摘要
目的探索耐阿霉素的乳腺癌细胞株MCF-7/Adr凋亡的可能途径。方法采用耐药株MCF-7/Adr和野生株MCF-7/wt2种细胞株进行对比,以噻唑蓝分析(MTT)法探索H2O2导致其线粒体功能下降的浓度,观察细胞形态学和线粒体超微结构的改变。应用DNA凝胶电泳、AnnexinⅤ和PI双标记流式细胞术检测细胞凋亡发生,Rh123共聚焦显微镜检测线粒体膜电位和P-gp功能,Western blot检测细胞色素C(Cyto C)的释放状况。结果4mmol/L H2O2引起MCF-7/Adr线粒体整体功能不可逆的进行性下降,线粒体空化,嵴肿胀。MCF-7/Adr凋亡率(96.52%)显著高于MCF-7/wt(18.57%,P<0.01)。凋亡DNA电泳仅见800、1600bp2个分子片段。MCF-7/Adr线粒体数量较MCF-7/wt减少,MCF-7/Adr膜电位下降。4mmol/L H2O2处理使MCF-7/Adr线粒体Cyto C释放至细胞质,MCF-7/wt仅少量Cyto C释放。结论P-gp阳性细胞因其线粒体的结构和功能改变的特点,较其野生株细胞更易经线粒体途径凋亡。提示以线粒体为靶点的药物有助于克服与P-gp表达有关的多药耐药。
Objective To investigate the possible apoptotic pathway in breast cancer daunorubicin-resistant subline, MCF-7/Adr cell line. Methods Drug-resistance cell line MCF-7/Adr expressing functional P-gp and its wild type MCF-7/wt cell line were used for comparison. The concentration of hydrogen peroxide （H2O2 ） that may lead to loss of mitochondrial function was determined using MTr assay. The cell morphology and the mitochondrial uhrasructure were observed by invert and transmission electron microscopy, respectively. The apoptosis of the two cell lines was determined with DNA gel electrophoresis and Annexin V and PI doublestained flow cytometry. Mitochondrial membrane potential （MMP） and the function of P-gp were assessed with rhodamine 123 （ Rh123 ） staining intensity by confocal microscopy. The release of cytoehrome C from mitochondria was observed by Western blotting. Results The mitochondrial function of MCF-7/Adr was irreversibly damaged at the concentration of 4 mmol/L H2O2. The mitochondria were vacuolated and their cristae were swollen. The percentage of MCF-7/Adr cell apoptosis was significantly higher than that of MCF-7/wt, 96.52% and 18.57%, respectively （P 〈0.01 ）. Only 800 and 1 600 Da DNA fragmentation was seen. The number of mitochondria and membrane potential of MCF-7/Adr mitochondria decreased as compared with those of MCF-7/wt. H2O2 at the concentration of 4 mmol/L induced release of cytochrome C from MCF-7/Adr mitochondria to cytoplasma, but little was detected in MCF-7/wt. Conclusion MCF-7/Adr cells expressing functional P-gp are more sensitive to H2O2-mediated mitoehondrial apoptosis. It may attribute to the changes of mitochondrial structure and function in MCF-7/Adr cells. These may suggest that some way which may modify mitochondrial characteristics may be benificial to overcome the muhidrug resistance related to P-gp.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第15期1475-1478,共4页
Journal of Third Military Medical University
关键词
肿瘤
多药耐药
凋亡
H2O2
线粒体
细胞色素C
tumor
multidrug resistance
apoptosis
hydrogen peroxide
mitochondria
cytochrome C
