased proteasome pathways contribute the decreased AR protein expression.

Reproductive outcomes of intracytoplasmic sperm injection using testicular sperm and ejaculated sperm in patients with AZFc microdeletions: a systematic review and meta-analysis.

Studies have explored the assisted reproductive technology (ART) outcomes of Y-chromosome azoospermia factor c (AZFc) microdeletions, but the effect of sperm source on intracytoplasmic sperm injection (ICSI) remains unknown. To determine the ART results of ICSI using testicular sperm and ejaculated sperm from males with AZFc microdeletions, we searched Embase, Web of Science, and PubMed to conduct a systematic review and meta-analysis. The first meta-analysis results for 106 cycles in five studies showed no significant differences in the live birth rate between the testicular sperm group and the ejaculated sperm group (risk ratio: 0.97, 95% confidence interval [CI]: 0.73-1.28, P = 0.82). The second meta-analysis of 106 cycles in five studies showed no difference in the abortion rate between the testicular sperm group and ejaculated sperm group (risk ratio: 1.06, 95% CI: 0.54-2.06, P = 0.87). The third meta-analysis of 386 cycles in seven studies showed no significant difference in clinical pregnancy rates between the testicular sperm group and the ejaculated sperm group (risk ratio: 1.24, 95% CI: 0.66-2.34, P = 0.50). Inevitable heterogeneity weakened our results. However, our results indicated that testicular sperm and ejaculated sperm yield similar ART outcomes, representing a meaningful result for clinical treatment. More properly designed studies are needed to further confirm our conclusions.

Effects of icariin on erectile function and expression of nitric oxide synthase isoforms in castrated rats.

AIM: To investigate the effect of icariin on erectile function and the expression of nitric oxide synthase (NOS) isoforms in castrated rats. METHODS: Thirty-two adult male Wistar rats were randomly divided into one sham-operated group (A) and three castrated groups (B, C and D). One week after surgery, rats were treated with normal saline (groups A and B) or oral icariin (1 mg/[kg.day] for group C and 5 mg/[kg.day] for group D) for 4 weeks. One week after treatment, the erectile function of the rats was assessed by measuring intracavernosal pressure (ICP) during electrostimulation of the cavernosal nerve. The serum testosterone (ST) levels, the percent of smooth muscle (PSM) in trabecular tissue, and the expression of mRNA and proteins of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and phosphodiesterase V (PDE5) in corpus cavernosum (CC) were also evaluated. RESULTS: ICP, PSM, ST and the expression of nNOS, iNOS, eNOS and PDE5 were significantly decreased in group B compared with those in group A (P 0.01). However, ICP, PSM and the expression of nNOS and iNOS were increased in groups C and D compared with those in group B (P 0.05). Changes in ST and the expression of eNOS and PDE5 were not significant (P 0.05) in groups C and D compared with those in group B. CONCLUSION: Oral treatment with icariin ( 98.6 % purity) for 4 weeks potentially improves erectile function. This effect is correlated with an increase in PSM and the expression of certain NOS in the CC of castrated rats. These results suggest that icariin may have a therapeutic effect on erectile dysfunction.

YAP is closely correlated with castration-resistant prostate cancer, and downregulation of YAP reduces proliferation and induces apoptosis of PC-3 cells.

Yes-associated protein 65 (YAP65) has been implicated as an oncogene, and its expression is increased in human cancer. Previous studies have demonstrated that alterations in YAP activity may result in tumourigenesis of the prostate. With androgen deprivation therapies becoming progressively ineffective, often leading to life­threatening androgen­resistant prostate cancer (CRPC). The present study aimed to analyse the role of YAP in prostate cancer (PCa), particularly in CRPC. YAP protein was detected using immunohistochemistry and western blot analysis in different prostatic tissues. In addition, three specific RNA interference vectors targeting the human YAP gene were synthesised, and PC­3 cells with a stable inhibition of YAP were obtained by transfection. MTT, flow cytometry, reverse transcription­quantitative polymerase chain reaction and western blot assays were used to analyse the effects of YAP inhibition on the proliferation and apoptosis of PC­3 cells. The frequency of cells that were positive for YAP protein in PCa (78.13%) was significantly higher, compared with para­PCa (26.67%; P=0.007) and benign prostatic hyperplasia (0%; P=0.002). The frequency of cells, which were positive for the expression of YAP exhibited a positive correlation (P=0.008) with the Gleason score, the tumour­node­metastasis staging (P=0.033) and the level of prostate specific antigens (P=0.0032) in PCa. The proliferative capacity of the transfected group was significantly lower, compared with the negative control group (P=0.022). The cell­cycle of the transfected group was arrested in the G1 stage, which was detected using flow cytometry, and there was a significant increase in the apoptosis of cells in the transfected group (P=0.002). The mRNA and protein levels of TEA domain family member 1 were inhibited in the transfected group (P=0.001 and P=0.00, respectively). Therefore, it was concluded that gene transcription and protein expression of YAP may be involved in the development of PCa, particularly CRPC, and may be a novel biomarker for investigation of the occurrence and progression of CRPC. However, the mechanism underlying the modulation of YAP in CRPC remains to be fully elucidated.

