Mechanism of hif-1α mediated hypoxia-induced permeability changes in bladder endothelial cells

This study aimed to investigate the mechanism of hypoxia-inducible factor-1 alpha (HIF-1α) mediated hypoxia-induced permeability changes in bladder endothelial cells. Models of in vitro hypoxic cell culture of bladder cancer, bladder cancer cells with low HIF-1α expression and HIF-1α RNA interference (RNAi) expression vector were established. Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to detect the expression of HIF-1α and vascular endothelial growth factor (VEGF) in each group. Bladder cell permeability was determined. Results showed that protein and mRNA expression of HIF-1α and VEGF at 3 and 12 h of hypoxia were significantly higher than normal control (P<0.05), and peaked at 12 h. HIF-1α and VEGF expression in the hypoxic group and hypoxic+3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) group were significantly higher than normal control (P<0.05), while expression in the hypoxic+YC-1 group was significantly lower than the hypoxic group (P<0.05). Bladder cell permeability in the hypoxic and hypoxic+YC-1 group were significantly increased compared to normal control (P<0.05), while in the hypoxic+YC-1 group was significantly decreased compared to the hypoxic group (P<0.05). Most of the cells in the stably transfected HIF-1α RNAi expression vector pcDNA6.2-GW/EmGFP-miR-siHIF-1α expressed green fluorescence protein (GFP) under fluorescence microscope. pcDNA6.2-GW/EmGFP-miR-siHIF-1α could significantly inhibit HIF-1α gene expression (P<0.05). HIF-1α and VEGF expression in the hypoxic group and siHIF-1α hypoxic group were significantly higher than normal group (P<0.05), while expression in the siHIF-1α hypoxic group was significantly lower than the hypoxic group (P<0.05). Findings suggest that HIF-1α is an important factor in the increase of bladder cancer cell permeability.

Expression, purification and biological effect of a novel single chain Fv antibody and protamine fusion protein for the targeted delivery of siRNAs to FGFR3 positive cancer cells

Background: Gain-of-function of fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies have focused on the potential usage of therapeutic single-chain Fv (ScFv) antibodies against FGFR3. RNA interference (RNAi) has been considered as a promising therapeutic method against cancer. A tool which can deliver small interference RNAs (siRNAs) into FGFR3 positive cancer cells is very promising for anti-tumor therapy. Results: In this study, a novel fusion protein R3P, which consists of FGFR3-ScFv and protamine, was generated in Escherichia coli by inclusion body expression strategy and Ni-NTA chromatography. Its yield reached 10 mg per liter of bacterial culture and its purity was shown to be higher than 95%. 1 µg of R3P could efficiently bind to about 2.5 pmol siRNAs and deliver siRNAs into FGFR3 positive RT112 and K562 cells. Annexin V staining results showed that R3P can deliver the amplified breast cancer 1 (AIB1) siRNAs to induce RT112 cell apoptosis. Conclusion: These results indicated that R3P was a promising carrier tool to deliver siRNAs into FGFR3 positive cancer cells and to exert anti-tumor effect.

Fibroblast Growth Factor Receptor 1 Overexpression Is Associated with Poor Survival in Patients with Resected Muscle Invasive Urothelial Carcinoma

PURPOSE: To examine the usefulness of various receptor tyrosine kinase expressions as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients. MATERIALS AND METHODS: We retrospectively analyzed the data of 98 patients with muscle invasive UC who underwent radical cystectomy between 2005 and 2010 in Yonsei Cancer Center. Using formalin fixed paraffin embedded tissues of primary tumors, immunohistochemical staining was done for human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 3 (FGFR3). RESULTS: There were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, significantly shorter median time to recurrence (TTR) (12.9 months vs. 49.0 months; p=0.008) and overall survival (OS) (22.3 months vs. 52.7 months; p=0.006) was found in patients with FGFR1 overexpression. By contrast, there was no difference in TTR or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% confidence interval: 1.27-3.90, p=0.006) in multivariate analysis. CONCLUSION: Our result showed that FGFR1 expression, but not FGFR3, is an adverse prognostic factor in muscle invasive UC patients after radical cystectomy. FGFR1 might be feasible for prognosis prediction and a potential therapeutic target after thorough validation in muscle invasive UC.

