联合阻断EGFR和mTOR信号通路对EGFR-TKI耐药鼻咽癌细胞的作用及其机制的研究

发布时间：2018-09-08 15:55

【摘要】：目的：体外研究表皮生长因子受体（Epidermal growth factor receptor，EGFR）抑制剂吉非替尼（Gefitinib）和雷帕霉素靶蛋白（Mammalian target of rapamycin，mTOR）抑制剂依维莫司（Everolimus）在gefitinib耐药鼻咽癌细胞中联合作用的效果及机制。方法：MTT法测定单药及联合用药对人鼻咽癌细胞株HONE1的增殖抑制；流式细胞术检测药物单独或联合作用于HONE1细胞株48h、72h后，各组细胞凋亡及周期分布的改变；Western Blot检测单药及联合用药48h后，HONE1细胞中磷酸化AKT（Phospho-AKT，p-AKT）和磷酸化S6K（Phospho-S6K，p-S6K）表达水平的变化。结果：MTT法结果显示gefitinib和everolimus对HONE1细胞的生长抑制均呈剂量依赖性，并由此计算出，gefitinib的IC50为17.92μmol/L，everolimus的IC50为2.46nmol/L，但二者的联合作用并没有表现出明显的协同效应（P0.05）；流式细胞术显示gefitinib及everolimus作用于HONE1细胞时，可诱导凋亡，引起G0/G1期细胞阻滞，，两种作用均具有时间依赖性，而二者联合的效果并未表现出明显的优势(P0.05)；Western Blot显示gefitinib对p-AKT抑制作用不明显，everolimus可下调p-S6K的表达，但同时上调了p-AKT的表达，联合用药对p-S6K和p-AKT表达的抑制作用并不明显强于单药。结论：mTOR抑制剂everolimus并不能逆转gefitinib耐药鼻咽癌细胞株HONE1的耐药性，两者联合的效果在鼻咽癌细胞中没有明显优势，EGFR/AKT信号通路与mTOR信号通路的关系及其在鼻咽癌细胞中的作用机制有待进一步研究。
[Abstract]:Aim: to investigate the combined effect and mechanism of epidermal growth factor receptor (Epidermal growth factor receptor,EGFR) inhibitor gefitinib (Gefitinib) and rapamycin target protein (Mammalian target of rapamycin,mTOR) inhibitor (Evimostr (Everolimus) in gefitinib resistant nasopharyngeal carcinoma (NPC) cells in vitro. Methods the proliferation of human nasopharyngeal carcinoma (NPC) cell line HONE1 was inhibited by single drug and combined drug, and apoptosis and cell cycle distribution were detected by flow cytometry after treated with drugs alone or in combination for 48 h or 72 h. The expression of phosphorylated AKT (Phospho-AKT,p-AKT) and phosphorylated S6K (Phospho-S6K,p-S6K) in HONE1 cells were detected by Western Blot. Results the results of gefitinib and everolimus showed that the growth inhibition of HONE1 cells was dose-dependent, and the IC50 of gefitinib was 17.92 渭 mol/L,everolimus and the IC50 of HONE1 was 2.46 nmol / L, but the combined effect of the two showed no significant synergistic effect (P0.05). Flow cytometry showed that gefitinib and everolimus could induce apoptosis and induce cell arrest in G0/G1 phase when HONE1 cells were treated with gefitinib and everolimus. Both of the two effects were time-dependent, but the combined effect did not show obvious advantage (P0.05). Western Blot showed that the inhibitory effect of gefitinib on p-AKT was not obvious. Gefitinib could down-regulate the expression of p-S6K, but at the same time up-regulated the expression of p-AKT. The inhibitory effect of combined treatment on p-S6K and p-AKT expression was not significantly stronger than that of single drug. Conclusion everolimus can not reverse the drug resistance of gefitinib resistant nasopharyngeal carcinoma cell line HONE1. The relationship between EGFR / AKT signaling pathway and mTOR signaling pathway and its mechanism in nasopharyngeal carcinoma cells need to be further studied.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

【参考文献】