二甲双胍调控AKT/MDR-1增强人结肠癌细胞HT-29对5-FU药物敏感性机制探讨

Metformin reserves the drug resistance of 5-FU in HT-29 human colon carcinoma cells through regulation of AKT/ MDR-1

目的:探究二甲双胍增强化疗药物5-氟尿嘧啶(5-FU)对人结肠癌细胞株HT-29细胞毒的相关机制。方法:体外培养人结肠癌细胞株HT-29,以5-FU联合或不联合二甲双胍干预细胞,MTT法检测细胞增殖情况;流式细胞仪检测细胞凋亡;Lipofectmin 2000转染AKT质粒;RT-PCR法检测AKT、MDR-1核酸表达;Western Blot法检测AKT、p-AKT、MDR-1蛋白表达。结果:二甲双胍能够增强5-FU对HT-29细胞增殖的抑制作用,单用5-FU与5-FU联合二甲双胍干预的细胞早期及晚期凋亡率分别为(50.12±5.96)%和(78.00±3.39)%,差异具有统计学意义(P<0.05)。二甲双胍可以在核酸及蛋白的水平上下调AKT、pAKT及MDR-1水平;过表达AKT单独5-FU处理较空白质粒组凋亡率降低(19.72±2.23)%,过表达联合用药组凋亡率较空白质粒组降低(20.30±3.15)%,差异具有统计学意义(P<0.05)。结论:二甲双胍通过调节AKT/MDR-1增强人结肠癌细胞HT-29对5-FU的药物敏感性。

Objective：To explore the mechanism of that mefformin enhances the cytotoxicity of 5 -fluorouracil （5 - FU ） to human colon cancer cell line HT - 29. Methods： Treating the HT - 29 cells with 5 - FU with or without mefformin. MTF assay to analyze the cell proliferation ,flow eytometry to detect apoptosis. Lipofectmin 2000 transfected AKT plasmid. RT - PCR to detect the nucleic acid expression level of AKT, MDR - 1. Western Blot to check the pro- tein expression level of AKT, p - AKT and MDR - 1. Results： Compared with the single - agent 5 - FU, mefformin can enhance 5 - FU on inhibiting the proliferation of HT - 29 cells. The apoptosis rates for single - agent and double - agent were （ 50.12 ± 5.96） % and （78.00± 3.39 ） %, respectively （ P 〈 0.05 ）. Mefformin may down - regulate the p - AKT and MDR - 1 levels in both nucleic acid and protein levels. While overexpressed of AKT,for the 5 - FU sin- gle - agent, apoptotic rate was decreased （ 19.72 ± 2.23 ） % than negative control plasmid,and for combination,it also decreased （20.30 ±3.15 ） % （ P 〈 0.05 ）. Conclusion： By regulating AKT/MDR - 1 pathway, metformin enhances the drug sensitivity of 5 -FU to human colon cancer cell line HT- 29.