Objective: To investigate the effect of metformin on the proliferation, apoptosis and energy metabolism of acute myeloid leukemia (AML) K562 cells and the possible mechanism.
Methods: 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.
Results: 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).
Conclusion: 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.
题目: 二甲双胍对K562细胞增殖、凋亡及糖酵解的影响.
目的: 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.
方法: 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.
结果: 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).
结论: 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.