Wenhao Shen

Celastrol suppresses tumor cell growth through targeting an AR-ERG-NF-κB pathway in TMPRSS2/ERG fusion gene expressing prostate cancer.

The TMPRSS2/ERG (T/E) fusion gene is present in the majority of all prostate cancers (PCa). We have shown previously that NF-kB signaling is highly activated in these T/E fusion expressing cells via phosphorylation of NF-kB p65 Ser536 (p536). We therefore hypothesize that targeting NF-kB signaling may be an efficacious approach for the subgroup of PCas that carry T/E fusions. Celastrol is a well known NF-kB inhibitor, and thus may inhibit T/E fusion expressing PCa cell growth. We therefore evaluated Celastrol’s effects in vitro and in vivo in VCaP cells, which express the T/E fusion gene. VCaP cells were treated with different concentrations of Celastrol and growth inhibition and target expression were evaluated. To test its ability to inhibit growth in vivo, 0.5 mg/kg Celastrol was used to treat mice bearing subcutaneous VCaP xenograft tumors. Our results show Celastrol can significantly inhibit the growth of T/E fusion expressing PCa cells both in vitro and in vivo through targeting three critical signaling pathways: AR, ERG and NF-kB in these cells. When mice received 0.5 mg/kg Celastrol for 4 times/week, significant growth inhibition was seen with no obvious toxicity or significant weight loss. Therefore, Celastrol is a promising candidate drug for T/E fusion expressing PCa. Our findings provide a novel strategy for the targeted therapy which may benefit the more than half of PCa patients who have T/E fusion expressing PCas.

Roles of stem cell factor on loss of interstitial cells of Cajal in bladder of diabetic rats.

OBJECTIVE To explore the roles of stem cell factor (SCF) on the loss of interstitial cells (ICCs) in the bladder of diabetic rats, which have not been investigated. METHODS The rats were assigned to 3 groups: normal control rats, diabetic rats, and SCF-treated diabetic rats. The diabetic rat model was created using a streptozotocin (60 mg/kg) intraperitoneal injection. The SCF and c-kit levels in bladder tissue were determined using reverse transcriptase-polymerase chain reaction and Western blot analysis. The quantity of ICCs as represented by c-kit-positive cells was examined by image analysis of immunofluorescence staining. RESULTS Compared with the control rats, the diabetic rats exhibited a significant decrease in the SCF levels and c-kit expression and number of ICCs in the bladder tissues. All these impaired parameters were effectively restored to the control level after exogenous SCF treatment. CONCLUSION These findings suggest that the loss of ICCs in the bladder tissue of diabetic rats can be attributed to a deficiency in endogenous SCF. The beneficial effect of exogenous SCF on diabetic depletion of ICCs could provide a theoretical rationale for the use of SCF as a potential therapeutic drug in treating patients with diabetes-related voiding dysfunction.

Cathepsin L is Associated with Proliferation and Clinical Outcome of Urothelial Carcinoma of the Bladder

Increasing evidence suggests that the activity of cysteine proteases, including cathepsin L, is important in cancer cell invasion and metastasis. This study was designed to investigate the clinicopathological and prognostic significance of cathepsin L in human urothelial carcinoma of the bladder (UCB). Standard immunohistochemistry was used to determine the presence of cathepsin L and Ki-67 (a marker of proliferation) in paraffin-embedded specimens of 82 human UCB cases. Cathepsin L protein was localized in the cytoplasm of the malignant UCB cells, and was significantly associated with the pathological tumour stage (invasiveness), tumour grade, survival, local tumour recurrence during follow-up, the occurrence of distant metastasis during follow-up and the presence of Ki-67 protein. Multivariate analysis using the Cox proportional hazards model revealed that cathepsin L immunopositivity and pathological tumour stage (invasiveness) were independent significant prognostic factors for overall survival. This study showed that cathepsin L provides significant prognostic information and that it might be a useful therapeutic target in the future.

Identification and Management of Emptying Failure in Male Patients With Orthotopic Neobladders After Radical Cystectomy for Bladder Cancer

OBJECTIVE To treat neobladder emptying failure after radical cystectomy in patients with bladder cancer. The etiology of neobladder emptying failure should be identified. METHODS We analyzed the outcome of neobladder emptying in 231 male patients who received neobladder reconstruction after radical cystectomy. The clinical characteristics, urodynamic evaluation, and treatment information were collected from all patients with emptying failure. RESULT The total occurrence of neobladder emptying failure was 37 of 231 (16%). Emptying failure was a result of mechanical obstruction in 25 (10.8%) patients; obstructions were caused by strictures of the neobladder-urethral anastomosis (13 cases, 5.6%), anterior urethral strictures (3 cases, 1.2%), obstructive mucosal valves (2 cases, 0.9%), primary cystolithiasis (1 case, 0.4%), mucus plugs (2 cases, 0.9%), urethral tumor recurrence (2 cases, 0.9%), and pelvic tumor recurrence (2 cases, 0.9%). In 21 of 25 patients with mechanical obstructions, bladder function was completely recovered via an endourological approach. However, in 12 of patients with dysfunctional voiding, 3 patients presented higher compliance of neobladder. Two patients were found with a narrower posterior urethral angle. Eventually, 10 patients of 12 with dysfunctional voiding performed intermittent self-catheterization. CONCLUSIONS The obstructive outlet was the primary cause of emptying failure in neobladders. Most of the patients with mechanical obstructions could obtain satisfactory neobladder emptying by a minimally invasive surgical approach. However, nearly all the patients with dysfunctional voiding will have to receive clean intermittent catheterization until the mechanisms causing failure are better understood.

