三阴性乳腺癌细胞中线粒体功能障碍对糖代谢途径的影响

目的:探讨线粒体功能损伤和膜通透性转换与乳腺癌细胞中葡萄糖有氧糖酵解途径的相关性。方法:分别采用MitoTracker Green、TMRE和钙黄绿素（Calcein AM）荧光染色检测乳腺上皮细胞MCF-10A和乳腺癌细胞MDA-MB-231、BT-20的线粒体形态、线粒体膜电位和膜通透性;通过检测试剂盒分析其葡萄糖消耗量、乳酸生成量、ATP水平，以及三羧酸循环中琥珀酸脱氢酶（SDH）和线粒体异柠檬酸脱氢酶（ICDHm）的活性;采用Western blot检测三组细胞中糖酵解相关蛋白葡萄糖转运受体1（GLUTI）、甘油醛-3-磷酸脱氢酶（GAPDH）、M2型丙酮酸激酶（PKM2）和乳酸脱氢酶A（LDHA）的表达情况。采用2.5 μmol/L环孢菌素A（CsA）处理MDA-MB-231和BT-20细胞24 h后检测其线粒体形态及糖代谢的相关指标。结果:与MCF-10A细胞比较，乳腺癌MDA-MB-231和BT-20细胞的线粒体呈短管状或点状，线粒体膜电位下降而线粒体膜通透性增加，葡萄糖消耗量和乳酸生成量均升高（P＜0.05）;细胞内GLUT1、GAPDH、PKM2和LDHA蛋白表达均显著升高而SDH和ICDHm酶活性降低（P＜0.05）;CsA处理后MDA-MB-231和BT-20细胞中线粒体呈长杆状，葡萄糖消耗量和乳酸生成量下降而ATP水平、SDH和ICDHm酶活性均升高（P＜0.05）。结论:乳腺癌MDA-MB-231和BT-20细胞中有氧糖酵解与线粒体异常相关，CsA可恢复线粒体结构和功能，重塑葡萄糖代谢途径。

Effect of mitochondrial dysfunction on glucose metabolism pathway in triple-negative breast cancer cells

Objective:To explore the relationship between mitochondrial dyfunction/permeability transition and glucose aerobic glycolysis pathway.Methods:Mitochondrial fluorescent probes MitoTracker Green,TMRE and Calcein AM were used to analyze the mitochondrial morphology,membrane potential and permeability of MCF-10A,MDA-MB-231 and BT-20 cells,respectively.Glucose consumption and lactic acid production and ATP levels were detected according to the detection kit,and the activities of SDH and ICDHm were also explored.The expression of glycolysis-related proteins GLUT1,GAPDH,PKM2 and LDHA was detected by Western blot.After MDA-MB-231 and BT-20 cells were treated with 2.5 μmol/L cyclosporine A for 24 hours,mitochondrial morphology and the related indexes of glucose metabolism were detected.Results:Compared with MCF-10A cells,the mitochondria of breast cancer MDA-MB-231 and BT-20 cells were short tubular or punctate,with decreased mitochondrial membrane potential and increased mitochondrial membrane permeability.The glucose consumption and lactate production were increased in MDA-MB-231 and BT-20 cells (P＜0.05).Protein levels of GLUT1,GAPDH,PKM2 and LDHA were significantly increased while enzyme activities of SDH and ICDHm were decreased (P＜0.05).After treatment of MDA-MB-231 and BT-20 cells with CsA,mitochondria became long rod-shaped,and glucose consumption and lactate production were decreased while ATP level,SDH and ICDHm enzyme activities were increased (P＜0.05).Conclusion:Aerobic glycolysis was associated with mitochondrial abnormalities in breast cancer MDA-MB-231 and BT-20 cells,and CsA could restore mitochondrial structure and function in the cells and modulated the glucose metabolism pathway.