Inhibition of c-FLIP by RNAi Enhances Sensitivity of the Human Osteogenic Sarcoma Cell Line U2OS to TRAILInduced Apoptosis

To study effects of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP)inhibition by RNA interference (RNAi) on sensitivity of U2OS cells to tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed andthen transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screenedfrom the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expressionof c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantlysuppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the controlcells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAILinducedcell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs)AT406, with or without 4 hrs pretreatment with rocaglamide, an inhibitor of c-FLIP biosynthesis, for 24 hrs.Cell death effects and apoptosis were measured by the methods of MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry, respectively. The results indicated that TRAIL-inducedcell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in thepresence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded throughcell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL weresimilar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA androcaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamidecan enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a goodtarget for anti-cancer therapy.