Differential expression of androgen, estrogen, and progesterone receptors in benign prostatic hyperplasia

This study aimed to identify the differential expression levels of androgen receptor (AR), estrogen receptors (ERα, ERβ), and progesterone receptor (PGR) between normal prostate and benign prostatic hyperplasia (BPH). The combination of immunohistochemistry, quantitative real-time reverse transcription polymerase chain reaction, and Western blotting assay was used to identify the distribution and differential expression of these receptors at the immunoactive biomarker, transcriptional, and protein levels between 5 normal human prostate tissues and 40 BPH tissues. The results were then validated in a rat model of BPH induced by testosterone propionate and estradiol benzoate. In both human and rat prostate tissues, AR was localized mainly to epithelial and stromal cell nuclei; ERα was distributed mainly to stromal cells, but not exclusively; ERβ was interspersed in the basal layer of epithelium, but sporadically in epithelial and stromal cells; PGR was expressed abundantly in cytoplasm of epithelial and stromal cells. There were decreased expression of ERα and increased expression of PGR, but no difference in the expression of ERβ in the BPH compared to the normal prostate of both human and rat. Increased expression of AR in the BPH compared to the normal prostate of human was observed, however, the expression of AR in the rat prostate tissue was decreased. This study identified the activation of AR and PGR and repression of ERα in BPH, which indicate a promoting role of AR and PGR and an inhibitory role of ERα in the pathogenesis of BPH.This study aimed to identify the differential expression levels of androgen receptor (AR), estrogen receptors (ERα, ERβ), and progesterone receptor (PGR) between normal prostate and benign prostatic hyperplasia (BPH). The combination of immunohistochemistry, quantitative real-time reverse transcription polymerase chain reaction, and Western blotting assay was used to identify the distribution and differential expression of these receptors at the immunoactive biomarker, transcriptional, and protein levels between 5 normal human prostate tissues and 40 BPH tissues. The results were then validated in a rat model of BPH induced by testosterone propionate and estradiol benzoate. In both human and rat prostate tissues, AR was localized mainly to epithelial and stromal cell nuclei; ERα was distributed mainly to stromal cells, but not exclusively; ERβ was interspersed in the basal layer of epithelium, but sporadically in epithelial and stromal cells; PGR was expressed abundantly in cytoplasm of epithelial and stromal cells. There were decreased expression of ERα and increased expression of PGR, but no difference in the expression of ERβ in the BPH compared to the normal prostate of both human and rat. Increased expression of AR in the BPH compared to the normal prostate of human was observed, however, the expression of AR in the rat prostate tissue was decreased. This study identified the activation of AR and PGR and repression of ERα in BPH, which indicate a promoting role of AR and PGR and an inhibitory role of ERα in the pathogenesis of BPH.

Long-Term Evaluation of Patients Undergoing Genitoplasty due to Disorders of Sex Development: Results from a 14-Year Follow-Up

Purpose. To summarize the experience in treating patients with genitoplasty due to disorders of sex development in China. Methods. The operative procedures, gender of rearing, surgical outcome, and psychosocial and family adjustments of 262 patients were reviewed retrospectively. Results. At initial diagnosis, the mean age was 14.3 ± 2.8 years (range: 2–38 years). There were 96 children, 133 adolescents, and 33 adults. Follow-up was done every 6 months. Patients with female sex assignment had no urinary incontinence or voiding difficulty. Five patients underwent the second surgery (3%); vaginal dilation was performed in 35 patients with postoperative vaginal stenosis; 12 patients (7.4%) were unsatisfactory with the outcome. For patients with male sex assignment, the median length of penis was 2.2 cm in prepubertal patients, 4.2 cm in pubertal patients, and 5.0 cm in adults; 39 patients developed postvoid dribbling (39%); 21 patients underwent a second surgery (21%); urethral dilation was done in 28 patients (28%) due to urethral stricture; 38 patients were unsatisfactory with the outcome (38%). In addition, 136 patients (83%) with female sex assignment and 54 (54%) with male sex assignment had favorable psychosocial adjustment. Conclusions. Patients with male sex assignment have more surgical complications and difficulties in psychosocial adjustment as compared to those with female sex assignment.

Platelet-derived growth factor-BB increases expression of connexin 43 in an extracellular-regulated protein kinase-dependent manner in bladder smooth muscle cells

To investigate whether platelet‐derived growth factor‐BB induces the upregulation of connexin 43 in bladder smooth muscle cells and to examine the involved signaling pathway.

