The Effects And Mechanism Of Ceramide In Head And Neck Squamous Cell Carcinoma

Purpose:Head and neck squamous cell carcinoma (HNSCC) accounts for90%of head and neck cancers, and is the fifth most common cancer diagnosed worldwide. Although treatment strategies, which are based on surgery and complementary with radiation and chemotherapy, have improved significantly in recent decades, the5-year survival rate remains low and patients’qualities of life haven’t been improved yet. So we did some research on the effects and mechanism of cytoprotection and cytotoxicity induced by C2-ceramide (C2-Cer) in HNSCC, and the impact on Yes-associated protein (YAP) expression by C2-Cer, to look out for critical protein in development of HNSCC, offer theoretical basis and unique direction to HNSCC-therapy study and discover a novel therapeutic target and method.Methods:1. The apoptosis of HNSCC cells was observed by microscope and flow cytometry after the treatment with C2-Cer and pan-caspase inhibitor Z-VAD-FMK. The impact of proliferation and viability of HNSCC cells was detected by CCK-8assay. And western blotting was taken to determine the protein expression of cleaved caspase-3. Then, the apoptosis induced by C2-Cer and the relative mechanism were discussed.2. The programmed necrosis (necroptosis) of HNSCC cells was observed by DNA agarose electrophoresis and immunofluorescence after the treatment with C2-Cer and necroptosis inhibitor Necrostatin-1(Nec-1), and CCK-8assay was taken to detected the effect of proliferation and viability of HNSCC cells under the drug exposure, demonstrating that the necroptosis was induced by C2-Cer in HNSCC cells.3. The expression of LC3-Ⅱ (effector protein of autophagy) and the formation of autophagic vacuole were observed by electron microscope and immunofluorescence after the treatment with C2-Cer in HNSCC cells. CCK-8assay was used to detect the influence of proliferation and viability of cells co-treated with autophagy inhibitor chloroquine (CQ). Before and after treated with C2-Cer and MEK specific inhibitor PD98059, the expression of autophagy relative protein LC3B-Ⅱ, Beclin-1, p-ERK, p-AMPK, p-Akt and p-mTOR was detected by western blotting. And CCK-8assay was taken. The protective autophagy induced by C2-Cer and relative mechanism were discussed further.4. The expression of Yes-associated protein (YAP, oncogenic protein in Hippo pathway) was detected by immunohistochemistry in HNSCC tissue sample and the data was processed statistically. The correlation between protein expression, pathology grade and gender was analyzed. Then western blotting was taken to determine the expression of YAP protein in HNSCC cells after exposure to C2-Cer. The correlation between YAP protein and pathogenesis was dicussed. And the effect of C2-Cer on YAP was studied.Results:1. C2-Cer induced apoptosis in HNSCC cells and significantly inhibited the proliferation of the cells, with no variation of cleaved caspase-3protein expression intracellular, and the apoptosis was not inhibited by Z-VAD-FMK, indicating that C2-Cer could effectively induce apoptosis in HNSCC cells via a caspase-3-independent pathway.2. Necrotic cells were observed after C2-Cer treatment and the necroptosis inhibitor could availably inhibit the generation of necrotic cells and the cytotoxicity induced by C2-Cer, indicating that C2-Cer could significantly induce necroptosis in HNSCC cells and collaborate with apoptosis to enhance the cytotoxicity function, while necroptosis inhibitor could effectively weaken the effect.3. Endogenous LC3-Ⅱ and p-ERK1/2levels increased in HNSCC cells after C2-Cer treatment in a concentration-dependent manner, while other autophagy relative protein levels had no significant change. LC3-Ⅱ levels were higher in cells co-treated with the autophagy inhibitor CQ compared with cells treated with C2-Cer alone. Specific autophagic vacuoles were observed in the cytoplasm via electron microscopy and immunofluorescence. Both autophagy inhibitor and MEK specific inhibitor could inhibit the autophagy induced by C2-Cer and enhance the cytotoxicity, indicating that C2-Cer could induce protective autophagy in HNSCC cells via ERK1/2signal pathway and autophagy inhibitor could increase the cytotoxicity.4. YAP protein levels were significantly increased in HNSCC tissues and cells compared to normal ones. But there was no obvious correlation between the protein level and pathological grade. C2-Cer could effectively inhibit the expression level of YAP. We were given a hint that the high levels of YAP protein might be connected with the development of HNSCC which might be a potential target of tumor therapy in HNSCC and C2-Cer might induce the antitumor effect via regulating the YAP level.