title: Inhibition of c-FLIP by RNAi enhances sensitivity of the human osteogenic sarcoma cell line U2OS to TRAIL-induced apoptosis.
abstract: 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)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed and then transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screened from the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expression of c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantly suppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the control cells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAIL- induced cell 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-induced cell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in the presence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded through cell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL were similar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA and rocaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamide can enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a good target for anti-cancer therapy.
authors.affiliation: Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China E-mail : kwenjenchang@yahoo.com, glwang83@gmail.com.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Zhang
authors.forename: Qing-Hong
authors.initials: QH
authors.lastname: Kong
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.forename: Guan-Lin
authors.initials: GL
authors.lastname: Wang
authors.forename: Kwen-Jen
authors.initials: KJ
authors.lastname: Chang
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D049109:Cell Proliferation; D004357:Drug Synergism; D005434:Flow Cytometry; D006801:Humans; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D020169:Caspases