Ultrastructural features and possible functional role of kit-positive interstitial cells in the guinea pig corpus cavernosum.

The objective of the present study was to identify kit-positive interstitial cells (ICs) in guinea pig corpus cavernosum and examine their relationships with adjacent smooth muscle cells (SMCs) and intramural nerves. In addition, we investigated the possible involvement of ICs in nitric oxide (NO)-mediated relaxation of corpus cavernosum smooth muscle (CCSM). ICs were identified by their immunoreactivity to the kit receptor, a cell surface marker encoded by c-kit proto-oncogene and specific for interstitial cells of Cajal. ICs were abundantly distributed in guinea pig corporal tissues. Ultrastructural investigation by conventional transmission electron microscopy revealed the ultrastructural features of ICs and gap junctions located between ICs and adjacent SMCs, furthermore, a close contact between ICs and intramural nerves for the first time. Western blot analysis of purified ICs by fluorescence-activated cell sorting revealed coexpression of soluble guanylate cyclase (sGC)α1, sGCβ1 and kit receptor tyrosine kinase protein in them. These observations imply that ICs express the NO-sensitive sGC molecule and may be involved in the NO-mediated relaxation of CCSM in the guinea pig corpus cavernosum.

Telomerase reverse transcriptase genetically modified adipose tissue derived stem cells improves erectile dysfunction by inhibiting oxidative stress and enhancing proliferation in rat model

Erectile dysfunction (ED) is considered to be incapable of obtaining or/and keeping a sufficient erection function to receive the satisfactory during the sexual intercourse. This study aims to investigate the effects of telomerase reverse transcriptase (hTERT) modified adipose tissue derived stem cells (ADSCs) autologously injected into cavernosa of the ED rats on erectile function. The ADSCs were isolated form the rat subcutaneous adipose tissue sample, and identified by examining the CD29 and CD44 molecule. The ED model was established by using 100μg/kg apomorphine (APO). The adenovirus expressing rat hTERT (Ade-hTERT vector) was established, and transfected into ADSCs and injected into ED rat model, respectively. Telomerase activity, cell growth, cell apoptosis were analyzed by using TRAP ELISA assay, CCK8 assay and flow cytometry assay, respectively. The trophic growth factors were examined by using enzyme-linked immunosorbent assay (ELISA). The mRNA and proteins were detected by using semi-quantitative PCR and western blot assay, respectively. Ade-hTERT vector was highly expressed in both ADSCs and ED rat mode. The hTERT expression enhanced the telomerase activity, inhibits cell apoptosis and enhances proliferation of ADSCs (P<0.05). hTERT expression triggers the secretory function of ADSCs and induces differentiative potential of ADSCs. hTERT expression inhibits apoptosis and increases eNOS and nNOS levels in older ED rats compared to the Ade-vector injected ED rats (P<0.05). In conclusion, the hTERT modification could enhance the ADSCs proliferation, and hTERT modified ADSCs could increase the anti-oxidative stress capacity in the ED rat model.

Synthesis and pharmacological evaluation of novel epidermal growth factor receptor inhibitors against prostate tumor cells

The aim of the present study was to investigate the activities of novel synthetic epidermal growth factor receptor (EGFR) inhibitors (ZINC05463076, ZINC2102846 and ZINC19901103) against prostate tumors, in vitro models and investigate the potential underlying mechanisms. A panel of prostate tumor cell lines (LNCaP, DU-145, PC-3 and LNCaP-AI cells) were used to evaluate antitumor activity of ZINC05463076, ZINC2102846, and ZINC19901103 in vitro. Cell growth and clonal formation were determined by MTT assay and Soft agar colony formation assay, respectively. An EGFR kinase assay following treatment of the compounds was performed by ELISA. Cell cycle-regulating proteins, including cyclin-dependent kinase (CDK)1, CKD2, CKD4 and inhibitory effects of these compounds on downstream signaling were analyzed by western blotting. Flow cytometry was performed to investigate apoptosis and cell cycle phases of the treated cells. It was revealed that all compounds synthesized in the present study demonstrated significant EGFR inhibition abilities, compared with approved EGFR inhibitor drug gefitinib. Treatment of LNCaP, DU-145, PC3 and LNCaP-AI cells with these compounds revealed cell proliferation inhibition and colony formation suppression dose-dependently in vitro. The agents impaired phosphorylation of EGFR and extracellular signal-regulated kinase 1/2 and suppressed their downstream signaling. In addition, these novel synthetic agents decreased the expression level of survivin, which may induce G1 cell cycle phase arrest and cell apoptosis in PCa cells subsequently. Collectively, ZINC05463076, ZINC2102846 and ZINC19901103 exhibited significant antitumor activity in human prostate tumors in vitro, by inhibiting EGFR and promoting apoptosis, which suggested a rationale for clinical development in prostate tumor therapy.