Effect of renewed SS-cream on spinal somatosensory evoked potential in rabbits.

AIM: The effect of a renewed SS-cream (RSSC) on the treatment of premature ejaculation (PE) was evaluated and compared with the original SS-cream (OSSC). METHODS: Sixty male white New Zealand rabbits, weighing 2.5 kg-3.0 kg, were divided at random into 3 groups: the RSSC, OSSC and placebo groups. The spinal somatosensory evoked potential (SSEP) elicited by electric stimulation of the glans penis with disk electrode was investigated with an electrophysiograph (Poseidomn, Shanghai, China) before and 10, 30 and 60 min after drug or placebo application on the glans. The Onset and the N1 latencies and the amplitude of SSEP were recorded and analyzed. RESULTS: There was no significant difference (P>0.05) in the mean Onset and N1 latency of SSEP among the 3 groups before drug application. Compared with the pre-application value, the mean Onset and N1 latencies in the RSSC and OSSC groups were significantly prolonged at 10, 30 and 60 min after treatment (P<0.05), while they were not significantly changed (P>0.05) in the placebo group. The mean Onset latency of RSSC at 10 and 30 min and that of OSSC at 30 min were significantly delayed (P<0.05) compared with the placebo group. The mean N1 latency of RSSC at 30 and 60 min and that of OSSC group at 30 min were also significantly delayed (P<0.05). CONCLUSION: RSSC delays the latencies of SSEP, suggesting a local desensitizing effect on the sensory receptor of the glans penis dorsal nerve, which provides the potential for PE treatment. The desensitizing effect of RSSC is higher than that of OSSC.

Sexual function of premature ejaculation patients assayed with Chinese Index of Premature Ejaculation.

AIM: To assess the psychometric properties of the Chinese Index of Premature Ejaculation (CIPE). METHODS: The sexual function of 167 patients with and 114 normal controls without premature ejaculation (PE) were evaluated with CIPE. All subjects were married and had regular sexual activity. The CIPE has 10 questions, focusing on libido, erectile function, ejaculatory latency, sexual satisfaction and difficulty in delaying ejaculation, self-confidence and depression. Each question was responded to on a 5 point Likert-type scale. The individual question score and the total scale score were analyzed between the two groups. RESULTS: There were no significant differences between the age, duration of marriage and educational level (P> 0.05) of patients with and without PE and normal controls. The mean latency of patients with PE and normal controls were 1.6 +/- 1.2 and 10.2 +/- 9.5 minutes, respectively. Significant differences between patients with (26.7 +/- 4.6) PE and normal controls (41.9 +/- 4.0) were observed on the total score of CIPE (P< 0.01). Using binary logistic regression analysis, PE was significantly related to five questions of the original measure. They are the so-called the CIPE-5 and include: ejaculatory latency, sexual satisfaction of patients and sexual partner, difficulty in delaying ejaculation, anxiety and depression. Receiver Operating Characteristic (ROC) curve analysis of CIPE-5 questionnaire indicated that the sensitivity and specificity of CIPE were 97.60 % and 94.74 %, respectively. Employing the total score of CIPE-5, patients with PE could be divided into three groups: mild (>15 point) 19.8 %, moderate (10-14 point) 62.8 % and severe (< 9 point) 16.7 %. CONCLUSION: The CIPE-5 is a useful method for the evaluation of sexual function of patients with PE and can be used as a clinical endpoint for clinical trials studying the efficacy of pharmacological intervention.

[Peroxisome proliferator-actived receptor-gamma ligand troglitazone induces apoptosis in renal cell carcinoma].

UNLABELLED: OBJECTIVE To investigate the expression of peroxisome proliferator-actived receptor-gamma (PPAR-gamma)and the inducement of apoptosis by PPAR-gamma ligand in renal cell carcinoma(RCC)-derived cell lines. METHODS: RT-PCR and Western blot analysis were performed to determined the expression of PPAR-gamma mRNA and protein in two RCC derived cell lines(786-O and A498) and two normal kidney(NK)-derived cell lines(HK-2 and HMCC). Two RCC cell lines were treated with 50 micromol/L troglitazoned for and evaluated for the effects of antidiabetic thiazolidinediones (TZDs) on the cells apoptosis by fluorescence microscopy and DNA ladder assay. The mutative expressions of Bcl-2 and Bax before and after TZDs treatment were also performed by western blot analysis. RESULTS: The expression of PPAR-gamma was observed to be stronger in 786-O and A498 cells than in HK-2 and HMCC cells by RT- PCR and Western blot analysis. Treated with 50 micromol/L troglitazone (for 48 h) it induced typical apoatosis in 786-O and A498 cells. After treatment, a decrease in Bcl-2 expression in RCC cells was observed by Western blot analysis,and the expression of Bax,however,was up-regulated. CONCLUSION: The results reveal that troglitazone has the tumor-suppressive effect on RCC cells. High-affinity PPAR-gamma ligands (TZDs) may be the candidates for a novel approach to the treatment of this refractory neoplasm.

