H2O2 Generation by bacillus Calmette-Guerin Induces the Cellular Oxidative Stress Response Required for bacillus Calmette-Guerin Direct Effects on Urothelial Carcinoma Biology

Purpose: Exposure of urothelial carcinoma cells to bacillus Calmette-Guerin affects cellular redox status and tumor cell biology but the mechanism(s) remain unclear. We examined free radical production by bacillus Calmette-Guerin 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. Materials and Methods: Using fluorescent probes we performed global profiling of reactive oxygen species/reactive nitrogen species in heat killed and viable bacillus Calmette-Guerin, and in the 253J and T24 urothelial carcinoma cell lines after exposure to the bacillus. Inhibition of bacillus Calmette-Guerin internalization and H2O2 pharmacological scavenging were studied for their effect on cellular reactive oxygen species/reactive nitrogen species generation and various physiological end points. Results: Viable bacillus Calmette-Guerin produced H2O2 and O-2(-) but nitric oxide was not generated. Loss of viability decreased H2O2 production by 50% compared to viable bacillus. Bacillus Calmette-Guerin 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-Guerin dependent alterations in tumor biology, including intracellular signaling, gene expression and cytotoxicity, depended on free radical generation. Conclusions: This study demonstrates the importance of free radical generation by bacillus Calmette-Guerin 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-Guerin represents a potential target to increase the efficacy of the bacillus.