乳酸对非小细胞肺癌细胞线粒体自噬的影响

Effect of lactate on autophagy of mitochondrion in non-small cell lung cancer cellsi

目的本研究旨在探讨乳酸对人非小细胞肺癌细胞线粒体自噬的影响。方法将人正常肺上皮细胞(BEAS-2B)和非小细胞肺癌细胞(A549)分别给予常氧(21%O_(2))和低氧(1%O_(2))处理,测定细胞在常氧和低氧条件下的乳酸生成情况;用外源乳酸(0、5、10mM)在常氧和低氧条件下分别处理BEAS-2B和A549细胞,应用qPCR法分析线粒体DNA拷贝数,用蛋白免疫印迹法检测LC3B、TOM20、HSP60蛋白水平;使用Mitotracker■Red(581/644nm)、Lyso View Blue法检测线粒体及溶酶体共定位情况。结果与常氧组相比,低氧组BEAS-2B和A549的乳酸生成量明显升高(P<0.05)。未用乳酸干预时,相比于常氧组,低氧组A549线粒体拷贝数明显增加(P<0.0001),BEAS-2B线粒体拷贝数明显减少(P<0.0001);用乳酸干预后,常氧组和低氧组A549线粒体拷贝数随乳酸浓度增加而减少,BEAS-2B线粒体拷贝数变化则呈相反趋势。与对照组相比,常氧组和低氧组经乳酸干预后,BEAS-2B、A549细胞中的LC3B-Ⅱ/Ⅰ比值增加,A549细胞中的HSP60、TOM20蛋白表达水平下降,而BEAS-2B细胞中的HSP60、TOM20蛋白表达水平无明显改变;在常氧和低氧条件下与对照组相比,Lac-10mM组A549细胞线粒体和溶酶体荧光共定位增强,差异有统计学意义(P<0.05)。结论乳酸可以增强非小细胞肺癌细胞线粒体自噬功能。

Objective The aim of this study was to investigate the effect of lactate on autophagy of mitochondrion in human non-small cell lung cancer cells.Methods Human normal lung epithelial cells(BEAS-2B)and nonsmall cell lung cancer cells(A549)were treated with normoxia(21%O_(2))and hypoxia(1%O_(2)),respectively.Lactate production was measured under normoxia and hypoxia conditions.BEAS-2B and A549 cells were treated with exogenous lactate(O,5,10 mM)under normoxia and hypoxia conditions,respectively.Mitochondrial DNA copy number was analyzed by qPCR,and protein levels of LC3B,TOM20 and HSP60 were detected by Western blotting.Mitotracker■ Red(581/644 nm)and LysoView Blue assay were used to detect the colocalization of mitochondria and lysosomes.Results Compared with normoxia group,lactate production was significantly increased in BEAS-2B and A549 hypoxia groups(P<0.05).Compared with the normoxia group,the A549 mitochondrial copy number was significantly increased in the hypoxia group without lactate intervention(P<0.0001),BEAS-2B mitochondrial copy number was significantly reduced(P<0.0001);After lactate intervention,the mitochondrial copy number of A549 decreased with the increase of lactate concentration in normoxia and hypoxia groups,but the change of mitochondrial copy number of BEAS-2B showed an opposite trend.Compared with the control group,the LC3B-Ⅱ/Ⅰ ratio in BEAS-2B and A549 cells was increased and the protein expression levels of HSP60 and TOM20 in A549 cells were decreased after lactic acid intervention in normoxia group and hypoxia group,but the protein expression levels of HSP60 and TOM20 in BEAS-2B cells were not significantly changed.Compared with the control group,the colocalization of mitochondria and lysosomes in A549 cells in the Lac-10 mM group was enhanced under normoxia and hypoxia conditions,and the difference was statistically significant(P<0.05).Conclusion Lactate can enhance the autophagy function of the mitochoncria in non-small cell lung cancer cells.