Method: CCK-8 and flow cytometry were used to examine the cytotoxicity and apoptosis effect of C2-ceramide on HNSCC cells. Immunofluorescence and electron microscope were used to examine the necroptosis and autophagy effect of C2-ceramide on HNSCC cells. Western blotting was used to examine the expression of cleaved-caspase-3, cleaved-PARP, LC3I/II, p-ERK1/2 and p-mTor in HNSCC cells. In order to examine the role of ERK, we decreased ERK expression in HNSCC cells by MEK inhibitor PD98059.
Result: C2-ceramide showed concentration-dependent cytotoxicity in HN4 and HN30 cell lines. Simultaneously, it induced caspase-3-independent apoptosis and programmed necrosis. C2-ceramide markedly increased the level of LC3 type II expression to cause protective autophagy. Autophagy inhibitor enhanced C2-Cer-mediated cytotoxicity while programmed necrosis inhibitor produced opposite effect. Further C2-ceramide up-regulated the phosphorylation of ERK1/2, but down-regulated its downstream substrate p-mTOR during the autophagy process.
Conclusion: These results suggested that C2-ceramide exerted anti-tumor effects by inducing programmed apoptosis and necrosis which autophagy inhibitor enhanced the cytotoxicity effect in HNSCC.