Pioglitazone mediates apoptosis in Caki cells via downregulating c-FLIP((L)) expression and reducing Bcl-2 protein stability

Pioglitazone is an anti-diabetic agent used in the treatment of type 2 diabetes, which belongs to the thiazolidinediones (TZDs) group. TZDs target peroxisome proliferator-activated receptor γ (PPARγ), which functions as a transcription factor of the nuclear hormone receptor. Pioglitazone has antitumor effects in several cancer types and could be a tool for drug therapy in various cancer treatments. Nevertheless, the molecular basis for pioglitazone-induced anticancer effects in renal cancer (RC) has not yet been elucidated. Thus, the aim of the present study was to investigate the detailed signaling pathway underlying pioglitazone-induced apoptosis in Caki cells derived from human clear cell renal cell carcinoma. As a result, it was demonstrated by flow cytometry analysis and Annexin V-propidium iodide staining that pioglitazone treatment induced apoptotic cell death in a dose-dependent manner in Caki cells. The protein expression levels of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP)((L)) and Bcl-2, which were determined by western blotting, decreased after pioglitazone treatment in Caki cells. Flow cytometry and western blot analyses demonstrated that pioglitazone-mediated apoptosis was blocked following pretreatment with the pan-caspase inhibitor, z-VAD-fmk, indicating that pioglitazone-induced apoptosis was mediated via a caspase-dependent signaling pathway. However, the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), did not affect pioglitazone-mediated apoptosis and degradation of c-FLIP((L)) and Bcl-2 protein. Of note, it was found by western blot analysis that Bcl-2 protein expression was downregulated by the decreased protein stability of Bcl-2 in pioglitazone-treated Caki cells. In conclusion, these findings indicated that pioglitazone-induced apoptosis is regulated through caspase-mediated degradation of FLIP((L)) and reduction of Bcl-2 protein stability, suggesting that pioglitazone is a feasible apoptotic agent that could be used in the treatment of human RC.