目的探讨二甲双胍联合奥沙利铂对人结肠癌细胞HCT-8迁移及侵袭的影响,并研究其可能的机制。方法将HCT-8细胞分为四组:对照组、二甲双胍组、奥沙利铂组和联合用药组,分别用不含药物、含20mmol/L二甲双胍、含20μg/ml奥沙利铂以及20mmol/L二甲双胍+20μg/ml奥沙利铂的培养基培养。应用MTT法检测二甲双胍联合奥沙利铂对人结肠癌细胞的生长抑制作用;划痕实验和侵袭实验观察结肠癌细胞迁移和侵袭能力的改变;Western blot实验检测迁移、侵袭相关因子基质金属蛋白酶(MMP)-2、MMP-9、血管内皮生长因子(VEGF)的蛋白表达情况;实时荧光定量聚合酶链式反应(Real-time PCR)检测结肠癌细胞中MMP-2 mRNA、MMP-9 mRNA的表达水平。结果MTT结果显示,二甲双胍及二甲双胍联合奥利沙铂均具有抑制HCT-8细胞增殖的作用,并呈时间-剂量依赖性,20mmol/L二甲双胍处理HCF-8细胞48h,抑制率为50%。依对照组→二甲双胍组→奥沙利铂组→联合用药组之序,OD值逐渐降低,HCT-8细胞的迁移和侵袭作用逐渐降低,MMP-2、MMP-9及VEGF蛋白表达量逐渐降低,MMP-2 mRNA、MMP-9 mRNA表达量逐渐降低,且两两比较差异有统计学意义(P<0.05)。结论二甲双胍联合奥沙利铂能够显著抑制人结肠癌细胞HCT-8的迁移和侵袭,两者具有协同作用。
Objective To investigate the effects of metformin combined with oxaliplatin on the migration and invasion of human colon cancer cell line HCT-8,and to study its possible mechanism.Methods HCT-8 cells were divided into four groups:control group,metformin group,oxaliplatin group and combination group,and they were cultured in medium containing of no drug,20 mmol /L metformin,20 μg /ml oxaliplatin and 20 mmol /L metformin plus 20 μg /ml oxaliplatin, respectively.MTT assay was used to detect the growth inhibition of metformin combined with oxaliplatin on human colon cancer cells.Scratch test and invasion test were used to observe the migration and invasion ability of colon cancer cells. Western blot was used to detect the migration and invasion related factors such as matrix metalloproteinase(MMP)-2,MMP- 9,and vascular endothelial growth factor (VEGF) protein.Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect MMP-2 and MMP-9 mRNA expression in colon cancer cells.Results MTT assay showed that metformin only and metformin combined with oxaliplatin could inhibit the proliferation of HCT-8 cells in a time-dosedependent manner.The inhibition rate was 50%,when 20 mmol /L metformin was used to treat HCT-8 cells for 48 h. According to the order of the control group→metformin group→oxaliplatin group→combination group,the OD value gradually decreased,the migration and invasion ability of HCT-8 cells gradually decreased,the expression levels of MMP-2, MMP-9 and VEGF protein gradually decreased,and the expression of MMP-2 mRNA and MMP-9 mRNA gradually decreased,the differences in each pairwise comparison among four groups were statistically significant (P < 0.05 ). Conclusion Metformin combined with oxaliplatin can significantly inhibit the migration and invasion of human colon cancer cell line HCT-8,and they have synergistic effects.
Chinese Journal of Clinical Research