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Exposure of urothelial carcinoma cells to bacillus Calmette-Guérin affects cellular redox status and tumor cell biology but the mechanism(s) remain unclear. We examined free radical production by bacillus Calmette-Guérin in tumor cells in response to the bacillus using global profiling of reactive oxygen species/reactive nitrogen species. The relationship between free radical generation and downstream cellular events was evaluated.Using fluorescent probes we performed global profiling of reactive oxygen species/reactive nitrogen species in heat killed and viable bacillus Calmette-Guérin, and in the 253J and T24 urothelial carcinoma cell lines after exposure to the bacillus. Inhibition of bacillus Calmette-Guérin internalization and H2O2 pharmacological scavenging were studied for their effect on cellular reactive oxygen species/reactive nitrogen species generation and various physiological end points.Viable bacillus Calmette-Guérin produced H2O2 and O2(-) but nitric oxide was not generated. Loss of viability decreased H2O2 production by 50% compared to viable bacillus. Bacillus Calmette-Guérin internalization was necessary for the bacillus to induce reactive oxygen species/reactive nitrogen species generation in urothelial carcinoma cells. Pharmacological H2O2 scavenging reversed reactive oxygen species/reactive nitrogen species mediated signaling in urothelial carcinoma cells. Bacillus Calmette-Guérin dependent alterations in tumor biology, including intracellular signaling, gene expression and cytotoxicity, depended on free radical generation.This study demonstrates the importance of free radical generation by bacillus Calmette-Guérin and intracellular generation of cellular oxidative stress on the urothelial carcinoma cell response to the bacillus. Manipulating the cellular oxidative stress induced by bacillus Calmette-Guérin represents a potential target to increase the efficacy of the bacillus.

Purpose: The attenuated mycobacterium bacillus Calmette-Guerin is widely used as intravesical immunotherapy for nonmuscle invasive urothelial carcinoma. Previous studies demonstrated that in the laboratory and clinical settings bacillus Calmette-Guerin viability is a variable that correlates with antitumor efficacy. We evaluated how loss of viability impacted a number of molecular and phenotypic intermediate end points that characterize and/or contribute to the direct effect of bacillus Calmette-Guerin on urothelial carcinoma cells.Materials and Methods: We studied the effect of loss of bacillus Calmette-Guerin viability on the tumor cell response to the treatment in 2 human urothelial carcinoma cell lines. The cellular response to bacillus Calmette-Guerin rendered nonviable by heat killing was compared to the response to viable bacillus. Response end points included the induction of oxidative stress, activation of intracellular signaling pathways, gene transactivation and phenotypic changes.Results: Loss...

Attenuated mycobacterium bacillus Calmette-Guérin is widely used as intravesical immunotherapy of nonmuscle invasive urothelial carcinoma. Currently there are limited data on the relationship between bacillus Calmette-Guérin dose intensity and tumor response. We evaluated the dose-response relationship of bacillus Calmette-Guérin to nonmuscle invasive bladder cancer in vitro using urothelial carcinoma cell lines and in vivo using an orthotopic mouse model.Two human urothelial carcinoma cell lines were used to study the effect of bacillus Calmette-Guérin dose on the tumor cell response. Internalization, activation of signaling pathways, gene transactivation, cell viability, lactate dehydrogenase and HMGB1 release were study end points. An orthotopic tumor model was used to compare the effect of different doses on the antitumor efficacy of bacillus Calmette-Guérin.Bacillus Calmette-Guérin internalization by urothelial carcinoma cells increased as a function of time and dose with a plateau at higher doses and/or long exposure times. Intracellular signaling demonstrated a similar direct, dose dependent increase. Cytokine expression by urothelial carcinoma cells as a function of dose was variable. Some genes increased progressively but others showed a decrease at the highest dose. While nonviable cell number increased in proportion to dose, the number of cells undergoing necrotic cell death decreased at higher doses. A higher dose of bacillus Calmette-Guérin (1:200) showed a better antitumor effect than a standard dose (1:50) (p0.01).Bacillus Calmette-Guérin dose has a direct impact on urothelial carcinoma cell biology. Increased dose intensity, particularly in nonresponders, may represent a strategy to increase bacillus Calmette-Guérin treatment efficacy.

