奥沙利铂诱导的细胞自噬对胃癌SGC7901细胞耐药的影响

doi:10.3760/cma.j.issn.1673-422X.2018.09.001

摘要/Abstract

摘要： 目的探讨奥沙利铂诱导的细胞自噬对胃癌细胞耐药的作用。方法体外培养胃癌SGC7901细胞，采用不同浓度的奥沙利铂作用于细胞，设置空白对照组（奥沙利铂0 μmol/L）、奥沙利铂处理组（奥沙利铂1、2、4 μmol/L）以及奥沙利铂联合多柔比星处理组（奥沙利铂0、1、2、4 μmol/L+多柔比星4 μmol/L），给药干预胃癌细胞24 h。Western blotting法及流式细胞仪检测自噬相关分子Beclin1的表达水平，四甲基偶氮唑蓝（MTT）法检测胃癌细胞活力，流式细胞仪分析SGC7901细胞摄取多柔比星药量。结果奥沙利铂处理组胃癌细胞均出现不同程度的Beclin1表达增高，1、2、4 μmol/L处理组的阳性细胞率分别为（9.51±0.27）%、（13.73±0.80）%、（20.17±1.03）%，空白对照组为（2.17±0.15）%，4组间差异有统计学意义（F=111.10，P<0.001），各处理组与对照组相比，Beclin1表达显著增加（P<0.05；P<0.001；P<0.001）。1、2、4 μmol/L奥沙利铂处理组摄取多柔比星的荧光强度分别为11 567±802、13 433±808、15 967±472，对照组为10 257±367，4组间差异有统计学意义（F=79.81，P<0.001），各处理组与空白对照组相比，多柔比星荧光强度均显著增加（P＜0.05；P<0.001；P<0.001）。奥沙利铂（1、2、4 μmol/L）联合多柔比星（4 μmol/L）处理胃癌细胞24 h后，细胞活力水平分别为（68.27±1.64）%、（51.72±1.93）%、（39.60±1.80）%，单独使用奥沙利铂（1、2、4 μmol/L）处理，细胞活力水平分别为（93.70±1.15）%、（76.53±1.10）%、（74.00±1.65）%，联合多柔比星较单独使用奥沙利铂更能降低细胞活力，差异均具有统计学意义（t=8.91，P<0.001；t=9.21，P<0.001；t=10.34，P<0.001）。经自噬抑制剂预处理的奥沙利铂（2 μmol/L）联合多柔比星（4 μmol/L）处理组的多柔比星荧光强度为16 898±105，奥沙利铂联合多柔比星处理组为22 245±168，单用多柔比星组为17 562±67，3组间差异有统计学意义（F=92.16，P<0.001），与自噬抑制剂预处理组相比，奥沙利铂联合多柔比星组细胞的多柔比星荧光强度显著增强（P<0.001）。上述3组胃癌细胞活力分别为（81.33±3.54）%、（65.00±2.61）%、（101.02±3.58）%，3组间差异有统计学意义（F=90.66，P<0.001），与自噬抑制剂预处理组相比，奥沙利铂联合多柔比星组的细胞活力显著降低（P<0.001）。结论奥沙利铂可以通过增加胃癌细胞的自噬水平从而降低胃癌细胞对化疗药的耐药，并提高化疗药物的治疗效果。

关键词: 抗肿瘤药, 自噬, 胃肿瘤, 抗肿瘤联合化疗方案

Abstract: ObjectiveTo evaluate the effect of oxaliplatin induced autophagy on drug resistance of gastric cancer cells. MethodsGastric cancer SGC7901 cells were cultured in vitro and treated with different concentrations of oxaliplatin. Control group (oxaliplatin 0 μmol/L), oxaliplatin treatment groups (oxaliplatin 1, 2, 4 μmol/L) and oxaliplatin combined with doxorubicin groups (oxaliplatin 0, 1, 2, 4 μmol/L + doxorubicin 4 μmol/L) were set up. Cells were treated with different conditions for 24 hours. Western blotting and flow cytometry were used to detect the expression of autophagyrelated molecule Beclin1. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell viability. Flow cytometry was used to analyze the uptake of doxorubicin in SGC7901 cells. ResultsThe expression of Beclin1 increased in gastric cancer cells treated with oxaliplatin. The percentages of positive cells treated with 1, 2 and 4 μmol/L oxaliplatin were respectively (9.51±0.27)%, (13.73±0.80)% and (20.17±1.03)%, control group was (2.17±0.15)%, and the difference was statistically significant (F=111.10, P<0.001). Compared with the control group, Beclin1 expression significantly increased in each treatment group (P<0.05; P<0.001; P<0.001). Different concentrations of 1, 2, 4 μmol/L oxaliplatin combined with doxorubicin were used to treat gastric cancer SGC7901 cells, fluorescence intensities of doxorubicin were 11 567±802, 13 433±808, 15 967±472, control group was 10 257±367, and the difference was statistically significant (F=79.81, P<0.001). Compared with the control group, the fluorescence intensity of doxorubicin significantly increased in each treatment group (P＜0.05; P<0.001; P<0.001). After treatment with oxaliplatin (1, 2, 4 μmol/L) and doxorubicin (4 μmol/L) for 24 hours, the cell viability levels were (68.27±1.64)%, (51.72±1.93)%, (39.60±1.80)% respectively. The cell viability levels of oxaliplatin treated with 1, 2 and 4 μmol/L were respectively (93.70±1.15)%, (76.53±1.10)%, (74.00±1.65)%. Compared with oxaliplatin alone, oxaliplatin combined with doxorubicin decreased cell viability more obviously, and the differences were statistically significant (t=8.91, P<0.001; t=9.21, P<0.001; t=10.34，P<0.001). The doxorubicin fluorescence intensity of oxaliplatin (2 μmol/L) combined with doxorubicin (4 μmol/L) group with pretreatment of autophagy inhibitor was 16 898±105, oxaliplatin combined with doxorubicin group was 22 245±168, and doxorubicin alone group was 17 562±67, and the difference was statistically significant (F=92.16, P<0.001). Compared with the pretreatment group, the doxorubicin fluorescence intensity of oxaliplatin combined with doxorubicin group was significantly increased (P<0.001). On the other hand, the cell viability levels of the above three groups were (81.33±3.54)%, (65.00±2.61)% and (101.02±3.58)%, and the difference was statistically significant (F=90.66, P<0.001). Compared with the pretreatment group, the cell viability level of oxaliplatin combined with doxorubicin group was significantly lower (P<0.001). ConclusionOxaliplatin can reduce the resistance of gastric cancer cells to chemotherapeutics by increasing the level of autophagy in gastric cancer cells, and improve the therapeutic effect of chemotherapy drugs.

Key words: Antineoplastic agents, Autophagy, Stomach neoplasms, Antineoplastic combined chemotherapy protocols

王刚,王黄震,薛挺. 奥沙利铂诱导的细胞自噬对胃癌SGC7901细胞耐药的影响[J]. 国际肿瘤学杂志, 2018, 45(8): 513-518.

Wang Gang, Wang Huangzhen, Xue Ting. Effect of oxaliplatin induced autophagy on drug resistance of SGC7901 gastric cancer cells[J]. Journal of International Oncology, 2018, 45(8): 513-518.

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