ZEB1-mediated vasculogenic mimicry formation associates with epithelial-mesenchymal transition and cancer stem cell phenotypes in prostate cancer.

The zinc finger E-box-binding homeobox 1 (ZEB1) induced the epithelial-mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid-Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1-induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT-related and CSC-associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p-Src527 level but not p-Src416 level, while ZEB1 knockdown also down-regulated the level of p-Src527 in PC3 and DU-145 cells. PP2 treatment also significantly reduced the expression of VE-cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.

Matrix metalloproteinase-9 is required for vasculogenic mimicry by clear cell renal carcinoma cells.

BACKGROUND: Vasculogenic mimicry (VM), a new pattern of tumor microcirculation system, has been proved to be important for tumor growth and progression and may be one of the causes of antiangiogenesis resistance. Matrix metalloproteinase-9 (MMP9) was shown to correlate with VM formation in some other cancers. However, the relationship between VM formation and MMP9 in renal cell carcinoma (RCC) has not been determined. METHODS: The VM formation and MMP9 expressions were analyzed by CD34/periodic acid-Schiff dual staining and immunohistochemistry in 119 RCC specimens. We used a well-established 3-dimention culture model to compare VM formation in 786-O, 769-P, and HK-2 cell lines in vitro. MMP9 expressions on either messenger RNA or protein levels were compared among the cell lines by quantitative polymerase chain reaction or Western blot. To determine further the relationship between MMP9 and VM in RCC, 786-O and 769-P were treated with specific MMP9 inhibitor or small interfering RNA. VM formation, cell migration, and invasion were subsequently assessed by 3-dimention culture, wound-healing, and transwell assays. RESULTS: Immunohistochemistry demonstrated both VM formation and MMP9 overexpression were positively associated with clinical staging, pathological grade, and metastasis (P<0.01). VM formation was closely correlated with MMP9 overexpression in RCC (r = 0.602, P<0.01). Lower MMP9 expression level was observed in normal kidney cell line HK-2, which was unable to form VM on Matrigel, whereas higher expression of MMP9 was found in VM-forming cancer cell lines 786-O and 769-P. Inhibition of MMP9 not only disrupted VM formation in 786-O and 769-P but also reduced cell migration and invasion. CONCLUSIONS: These results indicate an intimate relationship between MMP9 overexpression and VM formation in RCC. Treatments targeting VM formation by inhibiting the activity of MMP9 could be beneficial in RCC therapy.

The performance of transrectal ultrasound in the diagnosis of seminal vesicle defects: a comparison with magnetic resonance imaging.

Obstructive azoospermia (OA) is one of the most common causes of male infertility. Transrectal ultrasound (TRUS) has been used to diagnose OA for many years. From 2009 to 2013, we evaluated a prospective cohort of 1249 patients with suspected OA using TRUS. It was found that dilation of the ejaculatory duct (ED) (29.9%, 374/1249) was the most common cause of OA, followed by seminal vesicle (SV) abnormalities (28.5%, 356/1249). A total of 237 patients were diagnosed with congenital defects (agenesis and/or hypoplasia) of the SV, constituting more than half of the cases of SV disease in OA (19.0%, 237/1249). In contrast to ED, congenital defects of the SV could not be corrected with surgical treatment. Therefore, it is meaningful to compare TRUS and magnetic resonance imaging (MRI) for accurate diagnosis of SV defects. Among our patients, 30 with agenesis or/and hypoplasia of the SV on TRUS were further evaluated using pelvic MRI within 2 years, with the objective of verifying the TRUS results. The concordance rate for diagnosing congenital defects of the SV was 73.3% (22/30). We concluded that TRUS is a reliable and convenient method for diagnosing agenesis or hypoplasia of the SV in OA patients with a high concordance with MRI while MRI is useful in patients with inconclusive TRUS findings.

Genetic characterization and protein stability analysis of a Chinese family with Von Hippel-Lindau disease.

BACKGROUND: Von Hippel-Lindau disease (VHL), a heritable autosomal dominant disease characterized by neoplasia in multiple organ systems, has rarely been reported in Asia. We genetically investigated a unique Chinese family with VHL disease and performed an analysis of the VHL protein stability. METHODS: Genomic deoxyribonucleic acid (DNA) extracted from peripheral blood was amplified by polymerase chain reaction (PCR) to three exons of the VHL gene in 9 members of the Chinese family with VHL disease. PCR products were directly sequenced. We estimated the effects of VHL gene mutation on the stability of pVHL, which is indicated by the free energy difference between the wild-type and the mutant protein (ΔΔG). RESULTS: The Chinese family was classified as VHL type 1. Three family members, including two patients and a carrier, had a T to G heterozygotic missense mutation at nucleotide 515 of the VHL gene exon 1. This missense mutation resulted in the transition from leucine to arginine in amino acid 101 of the VHL protein. There was low stability of the VHL protein (the ΔΔG was 12.71 kcal/mol) caused by this missense mutation. CONCLUSIONS: We first reported a family with this VHL gene mutation in Asia. This missense mutation is predicted to significantly reduce the stability of the VHL protein and contribute to the development of the renal cell carcinoma (RCC) phenotype displayed by this family. The genetic characterization and protein stability analysis of families with VHL disease are important for early diagnosis and prevention of the disease being passed on to their offspring.

Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P.

Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells as compared with other four cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

Shorter recurrence-free survival time, higher risk of multiple recurrences: a retrospective study of non-muscle invasive bladder cancer after transurethral resection.

BACKGROUND: Multiple recurrences are common in non-muscle invasive bladder cancer, but the risk of multiple recurrences has not been fully described. Identifying patients at high risk of multiple recurrences will help to select an optimal therapeutic strategy and to improve prognosis. This study was conducted to identify the risk factors for multiple recurrences of non-muscle invasive bladder cancer. METHODS: We reviewed the clinical data of all patients with non-muscle invasive bladder cancer in our hospital between January 2003 and February 2010. Patients with at least one recurrence were included. Multivariate analysis was performed for theorized risk factors (age, gender, tumor stage, grade, size, location, number of lesions, adjuvant intra-vesical chemotherapy after transurethral resection, and recurrence-free survival after each resection) to clarify risk factors for multiple recurrences of non-muscle invasive bladder cancer. RESULTS: Of the 278 patients with non-muscle invasive bladder cancer, 84 were with at least one recurrence and a total of 222 recurrences among them were followed up for 6 - 70 months (mean, 36.1 months). Recurrence-free survival after initial resection predicted the overall frequency of bladder cancer recurrence (risk ratio (RR) = 37.83, 95% confidence interval (CI) = 3.45 - 396.13, P = 0.001) and second recurrence (RR = 6.15, 95%CI = 1.28 - 29.57, P = 0.023). Similarly, recurrence-free survival after a second resection was the only significant risk factor for third recurrence (RR = 31.08, 95%CI = 2.53 - 381.47, P = 0.007). Moreover, recurrence-free survival after initial resection was the only significant factor to predict later progression to muscle invasive bladder cancer (RR = 8.62, 95%CI = 1.47 - 58.34, P = 0.001). CONCLUSIONS: Recurrence-free survival after resection is an independent predictor of multiple recurrences of non-muscle invasive bladder cancer. The shorter the period between resection and recurrence is, the higher the risk of multiple recurrences.

Percutaneous ultrasound-guided radiofrequency ablation treatment and genetic testing for renal cell carcinoma with Von Hippel-Lindau disease.

Percutaneous ultrasound-guided radiofrequency ablation is increasingly being studied in the treatment of renal tumors. Because percutaneous ultrasound-guided radiofrequency ablation is a minimally invasive and nephron-sparing procedure, it is ideally suited for patients with a single kidney, multiple tumors, or contraindications to conventional surgery. We report on a patient with Von Hippel-Lindau (VHL) disease who had multicentric tumors in the single kidney that was successfully treated with percutaneous ultrasound-guided radiofrequncy ablation. The one-year follow-up showed that there was no local recurrence or metastasis. And genetic testing showed the patient had a T to G heterozygotic missense mutation at nucleotide 515 of VHL gene exon 1.

The value of contrast-enhanced transrectal ultrasound in predicting the nature of prostate diseases and the Gleason score of prostate cancer by a subjective blood flow grading scale.

OBJECTIVES: To investigate the value of contrast-enhanced transrectal ultrasound (CETRUS) in predicting the nature of prostate diseases and prostate cancer Gleason score. METHODS: 106 patients suspected of prostate cancer were evaluated with CETRUS followed by systematic biopsy. Prostate blood flow of CETRUS was graded using a subjective 5-point scale. The relationships between ultrasound findings and biopsy outcomes, as well as prostate cancer Gleason score were analyzed. Receiver operating characteristic (ROC) analysis was performed to evaluate the diagnostic performance of CETRUS. RESULTS: Biopsy revealed prostate cancer in 43 of 106 patients. The proportions of malignant histology in the groups with CETRUS scores of 1-5 were 0% (0/10), 8.3% (2/24), 31.7% (13/41), 88.9% (16/18) and 92.3% (12/13), respectively. The rate of prostate cancer with a Gleason score ≥7 in the groups with a CETRUS score of 2-5 were 0% (0/2), 15.4% (2/13), 37.5% (6/16) and 91.7% (11/12), respectively. The blood flow grading scale correlated with pathological outcomes and Gleason score significantly (r = 0.66, p < 0.001; and r = 0.61, p < 0.001, respectively). ROC analysis showed the area under the ROC curve to be 0.87. CONCLUSIONS: CETRUS-based blood flow grading scale is a reliable tool for predicting the pathological outcome of prostate diseases and prostate cancer Gleason score noninvasively.

