Dose-dependent effect of Resveratrol on bladder cancer cells: Chemoprevention and oxidative stress

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toxicogenomic activity of gemcitabine in two TP53-mutated bladder cancer cell lines: special focus on cell cycle-related genes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of genes related to apoptosis, cell cycle and signaling pathways are independent of TP53 status in urinary bladder cancer cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CD147 overexpression allows an accurate discrimination of bladder cancer patients' prognosis

Background: Urothelial bladder carcinoma (UBC) is a chemo-sensitive tumour, but the response to treatment is heterogeneous. CD 147 has been associated with chemotherapy resistance. We aimed to define tumours with an aggressive phenotype by the combined analysis of clinicopathological and biological parameters.Methods: 77 patients with T1G3 or muscle-invasive UBC treated by radical cystectomy were studied. Immunohistochemistry was performed to detect CD147, heparanase, CD31 (blood vessels identification) and D2-40 (lymphatic vessels identification) expressions. The immunohistochemical reactions were correlated with the clinicopathological and the outcome parameters. 5-year disease-free survival (DFS) and overall survival (OS) rates were estimated using the Kaplan-Meier method. Multivariate analysis was performed by Cox proportional hazards analysis.Results: The 5-year DFS and OS rates were significantly influenced by the classical clinicopathological parameters, and by the occurrence of lymphovascular invasion. CD 147 and heparanase immunoexpression did not affect patients' outcome. However, patients with pT3/pT4 tumours had a median OS time of 14.7 months (95% CI 7.1-22.3, p = 0.003), which was reduced to 9.2 months (95% CI 1.5-17.0, p = 0.008) if the tumours were CD147 positive. We developed a model of tumour aggressiveness using parameters as stage, grade, lymphovascular invasion and CD147 immunoexpression, which separated a low aggressiveness from a high aggressiveness group, remaining as an independent prognostic factor of DFS (HR 3.746; 95% CI 1.244-11.285; p = 0.019) and OS (HR 3.247; 95% CI 1.015-10.388, p = 0.047).Conclusion: CD 147 overexpression, included in a model of UBC aggressiveness, may help surgeons to identify patients who could benefit from a personalized therapeutic regimen. Additional validation is needed. (C) 2011 Elsevier Ltd. All rights reserved.

Anti-Angiogenic Effects of the Superantigen Staphylococcal Enterotoxin B and Bacillus Calmette-Guerin Immunotherapy for Nonmuscle Invasive Bladder Cancer

Purpose: We compared and characterized the effects of intravesical bacillus Calmette-Guerin and/or staphylococcal enterotoxin B for nonmuscle invasive bladder cancer.Materials and Methods: A total of 75 female Fisher 344 rats were anesthetized. of the rats 15 received 0.3 ml saline (control) and 60 received 1.5 mg/kg MNU (N-methyl-n-nitrosourea) intravesically every other week for 6 weeks. The rats were divided into 5 groups. The MNU and control groups received 0.3 ml saline. The bacillus Calmette-Guerin group received 10(6) cfu bacillus Calmette-Guerin. The staphylococcal enterotoxin B group received 10 mu g/ml staphylococcal enterotoxin B. The bacillus Calmette-Guerin plus staphylococcal enterotoxin B group received the 2 treatments simultaneously. Each group was treated intravesically for 6 weeks. At 15 weeks all bladders were collected for histopathological and immunological evaluation, and Western blot.Results: Papillary carcinoma (pTa) and high grade intraepithelial neoplasia (carcinoma in situ) were more common in the MNU group. Papillary hyperplasia was more common in the bacillus Calmette-Guerin and enterotoxin groups. Flat hyperplasia was more common in the bacillus Calmette-Guerin plus enterotoxin group. No significant toxicity was observed. The apoptosis and cellular proliferation indexes decreased in the bacillus Calmette-Guerin, enterotoxin and bacillus Calmette-Guerin plus enterotoxin groups compared to the MNU group. Intensified vascular endothelial growth factor, matrix metalloproteinase-9, Ki-67 and insulin-like growth factor receptor-1 immunoreactivity was verified in the MNU group, moderate in the bacillus Calmette-Guerin and enterotoxin groups, and weak in the bacillus Calmette-Guerin plus enterotoxin and control groups. In contrast, intense endostatin immunoreactivity was verified in the control and bacillus Calmette-Guerin plus enterotoxin groups.Conclusions: Bacillus Calmette-Guerin and staphylococcal enterotoxin B showed similar anti-angiogenic effects. Bacillus Calmette-Guerin plus enterotoxin treatment had additional activity compared to that of monotherapy. It was more effective in restoring apoptosis and balancing cellular proliferation, and it correlated with increased endostatin, and decreased vascular endothelial growth factor, matrix metalloproteinase-9, Ki-67 and insulin-like growth factor receptor-1 reactivity.

