二甲双胍对Eca109人食管癌细胞转移潜能影响及其机制探讨

Effects of antidiabetic drug metformin on migration and invasion of human esophageal squamous cancer cell Eca109and its mechanisms

目的:研究二甲双胍对人食管癌Eca109细胞凋亡、迁移及侵袭方面的潜在影响,并初步探讨可能机制。方法:人食管癌Eca109细胞常规培养,MTT法测0、1、5、10、20和40mmol/L二甲双胍培养基细胞生长曲线;细胞划痕实验检测10mmol/L二甲双胍对细胞迁移能力的影响(不加二甲双胍为对照);Boyden-Chamber分析法测定10mmol/L二甲双胍对细胞体外侵袭能力;流式细胞仪检测0、5、10和20mmol/L二甲双胍对食管癌细胞凋亡率的影响,并进行Caspase-3活性检测;抽提mRNA以RT-PCR(reverse transcription,PCR)检测相关基因的mRNA表达情况;蛋白质印迹法检测Bax和Bcl-2蛋白表达。结果:MTT结果显示,二甲双胍对Eca109细胞的增殖抑制呈剂量和时间依赖性,P<0.001,其中半数有效抑制率IC50约为10mmol/L;细胞划痕实验结果显示,10mmol/L二甲双胍和对照组划痕12(t=6.298,P<0.001)、24(t=5.637,P<0.001)和48h(t=8.981,P<0.001)细胞迁移指数差异均有统计学意义;侵袭实验显示,10mmol/L二甲双胍穿过ECM凝胶滤膜的细胞数量为21.3±6.2,对照组为106.8±8.1,差异有统计学意义,t=32.306,P<0.001;流式细胞仪检测结果显示,不同浓度二甲双胍凋亡率与对照组相比,差异有统计学意义,F=398.015,P<0.001;Caspase-3活性检测结果显示,二甲双胍组Caspase-3活性与对照组相比显著增加,F=247.849,P<0.001;PCR结果显示,与对照组相比,Bax和Caspase-3的mRNA的表达升高,Bcl-2、MMP-2和MMP-9的mRNA的表达降低,差异均有统计学意义;实验组凋亡相关蛋白Bcl-2/Bax灰度值的比值与对照组相比,差异有统计学意义,F=121.819,P<0.001,且呈浓度依赖性。结论:二甲双胍对食管癌Eca109细胞的增殖抑制呈剂量和浓度依赖性,并降低食管癌细胞迁移与侵袭能力,其可能与上调或下调某些相关基因和蛋白的表达有关。

OBJECTIVE：To evaluate the effects of metformin,a widely used antidiabetic agent,on cell apoptosis,mi- gration and invasion in human esophageal cancer cell line Ecal09 in vitro, and to explore its mechanisms underlying the an- titumor effects of metformin on esophageal cancer. METHODS： 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide（MTT） assay was used to detect cell relative viability after treatment with metformin for different concentration （0,1,5,10,20,40 retool/L）. Cell migration and invasion abilities were determined by scratch assay and Boyden-Chamber assay when the cells treated with metformin 10 mmol/L,without metformin as the control group, respectively. Apoptosis was examined by flow cytometry （FCM） after treatment with metformin for different concentration（0,5,10,20 mmol/L）. The activities of Caspase-3 were assayed with Caspase Colorimetric Assay kit. The expression of Bax, Bcl-2, MMP-2, MMP-9 and Caspase-3 mRNAs were detected by reverse transcription PCR（RT-PCR）. The levels of Bax and Bcl-2 were measured by western blot. RESULTS： Metformin significantly inhibited the proliferation of esophageal cancer cell line Ecal09 in a dose-and time-dependent manner（P^0. 001）and the ICs0 was 10 mmol/L. The scratch assay showed that the migration index was much slower than that in control after treatment with metformin for 12 h（t= 6. 298,P〈0. 001）, 24 h （t=5. 637,P〈0. 001） ,48 h（t=8. 981,P〈0. 001）. The invasion assay showed that the number of cell penetrating Ma- trigelgel was 21.3 zk 6.2,106.8 ＋ 8.1. The invasion ability of the group treatment with metformin 10 mmol/L was signifi- cantly decreased（t= 32. 306,P〈0. 001）. Apoptosis rate significantly increased with the concentration gradient compared with control （F=398. 015 ,P〈0. 001）. The activation of Caspase-3 increased compared with control （F= 247. 849, P〈 0. 001）. The expression of Bcl-2, MMP-2 and MMP-9 mRNAs was down-regulated, while the expression of Bax and Caspase-3 mRNA was up-regulated after metformin treatment. The ratio of expression of Bcl-2/Bax protein decreased with the concentration gradient compared with control（F= 121. 819 ,P〈0. 001）. CONCLUSION： Metformin inhibits cell prolif- eration,migration and decreases the invasion abilities, and promotes apoptosis in human esophageal cancer cell line Ecal09, which may be attributed to the down-regulation or the up-regulation of the related mRNA and protein.