[Transrectal ultrasonography in the etiological diagnosis of male obstructive azoospermia: analysis of 695 cases].

OBJECTIVE: To assess the role of transrectal ultrasonography (TRUS) in the etiological diagnosis of male obstructive azoospermia. METHODS: We retrospectively analyzed the clinical data and TRUS findings of 695 patients with obstructive azoospermia from January 2007 to May 2009. RESULTS: Concerning the etiology of obstructive azoospermia, the main TRUS findings included ejaculatory duct abnormality (29.2%), seminal vesicle abnormality (25.4%) and prostate midline cyst (18.5%). TRUS revealed 203 cases of ejaculatory duct dilation, 177 cases of seminal vesicle abnormality (including 108 with absence or agenesis and 51 with dilation of the seminal vesicle), and 128 cases of prostate midline cyst (including 75 with ejaculatory duct cyst and 39 with Müllerian cyst). Calcification of the verumontanum or ejaculatory duct was suspected to be the causes of obstructive azoospermia in 34 cases. However, no significant etiological abnormality was found in 153 cases. Obvious etiology was shown by TRUS in 78.0% of the patients. CONCLUSION: TRUS can clearly display the structural abnormality of the ejaculatory duct and seminal vesicle, and provide important information on the etiology of male obstructive azoospermia.

[Value of 3D 1H-MRS with body coil at 3T in the differential diagnosis of the prostate cancer].

OBJECTIVE: To assess the value of three dimensional proton magnetic resonance spectroscopy (3D 1H-MRS) with body coil at 3T in the differential diagnosis of prostate cancer. METHODS: Forty patients suspected of prostate cancer underwent MRI and MRS examinations, and then transrectal ultrasound guided prostate biopsy for pathological diagnosis. The MRI and MRS features of benign prostate hyperplasia, prostate cancer and prostatic intraepithelial neoplasia (PIN) were analyzed in comparison with the pathological reports, and the receiver operating characteristic curve was drawn for the diagnosis of cancer from peripheral zones. RESULTS: The examinations were accomplished for all the patients. The mean ratios of (Cho + Cre)/Cit in the interstitial and glandular hyperplasia tissues, the cancer tissue of the central and peripheral glands, the healthy peripheral gland and PIN were 0.75 +/- 0.23, 0.59 +/- 0.14, 1.79 +/- 0.90, 1.18 +/- 0.95, 0.46 +/- 0.18, and 0.97 +/- 0.10, respectively, with statistically significant differences between the cancer and normal prostate tissues (P < 0.01). The optimum threshold for the diagnosis of prostate cancer in the peripheral zone was 0.68 with a sensitivity of 88.6% and a specificity of 88.7%. CONCLUSION: The 3D 1H-MRS with body coil at 3T has a high sensitivity and specificity in the differential diagnosis of prostate cancer, and can provide valuable information for the diagnosis of PIN.

Utility of fluorescence in situ hybridization in the diagnosis of upper urinary tract urothelial carcinoma.

The purpose of this study was to evaluate the clinical utility of fluorescence in situ hybridization (FISH) assay as a non-invasive method for diagnosing and monitoring urothelial carcinoma (UC) in the upper urinary tract (UUT). Urine specimens from 21 consecutive patients with UUT-UC and 10 healthy controls were analyzed by means of cytology and FISH. For FISH analysis, labeled probes specific for chromosomes 3, 7, and 17 and for the p16 (9p21) gene were used to assess chromosomal abnormalities indicative of malignancy. Sensitivity and specificity of both techniques were determined and compared. The frequency of chromosomal aberrations of malignant cells from UUT was also determined. Overall sensitivity of FISH was significantly higher than that of urine cytology (85.7% vs. 23.8%, p = 0.0009). Specificities for both FISH and cytology were 100% (p = ns). Of 21 patients with UUT-UC, polysomies of chromosome 3, 7 and 17 were observed in 57.1%, 52.4% and 28.6%, respectively, and loss of the p16 gene in 47.6%. FISH has a higher sensitivity than cytology and a similar specificity in dectecting UUT-UC. It may be a promising non-invasive tool for the diagnosis and surveillance of UUT-UC.

[Testicular microlithiasis influences seminal profile and testicular blood flow in infertile men].