Clinical Significance of Substaging and HER2 Expression in Papillary Nonmuscle Invasive Urothelial Cancers of the Urinary Bladder

The study aimed to verify the prognostic utility, therapeutic application and clinical benefits of tumor substaging and HER2 status in papillary non-muscle invasive bladder cancer (NMIBC). Select NMIBC transurethral resection specimens from 141 patients were used to construct tissue microarrays for assessing the substaging, HER2 protein expression by immunohistochemistry (HER2-IHC) and gene amplification by dual-color silver in situ hybridization (HER2-SISH). Substages were identified by the differing depth of tumor invasion (pTa / pT1a / pT1b / pT1c). HER2 protein expression was semiquantitatively analyzed and grouped into negative (score 0, 1+) and positive (score 2+, 3+). Other clinicopathological variables were also investigated. For NMIBC, HER2-IHC and HER2-SISH showed positive results in 6/141 (4.3%) and 4/141 (2.8%) respectively, which correlated well with tumor substaging. In multivariate analysis, substaging, HER2-IHC, and HER2-SISH were found to be independent predictors of progression-free survival (P < 0.001, P < 0.001, P = 0.031). HER2-IHC was the sole independent predictor of recurrent free survival in NMIBC (P = 0.017). It is suggested that tumor substaging and HER2 status are independent predictive markers for tumor progression or recurrence, and thus could be included in diagnostic and therapeutic management for NMIBC.

Modulación del metabolismo de esfingolípidos en cáncer de vejiga como una potencial estrategia para optimizar resultados de terapias intravesicales para tumores de vejiga no musculoinvasores / Modulation of sphingolipid metabolism in bladder cancer as a potential strategy to optimize outcomes of intravesical therapies for non-muscle invasive bladder cancers

El cáncer de vejiga representa un problema de salud pública importante a nivel mundial. En Chile su relevancia es aún mayor en la región de Antofagasta. Este cáncer se caracteriza por una alta tasa de recurrencia, por lo que los pacientes requieren un seguimiento estricto que afecta su calidad de vida e implica elevados costos para los sistemas de salud. Esto explica la necesidad de optimizar los tratamientos actuales (quimioterapia e inmunoterapia con BCG intravesical) para reducir las tasas de recurrencia y progresión. Los esfingolípidos son lípidos bioactivos que a nivel celular cumplen funciones relacionadas con la regulación del crecimiento, proliferación, migración, invasión, resistencia a drogas y apoptosis. La evidencia disponible a la fecha sobre el rol de los esfingolípidos en cáncer de vejiga es escasa, pero sugiere que en este cáncer existe un metabolismo esfingolipídico desplazado hacia la reducción de los niveles intracelulares de ceramida y esfingosina (esfingolípidos pro-apoptóticos) y aumento de esfingosina 1-fosfato (esfingolípido anti-apoptótico). La manipulación del metabolismo esfingolipídico para invertir esta relación se propone en esta revisión como una estrategia que podría ayudar a optimizar el efecto de las terapias disponibles actualmente para reducir las recurrencias y progresiones de los tumores de vejiga no músculo-invasores...

Bladder cancer is an important health problem worldwide. In Chile it has particular relevance in the region of Antofagasta. This cancer is characterized by a high recurrence rate, for which patients need a strict follow up that impairs their quality of life and determines increased costs for health care systems. These facts explain the necessity of optimizing the actual treatments (chemotherapy and BCG immunotherapy) for reducing the rates of recurrence and progression. Sphingolipids are bioactive lipids that at a cellular level have roles related to the regulation of growth, proliferation, migration, invasiveness, drug resistance and apoptosis. To date, the available evidence about the role of sphingolipids in bladder cancer is scarce, but suggests that in this cancer there is a sphingolipid metabolism shifted towards a reduction of the intracellular levels of ceramide and sphingosine (pro-apoptotic sphingolipids) and an increase of sphingosine 1-phosphate (anti-apoptotic sphingolipid). In this review we propose that the manipulation of the sphingolipid metabolism to invert this balance can contribute to optimize the effect of the actual therapies to reduce the rates of recurrence and progression of non-muscle invasive bladder cancers...

