| TRAIL as a member of TNF superfamily possesses a unique capacity to induce apoptosis selectively in cancer cells in vitro and in vivo. Early clinical trials have confirmed the safety of TRAIL and agonistic antibodies of death receptors (DR). Moreover, most studies have shown that many cancer cells are resistant to TRAIL-induced apoptosis, because of the repression to cancer cell apoptosis by antiapoptotic proteins such as c-FLIP and IAPs. Recent researches demonstrated that the combined treatment of the down-regulating the expression of c-FLIP by siRNA and an IAP pan-antagonist AT406 profoundly enhanced the TRAIL induced apoptosis, suggesting that the above triple combination treatment may provide an ideal anti-cancer therapy.Osteosarcoma is the most common form of primary malignant bone tumor that mainly occurs in juvenile patients. It is a high-grade neoplasm with rapid growth and early metastasis. The mechanisms of formation and development of osteosarcoma have been studied for a long time. Recently, more evidences showed that c-FLIP plays important roles in regulating tumor growth.To study the sensitivity of U2OS cells with c-FLIP inhibition by RNA interference (RNAi) to TRAIL-induced apoptosis, we have done the following study:Objective:A stable osteosarcoma U2OS cell line of down-regulated c-FLIP by siRNA was constructed to understand the anti-apoptosis mechanism of cancer cells.Methods:pSUPER-c-FLIP-siRNA was constructed and then transfected into U2OS cells. Add 1μg/ml puromycin into the cells for 6 days, then add DMEM containing 20%FBS until the cell density to be 50% and add 0.25μg/ml purpmycin to continue to culture the cells. Finally, we got the clone cells downregulated the expression of c-FLIP and expand culturing the clone cells. During the screening, the state of cell death and growing should be observed by microscope. RT-PCR and Western blot were applied to measure the expression of c-FLIP at the levels of mRNA and protein. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for the TRAIL-induced cell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs) AT406, and with or without 4 hrs pretreatment of an inhibitor of c-FLIP biosynthesis rocaglamide for 24 hrs. The cell death effect and apoptosis were measured by the methods of MTT assay with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and the flow cytometry, respectively. Finally, statistical calculations were performed using SPSSv19 softwareResults:The stable transfection cell clones U2OS/pSUPER-c-FLIP-siRNA and U2OS/pSUPER were screened from the cells transfected with pSUPER-c-FLIP-siRNA that we have constructed and pSUPER. Western blot and RT-PCR indicated that the expression of c-FLIP was suppressed by the c-FLIP-siRNA. The MTT assay results indicated that TRAIL-induced cell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in the presence or absence of AT406. The flow cytometry indicated that TRAIL-induced cell death effects proceeded through cell apoptosis pathway. However, in the presence of rocaglamide, the cell death or apoptotic effects of TRAIL were similar and profound in both cell lines, suggesting that the mechanism of actions for both c-FLIP-siRNA and rocaglamide was identical.Conclusions:The inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamide can enhance the sensitivity of U2OS to TRAIL-induced apopotosis, which showed that inhibition of c-FLIP is a target for anti-cancer therapy. |
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