Low RKIP expression associates with poor prognosis in bladder cancer patients

Urothelial bladder cancer (UBC) is a heterogeneous type of disease. It is urgent to screen biomarkers of tumour aggressiveness in order to clarify the clinical behaviour and to personalize therapy in UBC patients. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor, and its downregulation is associated with metastatic events in an increasing number of solid tumours. We evaluated the clinical and prognostic significance of RKIP expression in patients with high risk of progression UBC. Using immunohistochemistry, we determined RKIP expression levels in a series of 81 patients with high-grade pT1/pTis or muscle-invasive UBC. Staining of CD31 and D2-40 was used to assess blood and lymphatic vessels, in order to distinguish between blood and lymphatic vessel invasion (LVI). We found that 90 % of pT1/pTis tumours, 94 % of non-muscle invasive papillary tumours and 76 % of the cases without LVI occurrence expressed RKIP in >10 % of cells. In this group, we observed a subgroup of tumours (42 %) in which the tumour centre was significantly more intensely stained than the invasion front. This heterogeneous pattern was observed in 63 % of the cases with LVI. Low RKIP expression was associated with poorer 5-year disease-free and overall survival rates, and remained as an independent prognostic factor for disease-free survival. Loss of RKIP expression may be an important prognostic factor for patients with high risk of progression bladder cancer. © 2013 Springer-Verlag Berlin Heidelberg.

Cell cycle kinetics, apoptosis rates, DNA damage and TP53 gene expression in bladder cancer cells treated with allyl isothiocyanate (mustard essential oil)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MRE11A and SKP2 genes are associated with the increased cytotoxicity induced by the synergistic effects of cisplatin and gemcitabine in bladder cancer cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

No Relationship between the Amount of DNA Damage and the Level of hMLH1 and RASSF1A Gene Expression in Bladder Cancer Cells Treated with Cisplatin and Gemcitabine

Tumor response to antineoplastic drugs is not always predictable. This is also true for bladder carcinoma, a highly recurrent neoplasia. Currently, the combination of cisplatin and gemcitabine is well accepted as a standard protocol for treating bladder carcinoma. However, in some cases, this treatment protocol causes harmful side effects. Therefore, we investigated the roles of the genes TP53, RASSF1A (a tumor suppressor gene) and hMLH1 (a gene involved in the mismatch repair pathway) in cell susceptibility to cisplatin/gemcitabine treatment. Two bladder transitional carcinoma cell (TCC) lines, RT4 (wild-type TP53) and 5637 (mutated TP53), were used in this study. First, we evaluated whether the genotoxic potential of cisplatin/gemcitabine was dependent on TP53 status. Then, we evaluated whether the two antineoplastic drugs modulated RASSF1A and hMLH1 expression in the two cell lines. Increased DNA damage was observed in both cell lines after treatment with cisplatin or gemcitabine and with the two drugs simultaneously, as depicted by the comet assay. A lack of RASSF1A expression and hypermethylation of its promoter were observed before and after treatment in both cell lines. On the other hand, hMLH1 downregulation, unrelated to methylation status, was observed in RT4 cells after treatment with cisplatin or with cisplatin and gemcitabine simultaneously (wild-type TP53); in 5637 cells, hMLH1 was upregulated only after treatment with gemcitabine. In conclusion, the three treatment protocols were genotoxic, independent of TP53 status. However, cisplatin was the most effective, causing the highest level of DNA damage in both wild-type and mutated TP53 cells. Gemcitabine was the least genotoxic agent in both cell lines. Furthermore, no relationship was observed between the amount of DNA damage and the level of hMLH1 and RASSF1A expression. Therefore, other alternative pathways might be involved in cisplatin and gemcitabine genotoxicity in these two bladder cancer cell lines.

Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phospho-mTOR in non-tumour and tumour bladder urothelium: Pattern of expression and impact on urothelial bladder cancer patients

Urothelial bladder carcinoma (UBC) is heterogeneous in its pathology and clinical behaviour. Evaluation of prognostic and predictive biomarkers is necessary, in order to produce personalised treatment options. The present study used immunohistochemistry to evaluate UBC sections containing tumour and non-tumour areas from 76 patients, for the detection of p-mTOR, CD31 and D2-40 (blood and lymphatic vessels identification, respectively). Of the non-tumour and tumour sections, 36 and 20% were scored positive for p-mTOR expression, respectively. Immunoexpression was observed in umbrella cells from non-tumour urothelium, in all cell layers from non-muscle-invasive (NMI) tumours (including expression in superficial cells), and in spots of cells from muscle-invasive (MI) tumours. Positive expression decreased from non-tumour to tumour urothelium, and from pTl/pTis to pT3/pT4 tumours; however, the few pT3/pT4 positive cases had worse survival rates, with 5-year disease-free survival being significantly lower. Angiogenesis occurrence was impaired in pT3/pT4 tumours that did not express p-mTOR. In conclusion, p-mTOR expression in non-tumour umbrella cells is likely a reflection of their metabolic plasticity, and extension to the inner layers of the urothelium in NMI tumours is consistent with an enhanced malignant potential. The expression in cell spots in a few MI tumours and absence of expression in the remaining tumours is intriguing and requires further research. Additional studies regarding the up- and downstream effectors of the mTOR pathway should be conducted.

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of ginger (Zingiber officinale Roscoe) on DNA damage and development of urothelial tumors in a mouse bladder carcinogenesis model

Extracts of the spice ginger (Zingiber officinale Roscoe) are rich in gingerols and shogaols, which exhibit antioxidant, anti-inflammatory, antifungal, anti mycobacterial, and anticarcinogenic proprieties. The present study evaluated the chemoprotective effects of a ginger extract on the DNA damage and the development of bladder cancer induced by N-butyl-N-(4-hydroxibutyl) nitrosamine (BBN)/N-methyl-N-nitrosourea (MNU) in male Swiss mice. Groups G1-G3 were given 0.05% BBN in drinking water for 18 weeks and four i.p. injections of 30 mg/kg body weight MNU at 1, 3, 10, and 18 weeks. Group G4 and G5 received only the BBN or MNU treatments, respectively, and groups G6 and G7 were not treated with BBN or MNU. Additionally, Groups G2, G3, and G6 were fed diets containing 1, 2, and 2% ginger extract, respectively, while Groups G1, G4, G5, and G7 were fed basal diet. Samples of peripheral blood were collected during the experiment for genotoxicity analysis; blood collected 4 hr after each MNU dose was used for the analysis of DNA damage with the Comet assay (assay performed on leukocytes from all groups), while reficulocytes collected 24 hr after the last MNU treatment of Groups G5-G7 were used for the micronucleus assay. At the end of the experiment, the urinary bladder was removed, fixed, and prepared for histopathological, cell proliferation, and apoptosis evaluations. Ginger by itself was not genotoxic, and it did not alter the DNA damage levels induced by the BBN/MNU treatment during the course of the exposure. The incidence and multiplicity of simple and nodular hyperplasia and transitional cell carcinoma (TCC) were increased by the BBN/MNU treatment, but dietary ginger had no significant effect on these responses. However, in Group G2 (BBN/MNU/2% ginger-treated group), there was an increased incidence of Grade 2 TCC. The results suggest that ginger extract does not inhibit the development of BBN-induced mouse bladder tumors.

Cytostatic in vitro Effects of DTCM-Glutarimide on Bladder Carcinoma Cells

Bladder cancer is a common malignancy worldwide. Despite the increased use of cisplatin-based combination therapy, the outcomes for patients with advanced disease remain poor. Recently, altered activation of the PI3K/Akt/mTOR pathway has been associated with reduced patient survival and advanced stage of bladder cancer, making its upstream or downstream components attractive targets for therapeutic intervention. In the present study, we showed that treatment with DTCM-glutaramide, a piperidine that targets PDK1, results in reduced proliferation, diminished cell migration and G1 arrest in 5637 and T24 bladder carcinoma cells. Conversely, no apoptosis, necrosis or autophagy were detected after treatment, suggesting that reduced cell numbers in vitro are a result of diminished proliferation rather than cell death. Furthermore previous exposure to 10 mu g/ml DTCM-glutarimide sensitized both cell lines to ionizing radiation. Although more studies are needed to corroborate our findings, our results indicate that PDK1 may be useful as a therapeutic target to prevent progression and abnormal tissue dissemination of urothelial carcinomas.