H_2O_2对肺腺癌A549细胞葡萄糖和谷氨酰胺代谢的影响  

The diferent effects of glucose and glutamine on lung adenocarcinoma A549 cells treated with H_2O_2

作　　者：俞小凤[1] 张腾[1] 黄辰[1] 张凡[1] 黄钢[1,2] 

机构地区：[1]上海交通大学医学院附属仁济医院核医学科,上海200127 [2]上海交通大学医学院健康科学研究所,上海200025

出　　处：《肿瘤》2014年第4期324-330,共7页Tumor

基　　金：国家自然科学基金资助项目(编号:81071180);"973"重大专项资助项目(编号:2012CB932604);重大新药创制资助项目(编号:2012ZX09506-001-005)

摘　　要：目的:探讨H2O2对肺腺癌A549细胞中葡萄糖和谷氨酰胺代谢途径依赖性的改变及可能的作用机制。方法:用不同浓度的H2O2处理A549细胞后,细胞计数试剂盒-8(cell counting kit-8,CCK-8)法检测细胞的存活率;荧光探针氯甲基-2’,7’-二氯二氢荧光素二乙酯[5-(and-6)-chloromethyl-2’,7’-dichlorol uorescein diacetate,acetyl ester,CM-H2DCFDA]染色法检测细胞内活性氧(reactive oxygen species,ROS)的水平;葡萄糖和乳酸测定试剂盒分别检测细胞中葡萄糖的摄取量和乳酸的产生量;CCK-8法检测葡萄糖代谢抑制剂2-脱氧-D-葡萄糖(2-deoxy-D-glucose,2-DG)对A549及H2O2处理过的A549细胞增殖的影响及谷氨酰胺对A549及H2O2处理过的A549细胞增殖的影响。实时荧光定量-PCR检测葡萄糖代谢通路和谷氨酰胺代谢通路上相关基因mRNA表达的变化;蛋白质印迹法检测不同浓度H2O2处理后对A549细胞中异柠檬酸脱氢酶1(isocitrate dehydrogenase1,IDH1)和IDH2蛋白表达的影响。结果:低毒性浓度H2O2(50和100μmol/L)作用2 h后,A549细胞的存活率未受明显影响,但细胞ROS水平增高,葡萄糖消耗增加,乳酸生成增高;葡萄糖代谢抑制剂2-DG对H2O2预处理后的A549细胞的增殖具有抑制作用,低浓度谷氨酰胺对H2O2预处理后的A549细胞有上调生存率的作用。与葡萄糖代谢通路相关基因mRNA的表达被上调,与谷氨酰胺代谢通路相关基因mRNA表达被下调,IDH1和IDH2蛋白的表达水平被下调。结论:H2O2可上调A549细胞对葡萄糖代谢通路的依赖性,而下调对谷氨酰胺代谢通路的依赖性,其作用机制可能与IDH1和IDH2蛋白表达的下调有关。Objective： To investigate the different reactions of dependence on glucose metabolic pathway and glutamine metabolic pathway of lung adenocarcinoma A549 cells after H2O2 treatment and to explore its possible mechanism. Methods： The survival rate of A549 cells treated with different concentrations of H2O2 was determined by cell counting kit-8 （CCK-8） assay. The change of cellular reactive oxygen species （ROS） level was detected by flow cytometry （FCM） using 5-（and-6）-chloromethyl- 2＇-7＇-dichlorofluorescein diacetate, acetyl ester （CM-H2DCFDA）. The glucose consumption and lactate production were detected by glucose and lactic acid detection kits. The effects of different concentrations of 2-deoxy-D-glucose （2-DG） and glutamine on survival rate of A549 cells treated with H2O2 were detected by CCK-8 assay. The mRNA expression levels of glucose metabolic pathway- and glutamine metabolic pathway-related genes in A549 cells were detected by real-time fluorescence quantitative-PCR. The protein expression levels of isocitrate dehydrogenase 1 and 2 in A549 cells without any treatment and in the A549 cells treated with H2O2 were detected by Western blotting. Results： The growth of A549 cells was not significantly inhibited by the low-dose H2O2 （50 and 100 μmol/L） for 2 hours. After 2-hour treatment of low-dose H2O2, the level of ROS was increased, and the glucose consumption and lactate production were also increased significantly. 2-DG could significantly inhibit the growth of A549 cells after H2O2 treatment. The survival rate of A549 cells receiving pretreatment with H2O2 was elevated after incubation under a low concentration of glutamine. The mRNA expression levels of glucose metabolic pathway-related genes in A549 cells after treatment with H2O2 were up-regulated, while those related to glutamine metabolic pathway were decreased slightly. The expression levels of isocitrate dehydrogenase 1 and 2 proteins were down-regulated. Conclusion： The cell line A549 seems to be more depend

关 键 词：肺肿瘤 活性氧 葡萄糖 谷氨酰胺 抗代谢药 抗肿瘤 

分 类 号：R734.2[医药卫生—肿瘤]