千金藤素通过调控p53信号介导的自噬逆转人非小细胞肺癌细胞的埃克替尼耐药性

Cepharanthine reverses icotinib resistance in human non-small cell lung cancer cells by regulating p53 signaling-mediated autophagy

目的探讨千金藤素对人非小细胞肺癌(NSCLC)细胞的埃克替尼耐药性的影响及可能的作用机制。方法采用埃克替尼(Icotinib)浓度递增的方式诱导人NSCLC PC-9细胞耐药建立埃克替尼耐药细胞PC-9/Icotinib resistance(IR)。MTT法筛选千金藤素的实验浓度,并检测千金藤素对PC-9/IR细胞埃克替尼敏感性的影响;取对数生长期PC-9/IR耐药细胞,分为PC-9/IR组(PC-9/IR细胞)、千金藤素组(PC-9/IR细胞+8μmol/L千金藤素)、埃克替尼组(PC-9/IR细胞+20μmol/L埃克替尼)、千金藤素+埃克替尼组(PC-9/IR细胞+20μmol/L埃克替尼+8μmol/L千金藤素)、3-MA组(PC-9/IR细胞+5 mmol/L的3-MA+20μmol/L埃克替尼+8μmol/L千金藤素)、PFT-α组(PC-9/IR细胞+40μmol/L的p53抑制剂Pifithrin-α+20μmol/L埃克替尼+8μmol/L千金藤素),流式细胞术检测细胞凋亡;MDC染色检测细胞自噬;透射电镜观察细胞超微结构变化;Western blot检测自噬相关蛋白表达[p53、雷帕霉素靶蛋白(mTOR)及其磷酸化蛋白(p-mTOR)、自噬相关蛋白Beclin-1、微管相关蛋白轻链3Ⅱ(LC3-Ⅱ)、微管相关蛋白轻链3Ⅰ(LC3-Ⅰ)]。结果千金藤素能增加PC-9/IR细胞的埃克替尼敏感性,与PC-9/IR组相比,单用千金藤素和埃克替尼对PC-9/IR细胞的凋亡、自噬无显著影响(P>0.05);与千金藤素组、埃克替尼组相比,千金藤素+埃克替尼组细胞凋亡率、绿色荧光的平均光密度、p53、Beclin-1、LC3-Ⅱ/LC3-Ⅰ表达显著增加(P<0.05),p-mTOR/mTOR的比值显著降低(P<0.05),自噬小体的数量增加;3-MA和PFT-α能明显抑制自噬和凋亡,逆转千金藤素对PC-9/IR细胞的埃克替尼耐药逆转作用。结论千金藤素可能通过激活p53介导的自噬,逆转PC-9/IR细胞的埃克替尼耐药性。

Objective To investigate the effect of cepharanthine on the icotinib resistance in human non-small cell lung cancer(NSCLC)cells and its possible mechanism.Methods The increasing concentration of icotinib was used to induce drug resistance in human NSCLC PC-9 cells to establish icotinib-resistant PC-9/Icotinib resistance(IR).MTT method was used to screen the experimental concentration of cepharanthine,and to detect the effect of cepharanthine on icotinib sensitivity of PC-9/IR cells.PC-9/IR cells in logarithmic growth phase were taken and divided into PC-9/IR group(PC-9/IR cells)and cepharanthine group(PC-9/IR cells+8μmol/L cepharanthine),icotinib group(PC-9/IR cells+20μmol/L icotinib),cepharanthine+icotinib group(PC-9/IR cells+20μmol/L icotinib+8μmol/L cepharanthine),3-MA group(PC-9/IR cells+5 mmol/L 3-MA+20μmol/L icotinib+8μmol/L cepharanthine),PFT-αgroup(PC-9/IR cells+40μmol/L p53 inhibitor Pifithrin-α+20μmol/L icotinib+8μmol/L cepharanthine).Flow cytometry was used to detect cell apoptosis;MDC staining was used to detect autophagy;transmission electron microscope was used to observe the cell ultrastructure changes,and western blot was used to detect the expression of autophagy-related protein[p53,mammalian target of rapamycin(mTOR)and its phosphorylated protein(p-mTOR),autophagy-related protein Beclin-1,microtubule-associated protein light chain 3Ⅱ(LC3-Ⅱ),microtubule-associated protein light chain 3Ⅰ(LC3-Ⅰ)].Results Cepharanthine could increase the sensitivity of PC-9/IR cells to icotinib.Compared with the PC-9/IR group,the single use of cepharanthine and icotinib had no significant effect on the apoptosis and autophagy of PC-9/IR cells(P>0.05);compared with the cepharanthine group and the icotinib group,the apoptotic rate,the average optical density of green fluorescence,and the expression of p53,Beclin-1,LC3-Ⅱ/LC3-Ⅰin the cepharanthine+icotinib group were significantly increased(P<0.05),the ratio of p-mTOR/mTOR was significantly decreased(P<0.05),and the number of autophagosomes was increased;3-MA and PFT-αcould significantly inhibit autophagy and apoptosis,and reverse the effect of cepharanthine on-resistance of PC-9/IR cells to icotinib.Conclusion Cepharanthine may reverse icotinib resistance in PC-9/IR cells by activating p53-mediated autophagy.