Gopitkumar Shah

iNOS expression and NO production contribute to the direct effects of BCG on urothelial carcinoma cell biology

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-Guérin (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 BCGs 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 BCGs 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.

Loss of Bacillus Calmette-Guérin Viability Adversely Affects the Direct Response of Urothelial Carcinoma Cells to Bacillus Calmette-Guérin Exposure

PURPOSE The attenuated mycobacterium bacillus Calmette-Guérin is widely used as intravesical immunotherapy for nonmuscle invasive urothelial carcinoma. Previous studies demonstrated that in the laboratory and clinical settings bacillus Calmette-Guérin 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-Guérin on urothelial carcinoma cells. MATERIALS AND METHODS We studied the effect of loss of bacillus Calmette-Guérin viability on the tumor cell response to the treatment in 2 human urothelial carcinoma cell lines. The cellular response to bacillus Calmette-Guérin 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 of viability resulted in a quantitative decrease in the tumor cell response relative to that of viable bacillus Calmette-Guérin for all measured end points. The decrease in response varied by cell line, ranging from 15% to 100% of the response to viable bacillus. While responses were quantitatively different, nonviable bacillus continued to induce responses that were qualitatively similar to those of bacillus Calmette-Guérin relative to that in untreated controls. CONCLUSIONS Bacillus Calmette-Guérin viability is an important variable influencing the direct tumor cell response to the treatment. Although the magnitude of its effects are attenuated, heat killed bacillus Calmette-Guérin remains active for the induction of bacillus Calmette-Guérin responsive biological end points.

MP34-04 THE DOSE-RESPONSE RELATIONSHIP OF BCG ON UROTHELIAL CARCINOMA CELL BIOLOGY

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.

MP34-05 THE OXIDATIVE STRESS RESPONSE OF UROTHELIAL CARCINOMA CELLS TO BCG EXPOSURE

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.