[Effects of icariin on intracavernosal pressure and systematic arterial blood pressure of rat].

OBJECTIVE: To realize the effect of icariin on erectile function of penis. METHODS: After the cavernous nerve (CN) of rat was isolated unilaterally, the corpora cavernosa (CC) and right carotid artery were exposed. A 26G needle catheter was inserted into the right CC to monitor the intracavernous pressure (ICP), and another 26G needle catheter was inserted into the left CC for drug administration. Another catheter was placed into the carotid artery to monitor mean systematic arterial blood press (MBp). Icariin of different concentrations was administrated intracavernosally, and the ICP and MBp were recorded during electric stimulation on CN. Sildenafil and papaveine were used as controls. The effects of nitric oxide syntheses (NOS) inhibitor N(omega)-nitro-L-arginine (LNNA), and soluble guanylate cyclase inhibitor H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (ODQ) on icariin (10(-4)mol/L) induced ICP changes were investigated also. RESULTS: Icariin, sidenafil and papaverine increased the ICP in a dose-depended manner (P < 0.01). Icariin and sildenafil did not influence the MBp (P > 0.05), however, papaverine significant influenced MBp (P < 0.01). EC(50) of Icariin, sildenafil and papaveine on ICP/MBp were 2.23 micro mol/L, 0.24 micro mol/L and 9.73 micro mol/L respectively. ODQ and LNNA significantly decreased ICP induced by icariin (10(-4)mol/L). CONCLUSION: Icariin increases ICP without influence on MBp and such effect is inhibited by LNNA and ODQ significantly. Icariin regulates the activity of NO-cGMP signal pathway on CC to enhance erectile function by oral therapy.

[Effects of icariin on the erectile function and expression of nitrogen oxide synthase isoforms in corpus cavernosum of arterigenic erectile dysfunction rat model].

OBJECTIVE: The study the effects of oral administered icariin on intracavernosal pressure (ICP) and on expression of the nitrogen oxide synthase (NOS) isoforms in corpus cavernosum (CC) of arteriogenic erectile dysfunction (A-ED) rat model. METHODS: Forty adult male Wistar rats were randomly divided into 4 groups of 10 rats: shame operated group (group A) and three A-ED model groups (group B, C and D). The internal pudendal arteries were isolated and ligated with 7-O nylon thread at both the main trunk and the penile branches to establish the A-ED model. ICP were tested after the operation to make sure the successful model establishment. The groups A and B were treated with saline: and the groups C and D were treated with icariin (5 mg/kg/day and 10mg/kg/day respectively) orally for 30 days. Then the ICP was measured again. The tissues of corpus cavernosum were taken and RT-PCR was used to detect the mRNA expression of nNOS, iNOS and eNOS in CC, and Western-blot was used to detect the protein expression of these NOS isoforms. RESULTS: The ICP in the group B was significantly decreased compared to the group A (P < 0.01), but the ICP values in the groups C and D were both increased compared to those in the group B (both P < 0.01). The expressions of the mRNA and protein of nNOS, iNOS, and eNOS were all decreased in the group B, however, the mRNA and protein expressions of eNOS were increased a in the groups C and D. In the group C, iNOS also increased. The expression of nNOS showed no obvious changes in the group C and group D. CONCLUSION: Chronic oral treatment with Icariin increases the erectile function (ICP) and restores the eNOS expression in CC of A-ED rats. Icariin may have a long-term therapeutic effect on ischemia/hypoxia induced ED.

Methylation­associated inactivation of LATS1 and its effect on demethylation or overexpression on YAP and cell biological function in human renal cell carcinoma.

