PO-074 Metformin-induced apoptosis facilitates degradation of the C-FLIPL protein through a caspase-dependent pathway in human renal cell carcinoma A498 cells

Introduction Renal cell carcinoma (RCC) is one of the most common cancers in adults. Previous studies have reported that the survival rate was significantly lower for renal cancer patients with diabetes than for those without diabetes. Metformin is a well-known anti-diabetic agent used for the treatment of type 2 diabetes mellitus (T2DM). It also inhibits cell proliferation and angiogenesis, and is known to have anti-tumour effects. However, the molecular mechanism for metformin-induced apoptosis in renal cell carcinoma is not understood. In the present study, treatment with metformin induced apoptosis in A498 cells in a dose-dependent manner. Material and methods Effect of metformin on A498 human renal cell carcinoma cells was studied. Morphological changes were visualised using a LM. Cellular DNA was stained by applying PI and the relative DNA contents of the stained cells were analysed using FACS. Proteins such as anti-cFLIPL, anti-PARP, anti-Bcl-2, anti-Bcl-xL, anti-Mcl-1, anti-cIAP-2, anti-XIAP and anti-actin antibodies were detected using by Imaging System. c-FLIPL mRNA expression was determined by RT-PCR. ROS generation was assessed by the dichlorofluoresence in fluorescence intensity of the cells using flow cytometer. Data were analysed using one-way ANOVA followed by post-hoc comparisons using the SPSS 8.0. Results and discussions We found that degradation of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (c-FLIP) and activation of procaspase-8 were associated with metformin-mediated apoptosis. In contrast, treatment with metformin did not affect the mRNA level of c-FLIPL in A498 cells. Treatment with benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone (z-VAD-fmk, a pan-caspase inhibitor) almost completely blocked metformin-induced apoptosis and degradation of c-FLIPL protein. However, N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, did not inhibit metformin-mediated apoptosis in A498 cells. Conclusion These results demonstrated that metformin-induced apoptosis was mediated by the degradation of c-FLIPL protein via activation of caspase-8 in A498 human renal cell carcinoma cells. This suggests that metformin can play the role of a chemotherapeutic agent for diabetes, as well as an anti-cancer agent.