[Effect of Metformin on Proliferation Capacity, Apoptosis and Glycolysis in K562 Cells].

To investigate the effect of metformin on the proliferation, apoptosis and energy metabolism of acute myeloid leukemia (AML) K562 cells and the possible mechanism. Different doses (0, 5, 10, 20 and 30 mmol/L) of metformin was added into the K562 cells, which were cultivated for 24 h, 48 h and 72 h. The inverted optical microscope was used to observe the cell growth, CCK 8 was used to detect the cell vitality. The appropriate metformin doses (0, 10, 20 and 30 mmol/L) and the best time (48 h) were selected for subsequent experiments. The flow cytometer with Annexin V-FITC /PI doulde staining was used to detect apoptosis; the glucose detection kit and lactate detection kit were used to detect glucose consumption and lactate production; fluorescence quantitative PCR was used to detect glycolysis-related gene expression, and Western blot was used to detect protein expression. Metformin inhibited the proliferation of K562 cells in a dose-dependent manner (r=0.92), and the relative survival in the 30 mmol/L group was as low as 19.84% at 72 h. When treated with metformin for 48 h, the apoptosis rates of 0, 10, 20 and 30 mmol/L groups were 5.14%, 12.19%, 26.29% and 35.5%, respectively. Compared with the control group, the glucose consumption and lactate secretion of K562 cells treated with metformin were significantly reduced (P<0.05), and showed a dose-dependent effect(r=0.94,r=0.93,respectively). Metformin inhibited the expression of GLUT1, LDHA, ALDOA, PDK1, and PGK1 genes of K562 cells (P<0.05) showing a dose-dependent manner(r=0.83，r=0.80，r=0.72，r=0.76，r=0.73,respectively). Metformin inhibited the expression of P-Akt, P-S6, GLUT1, LDHA proteins of K562 cells(P<0.05), showing a dose-dependent relationship(r=0.80，r=0.92，r=0.83，r=0.92,respectively). Metformin can inhibit the growth and proliferation of K562 cells and promote the apoptosis of K562 cells by inhibiting glycolysis energy metabolism. PI3K/Akt/mTOR signaling pathway may be one of the molecular mechanisms of metformin on k562 cells. 二甲双胍对K562细胞增殖、凋亡及糖酵解的影响. 探讨二甲双胍对K562细胞增殖、凋亡及糖酵解的影响及其可能的作用机制. 采用不同浓度二甲双胍(终浓度 0、 5、 10 、20和 30 mmol/L)作用于K562细胞，分别培养24、48 和72 h，倒置光学显微镜下观察细胞生长，应用CCK-8 检测细胞活力，筛选出合适的二甲双胍处理浓度(终浓度 0、10 、20和 30 mmol/L)及最佳作用时间（48 h）进行后续实验。应用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡，葡萄糖和乳酸试剂盒检测葡萄糖消耗和乳酸分泌，采用荧光定量PCR检测糖酵解相关基因表达，采用Western blot法检测蛋白表达. 与对照组比较，随着二甲双胍浓度的升高，K562细胞皱缩、密度减小、死亡增多；二甲双胍抑制K562细胞增殖，且呈剂量依赖性(r=0.92)，72 h时30 mmol/L组细胞相对存活率低至19.84%。二甲双胍处理K562细胞48 h, 0、10、20、30 mmol/L组细胞凋亡比例分别为5.14%、12.19%、26.29%和35.5%。与对照组比较，二甲双胍处理的K562细胞呈现葡萄糖消耗和乳酸分泌均明显减少(P<0.05)，且呈剂量依赖性(分别为r=0.94，r=0.93)。二甲双胍抑制K562细胞糖酵解相关基因GLUT1、LDHA、ALDOA、PDK1、PGK1基因的表达（P<0.05），且随着二甲双胍浓度升高，基因表达量减少，呈剂量依赖性(分别为r=0.83，r=0.80，r=0.72，r=0.76，r=0.73)。二甲双胍抑制K562细胞P-Akt、P-S6、GLUT1、LDHA蛋白的表达（P<0.05），呈剂量依赖性(分别为r=0.80，r=0.92，r=0.83，r=0.92). 二甲双胍可通过抑制糖酵解能量代谢，抑制K562细胞生长和增殖，促进K562细胞凋亡， PI3K/Akt/mTOR通路可能是二甲双胍作用K562细胞的分子机制之一.