2-D-脱氧葡萄糖对二甲双胍抑制人结肠癌细胞作用的影响及机制

2-DG enhances anti-tumor effect of metformin on human colon cancer cells

目的观察己糖激酶抑制剂2-D-脱氧葡萄糖(2-DG)对降糖药二甲双胍抑制人结肠癌细胞作用的影响,并探讨其机制。方法将不同浓度的2-DG、二甲双胍单药或联合作用于人结肠癌HT-29细胞,采用台盼蓝染色法测算细胞死亡率观察细胞活力,MTT掺入法测算细胞存活率观察细胞增殖能力,流式细胞术以Annexin V/PI双染色法测算细胞凋亡率,免疫印迹法检测细胞中的蛋白激酶B(AKT)/雷帕霉素靶蛋白(TOR)信号通路相关蛋白[AKT、磷酸化AKT(p-AKT)、核糖体p70S6激酶(p70S6K)、磷酸化核糖体p70S6激酶(p-p70S6K)]、自噬相关蛋白p62。结果不同浓度2-DG(1、5、10 mmol/L)和二甲双胍(5、10 mmol/L)联合处理24 h后,HT-29细胞死亡率均高于单药处理细胞(P均<0. 05);在2-DG、二甲双胍均为10 mmol/L时,HT-29细胞死亡率最高(P均<0. 01)。以10mmol/L 2-DG与不同浓度的二甲双胍(0、1、5、10、15、20 mmol/L)联合处理48 h,在二甲双胍浓度≥5 mmol/L后HT-29细胞存活率低于单药处理的细胞(P均<0. 05),10 mmol/L时HT-29细胞存活率最低(P均<0. 01),20 mmmol/L时HT-29细胞存活率未继续明显降低。不同浓度2-DG(5、10 mmol/L)和10 mmol/L二甲双胍联合处理24 h,仅10 mmol/L 2-DG与二甲双胍处理时HT-29细胞凋亡率高于单药处理细胞(P均<0. 05),且细胞中p-AKT、pp70S6K、p62蛋白相对表达量低于单药处理细胞(P均<0. 05)。结论 2-DG能增强二甲双胍抑制人结肠癌HT-29细胞活力、增殖和诱导细胞凋亡的作用,以10 mmol/L 2-DG时增强作用最明显;其作用机制可能与两者协同抑制AKT/mTOR信号通路及细胞自噬有关。

Objective To investigate the role of hexokinase inhibitor 2-deoxy-D-glucose(2-DG)in enhancing anti-tumor effect of metformin on human colon cancer cells and its mechanism.Methods HT-29 human colon cancer cells were treated with different dosages of 2-DG and metformin alone or jointly.The cell viability and proliferation rate were determined by trypan blue staining and MTT assay,respectively.The apoptotic rate was recorded by a double staining with Annexin V/PI followed by FACS analysis.Western blotting was used to detect the protein levels of several key components involved in AKT/mTOR signaling pathway and autophagy,including AKT,phosphorylated AKT(p-AKT),ribosomal p70S6 kinase(p70S6K),phosphorylated ribosomal p70S6 kinase(p-p70S6K),and p62.Results After combined treatment of 2-DG(1,5,and 10 mmol/L)and metformin(5 and 10 mmol/L)for 24 h,the cell mortality rates of HT-29 cells were much higher than those of cells receiving single drug treatment(all P<0.05),with the highest mortality rate at 10 mmol/L of both drugs(P<0.01).The combined treatment of 10 mmol/L 2-DG and various amount of metformin(0,1,5,10,15,and 20 mmol/L)for 48 h inhibited the cell proliferation as compared with the corresponding single drug treatment when the metformin was≥5 mmol/L(all P<0.05)and the cell viability was the lowest at 10 mmol/L metformin(P<0.01),however,when the concentration was 20 mmmol/L,the survival rate of HT-29 cells did not continue to decrease significantly.HT-29 cells were treated with different dosages of 2-DG(5 and 10 mmol/L)and 10 mmol/L of metformin for 24 h,but only under the combined treatment of 10 mmol/L 2-DG and metformin,the apoptosis rate of HT-29 cells was higher than that under the single drug treatment(all P<0.05),as well as the expression of p-AKT,p-p70S6K and p62 proteins.Conclusion2-DG enhances the efficacy of metformin in inhibiting the viability and proliferation of HT-29 cells and induces the apoptosis,with maximum effect at the dosage of 10 mmol/L,and the mechanism may be related to their synergistic inhibition of AKT/mTOR pathway and autophagy in tumor cells.