二甲双胍对直肠癌细胞放疗敏感性的影响

Effect of metformin on radiosensitivity of rectal cancer cells

目的探讨二甲双胍(DMBG)对直肠癌细胞放疗敏感性的影响及其可能的作用机制。方法体外培养直肠癌SW1463细胞,设对照组、DMBG组、照射组及DMBG+照射组,同时建立裸鼠移植瘤模型。MTT实验检测不同浓度(5 mmol/L、10 mmol/L、20 mmol/L、40 mmol/L、80 mmol/L)DMBG作用SW1463细胞的细胞活力,克隆集落形成实验检测细胞克隆形成能力,流式细胞仪检测细胞凋亡率及细胞周期,免疫印迹法检测DNA损伤蛋白γ-H2AX及DNA损伤修复蛋白DNA-PK的表达,同时观察各组裸鼠移植瘤重量及体积变化,计算抑瘤率,绘制肿瘤生长曲线。结果不同浓度(5 mmol/L、10 mmol/L、20 mmol/L、40 mmol/L、80 mmol/L)DMBG作用可抑制SW1463细胞活力(F=43.283,P=0.021)。DMBG+照射组在不同照射剂量(2 Gy、4 Gy、6 Gy、8 Gy)照射下的细胞存活率均低于DMBG组及照射组(P<0.05),细胞凋亡率明显高于DMBG组及照射组[(63.32±6.15)%vs(16.19±4.38)%,P<0.05;(63.32±6.15)%vs(17.24±5.17)%,P<0.05];DMBG+照射组G2/M期细胞比例增加到(61.50±5.25)%,G0/G1期细胞比例减少为(17.36±3.17)%,上调了γ-H2AX蛋白表达,并下调了DNA-PK蛋白表达,与DMBG组及照射组比较差异有统计学意义(P<0.05);裸鼠移植瘤体积和重量明显变小,抑瘤率亦明显高于DMBG胍组和照射组[(68.51±2.58)%vs(20.74±2.61)%,P<0.05;(68.51±2.58)%vs(31.52±3.43)%,P<0.05]。结论二甲双胍对直肠癌SW1463细胞及其裸鼠移植瘤具有放疗增敏作用,可能与DMBG联合放疗后改变肿瘤细胞周期分布,抑制肿瘤细胞对DNA损伤的自我修复能力有关。

Objective To investigate the effect of metformin(DMBG)on radiosensitivity of rectal cancer cells and its possible mechanism. Methods Rectal cancer SW1463 cells were cultured in vitro,control group,DMBG group,radiation group and metformin combined radiation group(DMBG+radiation group) were set up,a nude mouse xenograft model was also established. MTT assay and colony assay were used to detect cell ability of different concentrations(5,10,20,40,80 mmol/L) of DMBG on SW1463 cells. Flow cytometry was used to assess the apoptosis and cell cycle. The expression of DNA damage protein γ- H2AX and DNA repair protein DNA-PK were detected by Western Blot,The weight and volume of transplanted tumors were tested,the tumor inhibition rate was calculated,and the tumor growth curve was drawn. Results Different concentrations(5,10,20,40,80 mmol/L)of DMBG inhibited the ability of SW1463 cells(F=43.283,P=0.021). The cell ability of DMBG+radiation group under different radiation doses(2,4,6,8 Gy) was lower than that of DMBG group and radiation group(P<0.05),while the apoptosis rate was significantly higher than that of DMBG group and radiation group [(63.32±6.15)% vs (16.19±4.38)%,P<0.05;(63.32±6.15)% vs (17.24±5.17)%,P<0.05]. DMBG+ radiation group increased the proportion of G2 /M phase cells to(61.50±5.25)%,while the proportion of cells in G0/G1 phase decreased to (17.36±3.17)%,and the expression of γ-H2AX protein was up-regulated,the expression of DNA-PK protein was down-regulated,which were statistically significant compared with the DMBG group and radiation group(P<0.05). The volume and weight of the nude mice were significantly reduced,and the tumor inhibition rate was significantly higher than that of DMBG group and radiation group [(68.51±2.58)% vs (20.74±2.61)%,P<0.05;(68.51±2.58)% vs (31.52±3.43)%,P<0.05]. Conclusions DMBG has radiosensitizing effect on rectal cancer SW1463 cells and its xenografts in nude mice. The combination of metformin and radiotherapy may change the cell cycle distribution and inhibit the ability of DNA damage and repair.