Establishment of an Orthotopic Mouse Non-Muscle Invasive Bladder Cancer Model Expressing the Mammalian Target of Rapamycin Signaling Pathway

We established an orthotopic non-muscle invasive bladder cancer (NMIBC) mouse model expressing the mammalian target of the rapamycin (mTOR) signaling pathway. After intravesical instillation of KU-7-lucs (day 0), animals were subsequently monitored by bioluminescence imaging (BLI) on days 4, 7, 14, and 21, and performed histopathological examination. We also validated the orthotopic mouse model expressing the mTOR signaling pathway immunohistochemically. In vitro BLI photon density was correlated with KU-7-luc cell number (r2 = 0.97, P < 0.01) and in vivo BLI photon densities increased steadily with time after intravesical instillation. The tumor take rate was 84.2%, formed initially on day 4 and remained NMIBC up to day 21. T1 photon densities were significantly higher than Ta (P < 0.01), and histological tumor volume was positively correlated with BLI photon density (r2 = 0.87, P < 0.01). The mTOR signaling pathway-related proteins were expressed in the bladder, and were correlated with the western blot results. Our results suggest successful establishment of an orthotopic mouse NMIBC model expressing the mTOR signaling pathway using KU-7-luc cells. This model is expected to be helpful to evaluate preclinical testing of intravesical therapy based on the mTOR signaling pathway against NMIBC.

Prognostic Value of Sex-Hormone Receptor Expression in Non-Muscle-Invasive Bladder Cancer

PURPOSE: We investigated sex-hormone receptor expression as predicting factor of recurrence and progression in patients with non-muscle invasive bladder cancer. MATERIALS AND METHODS: We retrospectively evaluated tumor specimens from patients treated for transitional cell carcinoma of the bladder at our institution between January 2006 and January 2011. Performing immunohistochemistry using a monoclonal androgen receptor antibody and monoclonal estrogen receptor-beta antibody on paraffin-embedded tissue sections, we assessed the relationship of immunohistochemistry results and prognostic factors such as recurrence and progression. RESULTS: A total of 169 patients with bladder cancer were evaluated in this study. Sixty-threepatients had expressed androgen receptors and 52 patients had estrogen receptor beta. On univariable analysis, androgen receptor expression was significant lower in recurrence rates (p=0.001), and estrogen receptor beta expression was significant higher in progression rates (p=0.004). On multivariable analysis, significant association was found between androgen receptor expression and lower recurrence rates (hazard ratio=0.500; 95% confidence interval, 0.294 to 0.852; p=0.011), but estrogen receptor beta expression was not significantly associated with progression rates. CONCLUSION: We concluded that the possibility of recurrence was low when the androgen receptor was expressed in the bladder cancer specimen and it could be the predicting factor of the stage, number of tumors, carcinoma in situ lesion and recurrence.

Expression and role of miR-34a in bladder cancer.

To investigate the biological function of microRNA-34a (miR-34a) in bladder cancer, the expression of miR-34a was determined using quantitative real-time polymerase chain (qRT-PCR) reaction in 42 cases of bladder cancer. The relationship between the expression of miR-34a and development of bladder cancer was also studied. The mature mimics of miR-34a were chemically synthesized and transiently transfected into human bladder cancer T24 cells. The effects of miR-34a on apoptosis, cell cycle and proliferation in T24 cells were evaluated by flow cytometry and MTT, respectively. The results showed that the low expression rate of miR-34a was correlated with the malignancy and tumor size of bladder cancer. The up-regulation of miR-34a in T24 cells contributed to cell growth and cell cycle arrest, but not caspase-3 pathway. These findings suggest that the relative low expression of miRNA-34a might be involved in the tumorigenesis of bladder cancer.

Expression profile of microrna-145 in urothelial bladder cancer

Purpose Bladder cancer (BC) is the second most common malignancy of the urinary tract, with high mortality. The knowledge of the molecular pathways associated with BC carcinogenesis is crucial to identify new diagnostic and prognostic biomarkers. MicroRNAs (miRNAs) are short non-coding RNA molecules that play important roles in the regulation of gene expression by acting directly on mRNAs. miR-145 has been considered as a tumor suppressor, which targets the c-MYC, MUC-1 and FSCN1 genes. Our aim was to evaluate the expression profile of miR-145 in low-grade non-invasive and high-grade invasive bladder urothelial carcinomas. Materials and Methods We studied 30 specimens of low-grade, non-invasive pTa and 30 of pT2/pT3 high-grade invasive UC obtained by transurethral resection or radical cystectomy, followed over a mean time of 16.1 months. Normal controls were represented by five samples of normal bladder biopsy from patients who underwent retropubic prostatectomy to treat BPH. miRNA extraction and cDNA generation were performed using commercial kits. Analysis was performed by qRT-PCR, and miR-145 expression was calculated using the 2-∆∆ct method; we used RNU-43 and RNU-48 as endogenous controls. Results miR-145 was under-expressed in 73.3% and 86.7% of pTa and pT2/pT3, respectively, with expression means of 1.61 for the former and 0.66 for the last. There were no significant differences in miR-145 expression and histological grade, tumor stage, angiolymphatic neoplastic invasion and tumor recurrence. Conclusion miR-145 is under-expressed in low-grade, non-invasive and high-grade invasive urothelial bladder carcinoma and may play an important role in the carcinogenesis pathway, being an interesting candidate diagnostic marker. .

