[关键词]
[摘要]
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(2 μg/mL)组、臭椿酮中浓度(0.4 mg/mL)+DDP(2μg/mL)组、臭椿酮高浓度(0.8 mg/mL)+DDP(2 μg/mL)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(2 μg/mL)组、DDP(2 μg/mL)+Pifithrin-α(20 μmol/L)组、臭椿酮(0.8 mg/mL)+DDP组、臭椿酮+DDP(2μg/mL)+Pifithrin-α(20 μmol/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。
[Key word]
[Abstract]
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 (2 μg/mL 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 (20 μmol/L Pifithrin-α), ATE (0.8 mg/mL ATE)+DDP group, ATE+DDP+ Pifithrin-α group 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 LC3 Ⅱ, LC3 Ⅰ, 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, LC3 Ⅱ/LC3 Ⅰ value 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-α group and ATE+DDP+Pifithrin-α group was significantly higher than that of DDP group and ATE+DDP group, the apoptosis rate, LC3 Ⅱ/LC3 Ⅰ value 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.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(81903770)