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.