Prima-1 induces apoptosis in bladder cancer cell lines by activating p53

OBJECTIVES: Bladder cancer represents 3% of all carcinomas in the Brazilian population and ranks second in incidence among urological tumors, after prostate cancer. The loss of p53 function is the main genetic alteration related to the development of high-grade muscle-invasive disease. Prima-1 is a small molecule that restores tumor suppressor function to mutant p53 and induces cancer cell death in various cancer types. Our aim was to investigate the ability of Prima-1 to induce apoptosis after DNA damage in bladder cancer cell lines. METHOD: The therapeutic effect of Prima-1 was studied in two bladder cancer cell lines: T24, which is characterized by a p53 mutation, and RT4, which is the wild-type for the p53 gene. Morphological features of apoptosis induced by p53, including mitochondrial membrane potential changes and the expression of thirteen genes involved in apoptosis, were assessed by microscopic observation and quantitative real-time PCR (qRT-PCR). RESULTS: Prima-1 was able to reactivate p53 function in the T24 (p53 mt) bladder cancer cell line and promote apoptosis via the induction of Bax and Puma expression, activation of the caspase cascade and disruption of the mitochondrial membrane in a BAK-independent manner. CONCLUSION: Prima-1 is able to restore the transcriptional activity of p53. Experimental studies in vivo may be conducted to test this molecule as a new therapeutic agent for urothelial carcinomas of the bladder, which characteristically harbor p53 mutations.

Neoplasias uroteliais papilíferas superficiais da bexiga (pTa e pT1): correlação da expressão do p53, KI-67 E CK20 com grau histológico, recidiva e progressão tumoral / Superficial papillary urothelial neoplasms of the bladder (PTA E PT1): correlation of expression of P53, KI-67 and CK20 with histologic grade, recurrence and tumor progression

OBJETIVO: Investigar a expressão imunoistoquímica dos marcadores p53, Ki-67, CK20 em neoplasias uroteliais papilíferas superficiais da bexiga e correlacionar com o grau histológico, progressão tumoral e recidiva. MÉTODOS: Foram selecionadas amostras de 43 pacientes portadores de carcinoma de células transicionais superficiais da bexiga. Elas foram distribuídas em dois grupos, um denominado recorrente, de 18 indivíduos e outro não recorrente, com 25 casos. Foram confeccionados blocos multiamostrais. A técnica imunoistoquímica empregada foi de imunoperoxidase e os anticorpos foram: p53 (clone DO7), o Ki-67 (clone SP6) e CK20. RESULTADOS: A expressão do p53 foi observada em 11 casos, todos tumores de alto grau (p=0,0001). A progressão histológica ocorreu em seis indivíduos (p=0,0076). Dos 18 casos recorrentes, seis apresentaram imunorreação para o p53 e 12 foram negativos para este anticorpo (p=0,1715). O Ki-67 foi positivo em 17 dos 18 casos do grupo recorrente (p=0,0001) e dos 20 tumores de alto grau, 18 apresentaram reação para este anticorpo (p=0,0001). Dos 18 indivíduos que tiveram recorrência, 13 apresentaram expressão anômala para CK20 (p=0,0166). Nos carcinomas de alto grau, dos 20 casos, 16 apresentaram expressão anômala para este anticorpo, enquanto que 18 dos 23 indivíduos com tumores de baixo grau mostraram expressão habitual para a CK20 (p=0,0002). CONCLUSÃO: O p53 mostrou boa correlação com a progressão histológica e grau histológico. O Ki-67 apresentou forte associação com a recidiva e grau histológico, e a CK20 também associou-se com estas variáveis.