OBJECTIVE: To investigate the testicular blood flow in patients with testicular microlithiasis (TM) and its correlation with the seminal profile in infertile men. METHODS: We selected 88 infertile men and examined them by testicular color Doppler and routine seminal tests. RESULTS: Testicular microlithiasis was found in 19 (19.3%) of the patients, classic testicular microlithiasis (CTM) in 7 (8.0%), and limited testicular microlithiasis (LTM) in 10 (11.3%). No significant differences were observed in the age of onset, bilateral testicular volume, resistance index (RI) of bilateral testicular arteries, semen amount and the rate of teratospermia. The bilateral testicular peak systolic velocity (PSV), sperm count and sperm motility were significantly lower in the CTM than in the LTM group (P < 0.05), but showed no statistically significant difference between the LTM and the non-calcification group. CONCLUSION: TM may be one of the causes of poor sperm function in infertile men.

[Value of transrectal ultrasonography in the diagnosis of midline prostatic cysts].

OBJECTIVE: To assess the value of transrectal ultrasonography (TRUS) in the diagnosis of midline prostatic cysts. METHODS: We retrospectively analyzed the TRUS manifestations of 87 cases of midline prostatic cysts. RESULTS: Of the total number, 33 cases were diagnosed as Müllerian duct cysts, 21 cases ejaculatory duct cysts and the other 33 cases undifferentiated midline prostatic cysts; 19 cases had dilated seminal vesicles, 19 seminal vesicle agenesis, 9 seminal vesiculitis and 5 dilation of the ejaculatory duct. CONCLUSION: TRUS, convenient, sensitive, safe and non-invasive, is a desirable method for the diagnosis of midline prostatic cysts.

High expression of PSM-E correlated with tumor grade in prostate cancer: a new alternatively spliced variant of prostate-specific membrane antigen.

BACKGROUND: Prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) has been targeted for therapy and diagnosis of PCa. In the current study, PSMA cDNA was cloned from PCa tissue by RT-PCR. After sequencing, a new spliced variant of PSMA (PSM-E) was discovered and its specificity in PCa was evaluated. METHODS: PSM-E and PSMA mRNA were measured in LNCaP, PC-3 and prostate or nonprostatic malignancies. Following transfection of PC-3 with PSM-E cDNA in the pcDNA3.0 vector, PSM-E expression was measured by immunofluorescence and Western-blot. PSM-E and PSMA mRNA levels were quantified by a real-time PCR assay in normal prostate (n = 7), benign prostatic hyperplasia (BPH) (n = 22) and PCa (n = 41). The correlation between their levels and tumor grade was analyzed. RESULTS: PSM-E cDNA is identical to PSMA except for a 97-nucleotide region and a 93-nucleotide region. PSM-E and PSMA mRNA were detected in PCa and LNCaP, not in PC-3; PSMA could be detected in some nonprostatic tumors whereas PSM-E not. The expression of PSM-E protein was detected in transfected cells. Significant difference of PSM-E mRNA levels was observed among normal prostate, BPH and PCa (P < 0.001), and PSM-E levels increased with increasing Gleason score (r = 0.514, P < 0.001). PSMA mRNA levels were higher in BPH and PCa than in normal prostate (P < 0.001), but no difference between BPH and PCa, no significant correlation was observed between PSMA levels and Gleason score (r = 0.229, P = 0.057). CONCLUSIONS: PSM-E may be a potential prognostic indicator for PCa progression and may be a new target antigen for therapy of PCa.

Outcome of implanting penile prosthesis for treating erectile dysfunction: experience with 42 cases.

AIM: To report a short-time result of three-piece inflatable penile prosthesis (IPP) implantation on treating patients with organic erectile dysfunction (ED). METHODS: Three-piece IPPs were implanted in 42 Chinese patients with ED refractory to systemic treatment between May 2002 and May 2004. The etiologies of ED were neurogenic (28 with paraplegia and seven with traumatic nervi-erigentes injury); congenital venous leakage (5 cases), fibrosis of corpus cavernosum (1case) and Klinefelter's syndrome (1 case). The follow-up period ranged from 24 to 57 months. RESULTS: Implantation procedures were successfully performed upon all 42 patients. The length of implanted prosthesis was from 13 cm to 18 cm, and the diameter was 1 cm. The implanted prosthesis was made by the Medical Instrumentation Company of Muping (Muping, Shandong, China). Localized infection occurred in only one patient and mechanical complications occurred in five patients. Coitus could be performed in 41 cases (97.6%). Three patients with congenital venous leakage made their spouses pregnant after implantation. CONCLUSION: Implantation of three-piece IPP is an effective and safe modality for treating patients with ED. It can be well accepted by Chinese patients because of its efficacy.