榄香烯乳诱导线粒体凋亡途径逆转肺癌A549/DDP细胞株耐药

摘要

Objective:To explore the mechanism that elemene( ELE)reversed the multidurg resistance( MDR) of A549/DDP lung adenocarcinoma cell. Methods:MTT assay was used to determine the growth inhibition of human lung adenocarcinoma A549/DDP cells in vitro. Mitochondrial membrane potential( MMP)was monitored by JC-1 fluorescence probe with laser confocal scanning microscopy,the intercellular reactive oxygen species( ROS)level was measured by 2',7'-dichlorfluorescein-diacetate( DCFH-DA)staining and flow cytometry and the ratio of GSH/( GSSG﹢GSH)was calculated according to detection results of GSH kit. The expression of Cytochrome C,Caspase-3 and the Bcl-2 family proteins and in the case of cyclosporine A and DEVD-CHO,the expression of Caspase-3 expression were measured by Western blot. Results:MTT results showed that different concentrations ELE could in-hibit the proliferation of A549/DDP cells in a time-and dose-dependent manner. Intriguingly,ELE plus cisplatin enhanced the sensitivity of A549/DDP cells to cisplatin and reversed A549/DDP cells dury resistance. Different con-centrations ELE decreased mitochondrial membrane potential,increased intracellular ROS concentration and decreased GSH/( GSSG﹢GSH)ratio of A549/DDP cells in a time-and dose-dependent manner. Furthermore,the combina-tion with both ELE and cisplatin also enhanced the protein expression of Cytoplasmic C,Caspase-3 and Bad,and re-duced the protein levels of Bcl-2 and Pro-caspase-3 in the cisplatin-resistant A549/DDP cancer cells. Conclu-sion:ELE reversed the MDR of A549/DDP cell line may demage mitochondrial membrane,active intracellular redox system and induce the mitochondrial apopotosis pathway.%目的：探讨榄香烯乳（ elemene，ELE）逆转人肺腺癌耐顺铂（ cisplatin，DDP）细胞A549/DDP的耐药性及作用机制。方法：采用MTT法检测榄香烯乳单用的细胞毒作用及与DDP合用时耐药逆转作用。荧光探针JC-1结合激光共聚焦显微镜检测线粒体膜电位的变化。DCFH-DA荧光探针结合流式细胞仪检测细胞内活性氧（ reactive oxygen species，ROS）水平。用谷胱甘肽试剂盒结合分光光度法检测计算GSH/（ GSSG﹢GSH）比值。蛋白质印迹法检测胞质中Cyto C、Pro-caspase-3、Caspase-3和Bcl-2家族蛋白表达情况。结果：不同浓度榄香烯乳抑制A549/DDP细胞株生长，呈时间-剂量依赖性效应，联合顺铂能提高A549/DDP细胞株对顺铂的敏感性而逆转耐药。不同浓度榄香烯乳联合顺铂使A549/DDP细胞株线粒体膜电位下降，ROS浓度增加，GSH/（ GSSG﹢GSH）比值降低，上调胞质中Cyto C、Caspase-3、Bad蛋白表达，下调Pro-caspase-3、Bcl-2蛋白表达。结论：榄香烯乳逆转A549/DDP细胞株耐药性可能与其损伤线粒体膜，活化胞内氧化还原体系，诱导线粒体凋亡路径有关。