OBJECTIVE: To investigate the immunohistochemical expressions of p53, ki67, CK20 in superficial papillary urothelial neoplasms of the bladder and correlate them with histological grade, tumor progression and recurrence. METHODS: We selected samples of 43 patients with superficial transitional cell carcinoma of the bladder. They were divided into two groups, one called Recurrent (R), with 18 individuals, and other Non-Recurrent (NR), with 25. Multi-sampling blocks were prepared. The immunohistochemical technique employed was immunoperoxidase, and the antibodies were: p53: Novocastra (clone DO7) at a dilution of 1/100; Ki67: Spring (clone SP6) at a dilution of 1/100; and CK20: Dako (clone K20 .8) at a dilution of 1/50. RESULTS: The expression of p53 was observed in 11 cases, six in the Recurrent group and five in the Non-Recurrent, all high-grade tumors (p = 0.0001). The histological progression occurred in six patients (p = 0.0076). Of the 18 Recurrent cases, six showed immunoreactivity for p53 and 12 were negative for this antibody (p = 0.1715). Ki67 was positive in 17 of the 18 cases from the Recurrent group (p = 0.0001) and, from 20 high-grade tumors, 18 showed reaction to this antibody (p = 0.0001). Of the 18 individuals who had recurrence, 13 showed anomalous expression for CK20 (p = 0.0166). In high-grade carcinomas, of the 20 cases, 16 showed anomalous expression for this antibody, while 18 of the 23 patients with low-grade tumors showed normal expression for CK20 (p = 0.0002). CONCLUSION: The p53 showed good correlation with histological progression and histologic grade. Ki67 was strongly associated with recurrence and histological grade, and CK20 was also associated with these variables.

Expression of pRb, p53, p16 and Cyclin D1 and Their Clinical Implications in Urothelial Carcinoma

The aim of this study was to assess immunohistochemical expression of p53, pRb, p16, and cyclin D1, alone or in combination, as prognostic indicators and to investigate their correlation with clinocopathologic features of urothelial carcinoma. Immunohistochemical staining for p53, pRb, p16, and cyclin D1 was performed on a tissue microarray from 103 patients with urothelial carcinoma who underwent radical cystectomy. Of the patient samples analyzed, 36 (35%), 61 (59%), 47 (46%) and 30 (29%) had altered expression of p53, pRb, p16, and cyclin D1, respectively. Abnormal expression of p53 and pRb correlated with depth of invasion (P=0.040 and P=0.044, respectively). Cyclin D1 expression was associated with tumor stage and recurrence (P=0.017 and P=0.036, respectively). Altered pRb was significantly correlated with overall survival (P=0.040). According to the expression pattern of pRb and p53, p53/pRb (altered/normal) had worse survival than p53/pRb (normal/altered) (P=0.022). Alteration of all markers had worse survival than all normal (P=0.029). As determined by multivariate analysis, tumor stage, lymph node metastasis and the combined expression of p53 and pRb are independent prognostic factors. In conclusion, immunohistochemical evaluation of cell cycle regulators, especially the p53/pRb combination, might be useful in planning appropriate treatment strategies.

Effects of curcumin in an orthotopic murine bladder tumor model

Cigarette smoking (CS) is the main risk factor for bladder cancer development. There are more than 100 carcinogens present in cigarette smoke. Among the potential mediators of CS-induced alterations is nuclear factor-kappa (NF-êB), which is responsible for the transcription of genes related to cell transformation, tumor promotion, angiogenesis, invasion and metastasis. Curcumin is a polyphenol compound derived from Curcuma longa that suppress cellular transformation, proliferation, invasion, angiogenesis, and metastasis by down regulating NF-êB and its regulated genes. The aim of our study was to assess the effects of curcumin in bladder urothelial carcinoma. We studied the effects of curcumin in vitro and in vivo using the orthotropic syngeneic bladder tumor animal model MB49. Curcumin promotes apoptosis of bladder tumor cells in vitro. In vivo tumors of animals treated with curcumin were significantly smaller as compared to controls. Using immunohistochemistry, we demonstrated a decrease in the expression of Cox-2 by 8 percent and Cyclin D1 by 13 percent in the animals treated with curcumin; both genes regulated by NF-êB and related to cell proliferation. In this study, we showed that curcumin acts in bladder urothelial cancer, possibly dowregulating NF-êB-related genes, and could be an option in the treatment of urothelial neoplasms. The results of our study suggest that further research is warranted to confirm our findings.

