RIP-1/c-FLIP(L) Induce Hepatic Cancer Cell Apoptosis Through Regulating Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)

BACKGROUND: Almost all hepatic cancer cells have resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. c-FLIP(L) and RIP-1 are apoptotic negative regulatory factors. This study investigated the role of c-FLIP(L) and RIP-1 in hepatic cancer cell resistance to TRAIL-induced apoptosis. MATERIAL/METHODS: HepG2 cells were treated by TRAIL, RIP-1 siRNA, and/or BY11-7082. Cell viability was detected by MTT assay. Cell apoptosis was tested by flow cytometry. DISC component proteins, RIP-1, and p-p65 were measured by Western blot. Caspase-8 and caspase-3 were determined by spectrophotometry. RESULTS: Single TRAIL treatment showed no significant impact on cell proliferation and apoptosis. HepG2 cells expressed high levels of RIP1 and c-FLIP(L), while a high concentration of TRAIL upregulated RIP-1 and c-FLIP(L) expression but not DR4 and DR5. Single TRAIL treatment did not obviously activate caspase-8 and caspase-3. RIP-1 or c-FLIP(L) siRNA markedly induced cell apoptosis and enhanced caspase-8 and caspase-3 activities. Combined transfection obviously increased apoptotic cells. TRAIL markedly upregulated RIP-1 expression and enhanced p-p65 protein. Downregulating RIP-1 and/or BAY11-7082 significantly reduced NF-κB transcriptional activity, blocked cells in G0/G1 phase, weakened proliferation, elevated caspase-8 and caspase-3 activities, and promoted cell apoptosis. CONCLUSIONS: TRAIL can enhance RIP1 and c-FLIP(L) expression in HepG2 cells. High expression of RIP1 and c-FLIP(L) is an important reason for TRAIL resistance. Downregulation of RIP1 and c-FLIP(L) can relieve caspase-8 suppression, activate caspase-3, and promote cell apoptosis. TRAIL mediates apoptosis resistance through upregulating RIP-1 expression, enhancing NF-κB transcriptional activity, and weakening caspase activity.