Bacillus Calmette-Guerin Inhibits Apoptosis in Human Urothelial Carcinoma Cell Lines in Response to Cytotoxic Injury

Abstract

By inducing cell cycle arrest at the G1/S transition bacillus Calmette-Guerin has been shown to have a direct antiproliferative effect on urothelial carcinoma cell lines. In other systems cell cycle arrest has been shown to confer a relative state of apoptotic resistance. We assessed the effect of bacillus Calmette-Guerin on the susceptibility of urothelial carcinoma cells to apoptotic stimuli. The human UC cell lines T24 and 253J (American Type Cell Culture, Rockville, Maryland) were used to evaluate the effect of bacillus Calmette-Guerin or antibody mediated alpha5beta1cross-linking on apoptosis and apoptotic sensitivity. Following treatment baseline apoptosis and the response to the apoptotic inducing agent camptothecin was evaluated using assays for caspase 3 activation and DNA fragmentation. Pharmacological blockade of signaling pathways known to be activated in response to bacillus Calmette-Guerin/alpha5beta1 cross-linking was used to assess the role of these pathways in the bacillus Calmette-Guerin apoptotic response. A final series of experiments used the MTT assay to study the impact of bacillus Calmette-Guerin pretreatment on the cytotoxicity of the antineoplastic agent mitomycin C. Treatment with bacillus Calmette-Guerin failed to induce apoptosis, as measured by caspase 3 activation or DNA laddering. Bacillus Calmette-Guerin pretreatment significantly inhibited the induction of apoptosis in response to camptothecin. These effects were reproduced by antibody mediated cross-linking of alpha5beta1 integrin. Pharmacological inhibition of nuclear factor kappaB and/or AP1 signaling pathways reversed the anti-apoptotic effect of bacillus Calmette-Guerin. Mitomycin C cytotoxicity was significantly decreased by bacillus Calmette-Guerin pretreatment. Bacillus Calmette-Guerin exerts a direct anti-apoptotic effect on human urothelial carcinoma cell lines. The ability of antibody mediated cross-linking to reproduce the effect and the ability of signal transduction inhibitors to block it are consistent with a mechanism involving integrin mediated signaling. Apoptotic resistance represents a therapeutic target for modulating the response to bacillus Calmette-Guerin and it may have clinical implications in the sequencing of intravesical therapies.