Costimulatory molecule B7-H1 on the immune escape of bladder cancer and its clinical significance / 华中科技大学学报（医学）（英德文版）

B7-H1, a recently described member of the B7 family of costimulatory molecules, is thought to be involved in tumor immune escape by inducing T-cell apoptosis. In order to investigate the relationship between B7-H1 and immune escape of bladder cancer, B7-H1 expression in 50 cases of bladder cancer was detected by using immunohistochemical method. Survival curves were constructed using the Kaplan-Meier method and independent prognostic factors were evaluated using the Cox regression model. Our results showed that the positive rate of B7-H1 immunostaining in normal bladder tissue and bladder cancer was 0 and 72% respectively. The expression of B7-H1 was strongly associated with the pathological grade, clinical stage and recurrence (P<0.05). The survival rate was significantly lower in patients with B7-H1 positive group than in those with B7-H1 negative group and multi-variable analysis revealed that B7-H1 could be regarded as an independent factor in evaluating the prognosis of bladder cancer. It is concluded that the expression of B7-H1 is strongly associated with neoplastic progression and prognosis of bladder cancer. The manipulation of B7-H1 may become a beneficial target for immunotherapy in human bladder cancer.

Forkhead box O-class 1 and Forkhead box G1 as Prognostic Markers for Bladder Cancer

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.

Forkhead box O-class 1 and Forkhead box G1 as Prognostic Markers for Bladder Cancer

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.

Relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene in bladder tumor / 华中科技大学学报（医学）（英德文版）

To investigate the relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene, RASSF1A protein expression was measured by Western blotting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carcinoma (BTCC). The promoter methylation in BTCC and normal bladder tissues was detected by methylation-specific PCR (MSP). The results showed that the expression level of RASSF1A protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not correlated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF1A protein. It is concluded that RASSF1A gene promoter methylation may contribute to the low level or loss of RASSF1A protein expression, the inactivation of RASSF1A gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.

Expression and prognostic significance of survivin in the progression of bladder transitional cell cancer / 华中科技大学学报（医学）（英德文版）

The expression of survivin, a member of inhibitor of apoptosis (IAP) family, was examined in bladder transitional cell cancer (BTCC) tissue and adjacent normal tissues to examine its clinical implication in the development of BTCC. Thirty specimens of bladder cancer were detected for the expression of survivin by using immunohistochemistry and real-time quantitative reverse transcription polymerase chain reaction (RT-QPCR) in BTCC tissue and adjacent normal tissues. Our results showed that the positive rate of survivin immunostaining specimen were 0 and 60% (18/30) in the adjacent normal tissues, bladder cancer, respectively. The-DeltaDeltaCT value of survivin in bladder cancer tissue was 10.2829 (9.0034-11.5624) times that in the adjacent normal tissues. The expressions of survivin were correlated with the pathological grades of tumor and clinical stages. It is concluded that there was only weak expression of survivin mRNA in the adjacent normal tissues, but the expression of survivin mRNA in bladder cancer tissue was much higher than that in the adjacent normal tissues and the expression of survivin was correlated with pathological grades and clinical stages of tumor.

Survivin protein expression positively correlated with proliferative activity of cancer cells in bladder cancer.

CONTEXT: Survivin is an inhibitor of apoptosis that is selectively over-expressed in common human cancers, but not in normal tissues, and that correlates with aggressive disease and unfavorable outcomes. AIMS: To identify the role of survivin in bladder carcinogenesis and the correlation between survivin protein expression and the occurrence of spontaneous apoptosis, proliferative activity of cancer cells. SETTINGS AND DESIGN: Retrospective analysis. METHODS AND MATERIAL: Bladder transitional cell cancer (BTCC) tissue samples for 128 patients were investigated, with normal bladder tissues serving as controls. From these tumor samples, 42 (32.8%) were grade I, 59 (46.1%) were grade II, and 27 (21.1%) were grade III; 72 (56.2%) were superficial, 56 (43.8%) were invasive. The survivin protein level was quantified by Western blot analysis. The apoptotic index (AI) using in situ labeling apoptotic DNA fragment kit and the Ki-67 labeling index (Ki-67LI) with an anti-Ki-67 monoclonal antibody were analyzed in these tumors, respectively. STATISTICAL ANALYSIS USED: Differences in the S/beta ratio between tumor grade and stage were evaluated by using unpaired t-test and F-test. The relationships between the S/beta ratios and AIs, Ki-67LIs of tumors were evaluated by Pearson correlation coefficient. RESULTS: High survivin levels were detected by Western blot analysis in tumor tissue extracts. None of the expression of survivin protein was found in normal bladder tissues. Survivin levels were significantly different from different clinical stages and pathological grades of the tumors (P > 0.05, respectively). Spearman rank correlation test revealed a positively correlation between survivin protein level and the proliferative activity (P < 0.001) and failed to find significant correlation between AI and survivin protein level (P > 0.05). CONCLUSIONS: Survivin protein expression played an important role in the malignant progression of BTCC.