JNK activation is required for TNFα-induced apoptosis in human hepatocarcinoma cells

Background: A frequent feature of hepatocellular carcinoma is resistance to apoptosis induced by a variety of agents, including the pleiotropic cytokine tumor necrosis factor-α (TNF). Compared with other cell types, hepatocytes and hepatoma-derived cell lines are resistant to TNF-induced apoptosis. Resistance to TNF is largely ascribed to activation of the prosurvival transcription factor NF-B and can be overcome by co-administration of low doses of protein synthesis inhibitors or other drugs. Aims: This study analyses the molecular mechanisms by which TNF, in combination with cycloheximide (CHX), induces apoptosis in human hepatoma-derived Huh7 cells, focusing on the role played by JNK. Methods: Huh7 cell cultures were treated with TNF+CHX in the presence or absence of either the pancaspase inhibitor zVADfmk or the JNK inhibitor SP600125, or after suppression of JNK expression by RNAi. Apoptosis was assessed by both light microscopy and flow cytometry. JNK and caspase activation were assessed by western blotting and/or enzymatic assay. Results: TNF+CHX-induced death of Huh7 cells was partially prevented when JNK activity or expression was suppressed. Moreover, apoptosis was significantly reduced by zVADfmk. The combination of SP600125 and zVADfmk completely abrogated cell death. Conclusions: These results demonstrate a causal role for JNK and caspases in TNF+CHX-induced apoptosis of Huh7 human hepatoma cells. Strategies aimed at enhancing both pathways may be useful in overcoming the resistance of hepatocarcinoma cells to TNF-dependent apoptosis.