for induction of protective T cell immunity. In vivo studies have shown that TDM injection resulted in suppression of tumor growth in hepatoma and ocular squamous cell carcinoma. To date, the effect of TDM on Urothelial carcinoma (UC) cells has not been studied. The objectives of this study were to measure the cytotoxic effects of TDM on BCG sensitive and BCG resistant UC cell lines and determine its mechanism of action relative to BCG. METHODS: BCG sensitive (253J, T24) and BCG resistant (RT4) UC cell lines were used to study the effect of TDM. The biologic response to TDM was compared with the response to BCG. Response endpoints included viability, LDH release, HMGB1 release, caspase-3 activity, intracellular signaling pathways (NF-kB), gene transactivation and global profiling of reactive oxygen species(ROS) and nitrogen species (RNS). RESULTS: TDM cytotoxicity was similar in both BCG sensitive and resistant cell lines. TDM significantly decreased viability (T24, p

INTRODUCTION The attenuated mycobacterium Bacille Calmette Guerin is widely utilized as intravesical immunotherapy for the treatment of non-muscle invasive urothelial carcinoma. At present, there is limited data on the relationship between BCG dose intensity and tumor response. This study evaluated dose-response relationship of BCG to NMIBC in vitro using urothelial carcinoma cell lines and in vivo using orthotopic mouse model.

we performed in silico analyses using two independent algorithms, TargetScan, and GeneCodis3, and public microarray expression data approved by Gene Expression Omnibus (GEO). In these microarray expression data, we examined 90 BCs and 6 NBEs. RESULTS: Among the known miRNAs, a total of 60 miRNAs were significantly downregulated in BC. We also found that several miRNAs, such as miR-1/133a, miR-206/133b, let-7c/miR-99a, miR-143/ 145 and miR-195/497, were located close together at five distinct loci and constituted clustered miRNAs. Among these clustered miRNAs, we focused on the miR-195/497 cluster because this clustered miRNA had not been fully analyzed in BC. Transfection of mature miR-195 or miR-497 in two BC cell lines (BOY and T24) significantly inhibited cancer cell proliferation, migration and invasion, suggesting that the miRNAs functioned as tumor suppressors in BC. The TargetScan algorithm showed that 6,730 genes were putative miR-195/497 targets, and 113 significantly enriched signaling pathways were identified in this analysis. The € ugPathways in cancer€ uh category was the most enriched, involving 104 candidate target genes. Gene expression data revealed that 27 of 104 candidate target genes were actually upregulated in BC clinical specimens. To confirm this analysis, we selected the top two upregulated genes (BIRC5 and WNT7A), and validated whether these miRNAs were regulated by miR-195/497. Luciferase reporter assays and western blotting demonstrated that BIRC5 and WNT7A were directly targeted by miR-195/497. CONCLUSIONS: We constructed miRNA expression signatures of clinical BC specimens using deep sequencing. Identification of the tumor-suppressive miR-195/497 cluster in human BC could provide new information on potential therapeutic targets in the treatment of BC.

with the APIMpeptide ATX-101, and thereby attenuating cellular stress responses including DNA repair, a potentiation in the cytostatic effect of both bleomycin and MMC is noted. The absence of tumor also after 41 days suggests complete eradication after a single treatment. Increasing cellular sensitivity to cytostatic agents can potentially increase efficacy of intravesical chemotherapy for NMIBC patients.

Objectives Evidence suggests that oxidative stress occurring as a consequence of inducible nitric oxide synthase/nitric oxide (iNOS/NO) contributes to the biologic effects of bacille Calmette-Guerin (BCG). Objective of this study is to examine iNOS expression, NO production, and the biologic effect of NO on established intermediate end points for the human urothelial carcinoma cell response to BCG. Materials and methods Quantitative reverse transcriptase-polymerase chain reaction and real-time measurement of NO was used to assess iNOS and NO production, respectively, in 2 human urothelial carcinoma (UC) cell lines, in response to BCG. The effect of blocking NO production using the specific iNOS inhibitor 1400W was determined for multiple intermediate end points characterizing BCG’s direct effects on tumor cell biology. Activation of nuclear factor kappa B and nuclear factor (erythroid-derived 2)–like 2 signaling pathways, transactivation of genes, including p21, CD54, IL6, IL8, CXCL1, CXCL3, CCL20, and cytotoxicity, as measured by vital dye exclusion, lactate dehydrogenase release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were measured in response to BCG with and without iNOS inhibition. Results Exposure of UC cells to BCG significantly increased both iNOS expression and NO production. Inhibition of iNOS activity with 1400W significantly inhibited BCG’s direct biologic effect on UC cells for all of the end points evaluated. Conclusions iNOS expression, NO production, and the associated oxidative stress play a central role in the response of UC cells to BCG exposure. Manipulation of oxidative stress may afford an opportunity to enhance the antitumor effects of BCG.