柠檬苦素对DDP耐药胃癌细胞化疗敏感性的影响

Effect of limonin on chemosensitivity of DDP-resistant gastric cancer cells

目的探讨柠檬苦素对胃癌细胞化疗敏感性的影响及其机制。方法将对顺铂(DDP)耐药的SGC-7901细胞株(SGC-7901/DDP)分为4组:对照组、柠檬苦素组、DDP组和柠檬苦素+DDP组。对照组为正常培养的SGC-7901/DDP细胞,柠檬苦素组SGC-7901/DDP细胞采用40μmol/L柠檬苦素处理,DDP组SGC-7901/DDP细胞采用16μmol/L DDP处理,柠檬苦素+DDP组SGC-7901/DDP细胞采用40μmol/L柠檬苦素+16μmol/L DDP处理。通过MTT法和集落形成实验检测细胞增殖,流式细胞仪检测细胞凋亡,免疫荧光染色检测细胞中γ-H2AX的表达,Western blot检测细胞中Bax、Bcl-2、cleaved Caspase-3、cleaved PARP1、γ-H2AX和53BP1的蛋白表达。通过对小鼠左腋皮下注射200μl SGC-7901/DDP细胞(2×106)构建SGC-7901/DDP细胞移植瘤BALB/c-nu小鼠模型,然后将小鼠随机分为4组(n=6):模型组、柠檬苦素组、DDP组和柠檬苦素+DDP组。模型组小鼠不进行治疗。柠檬苦素给药剂量为每天10 mg/kg,DDP给药剂量为每天5 mg/kg,给药体积均为0.1 ml。连续治疗7 d。每隔1周统计肿瘤体积,并分析18个月随访期内的小鼠存活率。结果体外实验中,与柠檬苦素组和DDP组相比,柠檬苦素+DDP组的细胞存活率和集落数量均降低,而细胞凋亡率升高(均P<0.05)。与柠檬苦素组和DDP组相比,柠檬苦素+DDP组的Bax和cleaved Caspase-3蛋白表达水平均升高,Bcl-2蛋白表达水平均降低(均P<0.05)。与柠檬苦素组和DDP组相比,柠檬苦素+DDP组γ-H2AX阳性率升高,cleaved PARP1、γ-H2AX和53BP1蛋白表达水平均升高(均P<0.05)。在体实验中,与柠檬苦素组和DDP组相比,柠檬苦素+DDP组的肿瘤体积降低(P<0.05)。随访18个月内,各组小鼠的生存率存在显著差异(P<0.05)。结论柠檬苦素通过抑制DNA损伤修复来增强胃癌细胞的化疗耐药性。

Objective To investigate the effect of limonin on the chemotherapy sensitivity of gastric cancer cells and its mechanism.Methods The cisplatin(DDP)-resistant SGC-7901 cell line(SGC-7901/DDP)was divided into four groups:control group,limonin group,DDP group and limonin+DDP group.SGC-7901/DDP cells were cultured normally in control group,and SGC-7901/DDP cells were treated with 40μmol/L limonin in limonin group,16μmol/L DDP in DDP group,and 40μmol/L limonin+16μmol/L DDP in limonin+DDP group.Cell proliferation was detected by MTT method and colony formation experiment,cell apoptosis was detected by flow cytometry,γ-H2AX expression in cells was detected by immunofluorescence staining,and protein expression levels of Bax,Bcl-2,cleaved Caspase-3,cleaved PARP1,γ-H2AX and 53BP1 were detected by Western blot.SGC-7901/DDP cell transplantation tumor BALB/c-nu mouse model was constructed by subcutaneously injecting 200μl SGC-7901/DDP cells(2×106)into the left axilla of mice,and then the mice were randomly divided into four groups(n=6):model group,limonin group,DDP group and limonin+DDP group.The mice in model group were not treated.The dose of limonin was 10 mg/kg per day,the dose of DDP was 5 mg/kg per day,and the volume of administration was 0.1 ml.The mice were continuously treated for 7 d.The tumor volume was counted every other week,and the survival rate of mice during the 18-month follow-up period was analyzed.Results Compared with limonin group and DDP group,the cell survival rate and the number of colonies in limonin+DDP group were decreased,while the apoptosis rate was increased(all P<0.05).Compared with limonin group and DDP group,the protein expression levels of Bax and cleaved Caspase-3 were increased in limonin+DDP group,while the Bcl-2 protein expression level was decreased(all P<0.05).Compared with limonin group and DDP group,theγ-H2AX positive rate was increased in limonin+DDP group,while the protein expression levels of cleaved PARP1,γ-H2AX and 53BP1 were increased(all P<0.05).Compared with limonin group and DDP group,the tumor volume was reduced in limonin+DDP group(P<0.05).Within 18 months of follow-up,the survival rate of mice was significantly different among groups(P<0.05).Conclusion Limonin may enhance the chemotherapy resistance of gastric cancer cells by inhibiting DNA damage repair.