Large tumor suppressor 1 (LATS1) gene is one of the key factors in Hippo signaling pathway. Inactivation of LATS1 by promoter methylation was found in colorectal cancer (CRC), head and neck squamous cell carcinoma (HNSCC), astrocytoma, breast cancer and it was proved to be a tumor suppressor. However, its role is unclear in renal cell carcinoma (RCC). In this study, the expression of LATS1 was determined by reverse transcription polymerase chain reaction (RT­PCR) and immunohistochemistry in 30 pairs of RCC tissues and matched normal kidney tissues and RCC cells. We found that the expression of LATS1 was markedly reduced in RCC tissues and cells, in the RCC tissue in 46.7% (14/30), while in the normal kidney tissues in 76.7% (23/30), and was associated with pathological grade and clinical stage of RCC. We detected methylation status of LATS1 by bisulfite sequence-PCR (BSP) in renal cancer cell line 786-O which lowers expression of LATS1, and we found it hypermethy-lated (in 97.5%). In addition, pharmacological demethylation using 5-Aza-2'-deoxycytidine (5-Aza) restored the expression of LATS1 mRNA and protein in 786-O cells, both LATS1 demethylation and overexpression of LATS1 downregulated the expression of Yes-associated protein (YAP), inhibited cell proliferation, induced cell apoptosis and cell cycle G1 arrest in 786-O cells. Thus, this report for the first time demonstrates the inactivation of LATS1 by promoter methy-lation and it is a tumor suppressor in kidney cancer. LATS1 may serve as a biomarker for possible early diagnosis and as a potential therapeutic target for human RCC.

YAP is overexpressed in clear cell renal cell carcinoma and its knockdown reduces cell proliferation and induces cell cycle arrest and apoptosis.

Yes-associated protein (YAP) has been reported to be an oncogene in a number of malignancies. It constitutes an important regulatory mechanism for the Hippo pathway, a key regulator of cell growth and apoptosis. The present study aimed to investigate the clinical significance and the role of YAP in the development of clear cell renal cell carcinoma (ccRCC). YAP expression levels were compared between ccRCC and adjacent normal renal tissues by RT-PCR and immunohistochemistry, respectively. YAP expression levels were then detected in ccRCC cell lines 786-0 and ACHN, as well as in human embryonic kidney 293 cells (HEK-293) using western blotting. Three specific YAP-shRNA lentiviral vectors were constructed and transfected into 786-0 cells, and then the mRNA and protein levels of YAP and downstream transcription factor TEAD1 were detected. Finally, the effects of YAP silencing on proliferation and the cell cycle distribution of 786-0 cells were detected by Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM), respectively. The apoptosis rate was also analyzed by FCM. It was observed that the expression levels of YAP mRNA and protein in ccRCC tissues were higher than these levels in the adjacent normal renal tissues. The expression of YAP protein in ccRCC tissues was significantly correlated with clinical stage and differentiation. The YAP protein levels in the two ccRCC cell lines 786-0 and ACHN were significantly higher than that in the HEK-293 cells. Additionally, treatment of 786-0 cells with YAP-shRNA lentiviral vectors significantly reduced the expression levels of YAP and TEAD1 mRNA and protein. Further analyses in 786-0 cells in which YAP was decreased, revealed that cell proliferation was inhibited, cell cycle was arrested at the G1 phase and apoptosis was increased. These results indicate that YAP is an underlying oncogene in ccRCC and it may be a promising biomarker and therapeutic target of ccRCC.

Isolation and enrichment of PC-3 prostate cancer stem-like cells using MACS and serum-free medium.

Prostate cancer stem-like cells (PCSLCs) are considered to be the 'seed' of prostate cancer. The aim of this study was to confirm that the PC-3 cells, which we isolated and enriched from PC-3 cells through magnetic bead cell sorting (MACS) and serum-free medium (SFM) culture, were PCSLCs. Combinations of MACS, flow cytometry (FCM), SFM and immunocytochemistry (ICC) were used to ensure the positive expression of CD133 and CD44 on PC-3 and sphere-forming cell membranes. Self-renewal, multi-potential differentiation, unlimited proliferation and permanency assays were also applied to indentify whether the PC-3 cells exhibited the characteristics of cancer stem cells (CSCs). As a result, there was a low proportion of PCSLCs in the PC-3 cells. In the FCM assay, the proportion of cells expressing CD133 or CD44 in the PC-3 cells was 0.51 and 0.31%, respectively. In addition, we found that the proportion of PC-3 cells sorted by MACS that expressed CD133 was significantly increased compared with that of the sphere-forming cells cultured in SFM (99.09 vs. 84.80%, P<0.05), while no difference was observed in the proportion of cells expressing CD44 between them (99.88 vs. 99.82%, P>0.05). The expression of PAP and AR as detected by western blot analysis of induced PCSLCs was significantly increased compared with that of uninduced PCSLCs (P<0.05); the proliferation capacity of PCSLCs was significantly higher than that of both the PC-3 cells (P<0.05) and induced PCSLCs (P<0.05). Furthermore, the PCSLCs that were isolated from SFM and MACS both demonstrated certain characteristics of stem cells and should be considered as stem cell-like. These data may hold potential for further exploring the role of PCSLCs.