基于p53介导自噬通路探讨臭椿酮对顺铂耐药胃癌细胞株耐药性的影响

Effect of ailanthone on cisplatin-resistant gastric cancer cell line based on p53-mediated autophagy pathway

目的探讨臭椿酮对顺铂(DDP)耐药胃癌细胞株SGC-7901/DDP耐药性的影响及其机制。方法采用噻唑蓝(MTT)法检测不同质量浓度臭椿酮对SGC-7901/DDP细胞活力的影响以筛选无毒作用浓度。将体外培养的SGC-7901/DDP细胞分为臭椿酮低浓度(0.2 mg/mL)+DDP(2μg/m L)组、臭椿酮中浓度(0.4 mg/mL)+DDP(2μg/m L)组、臭椿酮高浓度(0.8mg/mL)+DDP(2μg/m L)组考察臭椿酮对SGC-7901/DDP细胞DDP敏感性的影响;将体外培养的SGC-7901/DDP细胞分为对照组、DDP(2μg/m L)组、DDP(2μg/mL)+Pifithrin-α(20μmol/L)组、臭椿酮(0.8 mg/mL)+DDP组、臭椿酮+DDP(2μg/m L)+Pifithrin-α(20μmol/L)组考察p53信号通路与药物作用的关系;采用MTT法检测SGC-7901/DDP细胞活力,流式细胞仪检测SGC-7901/DDP细胞凋亡率,免疫印迹法检测SGC-7901/DDP细胞中LC3Ⅱ、LC3Ⅰ、Beclin1和p53蛋白表达水平。结果0.2、0.4、0.8 mg/mL臭椿酮对SGC-7901/DDP细胞未产生明显细胞毒性。与DDP组相比,臭椿酮低浓度+DDP组、臭椿酮中浓度+DDP组、臭椿酮高浓度+DDP组细胞活力明显降低,凋亡率、LC3Ⅱ/LC3Ⅰ值和Beclin1、p53蛋白表达水平明显升高(P<0.05),且呈浓度依赖性;而DDP组和对照组之间差异无统计学意义(P>0.05);给予Pifithrin-α作用后的DDP+Pifithrin-α组和臭椿酮+DDP+Pifithrin-α组细胞活力较相应对照DDP组、臭椿酮+DDP组明显升高,而细胞凋亡率、LC3Ⅱ/LC3Ⅰ值和Beclin1、p53蛋白表达水平较DDP组、臭椿酮+DDP组明显降低(P<0.05)。结论臭椿酮可通过p53通路诱导自噬逆转SGC-7901/DDP细胞对DDP的耐药性。

Objective To investigate the effect of ailanthone(ATE)on the drug resistance of gastric cancer cell line SGC-7901/DDP to cisplatin and its mechanism.Methods Methyl thiazolyl tetrazolium(MTT)assay was used to detect the effects of different concentrations of ATE on SGC-7901/DDP cell viability to screen the non-toxic concentration.SGC-7901/DDP cells cultured in vitro were divided into low concentration ATE(0.2 mg/mL ATE)+DDP(2μg/m L cisplatin),medium concentration ATE(0.4 mg/mL ATE)+DDP group,high concentration ATE(0.8 mg/mL ATE)+DDP group to investigate the effect of ailanthone on DDP sensitivity of SGC-7901/DDP cells.SGC-7901/DDP cells cultured in vitro were divided into control group,DDP group,DDP+Pifithrin-α(20μmol/L Pifithrin-α),ATE(0.8 mg/mL ATE)+DDP group,ATE+DDP+Pifithrin-αgroup to investigate the relationship between p53 signaling pathway and drug action.MTT assay was used to detect the viability of SGC-7901/DDP cells,flow cytometry was used to detect the apoptosis rate of SGC-7901/DDP cells,Western blotting was used to detect the protein expression levels of LC3Ⅱ,LC3Ⅰ,Beclin1,and p53 in SGC-7901/DDP cells.Results 0.2,0.4,and 0.8 mg/mL ATE had no cytotoxicity on SGC-7901/DDP cells.Compared with those in DDP group,the cell viability of low concentration ATE+DDP group,medium concentration ATE+DDP group and high concentration ATE+DDP group were significantly lower,the apoptosis rate,LC3Ⅱ/LC3Ⅰvalue and Beclin1,p53 protein expression levels were significantly higher(P<0.05),and it was concentration dependent.There was no significant difference between DDP group and control group(P>0.05),after treatment with Pifithrin-α,the cell viability of DDP+Pifithrin-αgroup and ATE+DDP+Pifithrin-αgroup was significantly higher than that of DDP group and ATE+DDP group,the apoptosis rate,LC3Ⅱ/LC3Ⅰvalue and Beclin1,p53 protein expression levels were significantly lower(P<0.05).Conclusion ATE can induce autophagy and reverse cisplatin resistance of SGC-7901/DDP cells by p53 pathway.