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目的考察西达本胺对结肠癌细胞HCT-8和HT-29能量代谢调节的作用。方法西达本胺5,10和20μmol·L~(-1)分别处理HCT-8和HT-29细胞,普通光学显微镜观察细胞形态变化,MTT法检测细胞存活,荧光细胞活性试剂盒检测ATP含量,糖酵解压力试剂盒检测代谢变化,荧光定量PCR和Western蛋白印迹法分别检测糖酵解关键酶乳酸脱氢酶A(LDH-A)在m RNA水平和蛋白水平的表达。结果与对照组相比,西达本胺处理组HCT-8和HT-29细胞形态不规则,出现变形、皱缩和细胞碎片,增殖抑制率显著升高(P<0.05);西达本胺5和10μmol·L~(-1)处理16 h,HCT-8和HT-29细胞内ATP总含量均无差异,而20μmol·L~(-1)处理组ATP总含量显著降低(P<0.05)。西达本胺20μmol·L~(-1)处理HCT-8和HT-29细胞16 h,有氧呼吸水平均无差异,而糖酵解ATP产生速率分别降低30.7%和37.9%(P<0.05);LDH-A m RNA水平无差异,蛋白水平显著下调(P<0.01)。结论西达本胺具有抑制结肠癌细胞糖酵解能力的作用,可能与下调LDH-A有关。
AIM To investigate the effects of cedostain on the energy metabolism of colon cancer cells HCT-8 and HT-29. METHODS: HCT-8 and HT-29 cells were treated with 5, 10 and 20 μmol·L -1 cedostine respectively. The morphological changes of cells were observed under light microscope. The cell viability was measured by MTT assay. The ATP content Metabolic changes were detected by glycolytic stress kit. The expression of lactate dehydrogenase A (LDH-A), a key glycolytic enzyme, at m RNA and protein levels was detected by quantitative PCR and Western blotting. Results Compared with the control group, the morphology of HCT-8 and HT-29 cells in the treated group was irregular, with deformation, shrinkage and cell debris, and the proliferation inhibition rate was significantly increased (P <0.05) The total ATP content in HCT-8 and HT-29 cells was not significantly different at 5 and 10 μmol·L -1 for 16 h, while the ATP content in 20 μmol·L -1 treatment group was significantly decreased (P <0.05 ). Western blotting showed no difference in aerobic respiration, but decreased glycolytic ATP production by 30.7% and 37.9% (P <0.05, respectively) when treated with 20mgol·L -1 cedostine for 16 h ). There was no difference in LDH-A m RNA levels, and protein levels were significantly down-regulated (P <0.01). Conclusion The effect of cedostine on inhibiting glycolytic activity of colon cancer cells may be related to the downregulation of LDH-A.