CDODA-Me decreases specificity protein transcription factors and induces apoptosis in bladder cancer cells through induction of reactive oxygen species

Objective The objective is to determine whether methyl 2-cyano-3,11-dioxo-18b-olean-1,12-dien-30-oate (CDODA-Me) has therapeutic potential in bladder cancer. We investigated the effects of CDODA-Me on the growth and survival of bladder cancer cells, and expression of specificity protein (Sp) transcription factors that regulate genes associated with cancer cell proliferation and survival. Methods J82, RT4P, and 253JB-V bladder cancer cell lines were treated with vehicle alone or with CDODA-Me with or without the antioxidant L-glutathione. Cell viability and DNA fragmentation were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide-fluorescence-activated cell sorting (FACS) analysis, respectively. Intracellular reactive oxygen species (ROS) were measured by 2′,7′-dichlorofluorescin diacetate–FACS analysis. We assessed CDODA's effects on the levels of Sp and Sp-regulated proteins and induction of apoptosis in bladder cancer cells by Western blotting. We also assessed the anticancer effects of CDODA-Me in nude mice bearing RT4v6 bladder cancer. Results 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and FACS analysis revealed that CDODA-Me inhibited the proliferation and survival of the 3 bladder cancer cell lines in a dose-dependent manner. FACS analysis also indicated that CDODA-Me–induced intracellular ROS, and Western blot analysis indicated that CDODA-Me decreased levels of Sp and Sp-regulated proteins and induced apoptosis in a dose-dependent and time-dependent manner. L-Glutathione attenuated CDODA-Me's down-regulation of Sp and Sp-regulated proteins. Compared with the control treatment, CDODA-Me substantially inhibited tumor growth in vivo. Conclusions CDODA-Me has antineoplastic activity in bladder cancer cells by inducing ROS, which down-regulate Sp and Sp-regulated proteins. Thus, CDODA-Me has therapeutic potential in bladder cancer, and additional studies of the agent's efficacy and mode of action are warranted.