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 antitumor 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 10mmol/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-p70S6 K and p62 proteins. Conclusion 2-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.
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