二甲双胍通过mTOR信号通路促进结肠癌细胞凋亡的实验研究

Metformin promotes colon cancer cell apoptosis through mTOR signalling pathway

目的探讨二甲双胍对结肠癌细胞增殖以及凋亡的作用及其潜在机制。方法将HCT8细胞随机分为4组:对照组和二甲双胍组(5、10以及20 mM),对照组不接受任何干预措施。MTT法观察4组细胞增殖及对结肠癌细胞的抑制率,AnnexinV-FITC细胞凋亡检测试剂盒观察细胞凋亡情况,同时应用PCR以及Western-blot检测mTOR通路相关基因与蛋白的表达量。结果与对照组比较,不同浓度二甲双胍对HCT8均有抑制作用,并且呈剂量依赖性,20 mM二甲双胍对结肠癌HCT8细胞抑制作用最明显,与5、10 mM以及对照组差异均有统计学意义(P<0.05)。另外,随着时间的增加,二甲双胍对结肠癌HCT8的抑制作用增加,72 h后抑制率与24 h以及48 h抑制率差异均有统计学意义(P<0.05)。凋亡结果显示5、10以及20 mM二甲双胍作用于结肠癌HCT8细胞后凋亡率分别是(3.16±0.16)%、(5.12±0.39)%以及(6.11±0.15)%,均高于对照组凋亡率(1.5±0.28)%,各组之间凋亡率差异均有统计学意义(P<0.05)。二甲双胍干预后结肠癌细胞4EBP1、S6K1以及mTOR的mRNA表达量以及蛋白表达量均低于对照组,差异有统计学意义(P<0.05)。结论二甲双胍通过抑制mTOR信号通路抑制结肠癌细胞增殖以及促进结肠癌细胞凋亡。

Objective To evaluate the effect and mechanism of mefformin in promoting the colon cancer cells apoptosis. Methods HCT8 ceils were randomly divided into 4 groups, control group, mefformin group （ 5,10 and 20 mM ） , and the control group did not accept any intervention. Cell proliferation and the inhibition rate of colon cancer cells were identified by MTT methods, apoptosis of colon cancer ceils was identified by AnnexinV-FITC cell apoptosis detection kit. Mean- while, PCR and Western blot was performed to detection roTOR pathways related gene and protein expression respectively. Results Compared with the control group, different concentrations of mefformin had inhibitory effect on HCTS, and dose dependently. Twenty mM mefformin had the most significant inhibitory effect on colon cancer HCT8 cells,with 5 mM, 10 mM and the control group were statistically significant （ P 〈 0.05 ）. In addition, with the increase of time, the inhibitory effect of mefformin on colon cancer HCT8 increased,72 h inhibition rate with 24 h and 48 h inhibition rate were statisti- cally significant（P 〈 0.05 ）. The results showed that apoptosis rates of 5 mM, 10 mM and 20 mM metformin effecting on colon cancer HCT8 cells were （3.16 ±0.16 ） %, （5.12± 0.39 ） % and （6.11 ± 0.15 ） %, respectively, and they were all higher than that of in the control group（ 1.5 ± 0.28 ） %, and the differences of apoptosis rate between each group was sta- tistically significant （ P 〈 0.05 ）. When treating with mefformin, the mRNA and protein expression of 4EBP1, was less than the control group ,the difference was statistically significant（P 〈 0.05 ）. Conclusion Mefformin inhibits the colon cancer cell proliferation and promote apoptosis of colon cancer cells through inhibiting mTOR signaling pathway.