摘要
探究去甲斑蝥素(norcantharidin,NCTD)对三阴性乳腺癌MDA-MB-231细胞增殖和凋亡的影响。采用Western blot实验检测NCTD对MDA-MB-231细胞中凋亡相关蛋白Bax/Bcl-2、cleaved-PARP/PARP、cleaved-caspase-9、cleaved-caspase-3和MCL-1表达水平的影响;采用Western blot实验检测NCTD对MDA-MB-231细胞中自噬相关蛋白LC3-II/LC3-I,Parkin和PINK1表达水平的影响;采用流式细胞术检测NCTD对MDA-MB-231细胞线粒体膜电位及线粒体活性氧(reactive oxygen species,ROS)变化情况的影响;采用共聚焦显微镜检测NCTD对表达mCherry-EGFP-LC3的MDA-MB-231细胞自噬流的影响;采用流式细胞术检测NCTD联合使用氯喹(chloroquine,CQ)或3-甲基腺嘌呤(3-methyladenine,3-MA)后,对MDA-MB-231细胞凋亡情况的影响。实验结果显示,NCTD可显著上调Bax/Bcl-2、cleaved-PARP/PARP、cleaved-caspase-9、cleavedcaspase-3以及LC3-II/LC3-I蛋白的表达水平,促进Parkin的线粒体易位,阻断自噬流。此外,NCTD联合使用CQ加剧了细胞凋亡,而NCTD联合使用3-MA则减少了细胞凋亡。研究结果表明,NCTD可以诱导自噬体积累并导致MDA-MB-231细胞凋亡。
The aim of this study was to investigate the effect of norcantharidin(NCTD)on the proliferation and apoptosis of triple-negative breast cancer cell line MDA-MB-231.Western blot was used to detect the effect of NCTD on the expression levels of apoptosis-related proteins Bax/Bcl-2,cleaved-PARP/PARP/PARP,cleved-caspase-9,cleaved-caspase-3 and MCL-1 in MDA-MB-231 cells.Also,the expression levels of autophagy-related proteins LC3-II/LC3-I,Parkin and PINK1 in MDA-MB-231 cells were measured by Western blot.Flow cytometry was used to measure the effect of NCTD on the changes of mitochondrial membrane potential and mitochondrial reactive oxygen species(ROS).The effect of NCTD on autophagy flow in cells expressing mCherry-EGFP-LC3 was detected by a confocal microscope.Moreover,the effects of NCTD combined with chloroquine(CQ)or 3-methyladenine(3-MA)on the apoptosis of MDA-MB-231 cells were detected by flow cytometry.The results showed that NCTD significantly increased the expression levels of Bax/Bcl-2,cleaved-PARP/PARP,cleaved-caspase-9,cleasved-caspase-3 and LC3-II/LC3-I proteins,and promoted the mitochondrial translocation of Parkin,and blocked the autophagic flow in MDA-MB-231 cells.Moreover,NCTD combined with CQ accelerated apoptosis,while NCTD combined with 3-MA decreased apoptosis.These results suggest that NCTD can induce autophagy accumulation and lead to apoptosis of MDA-MB-231 cells.
作者
夏源
姜庆玲
王晓婷
李敏敬
郑秋生
李德芳
XIA Yuan;JIANG Qingling;WANG Xiaoting;LI Minjing;ZHENG Qiusheng;LI Defang(Key Laboratory of Xinjiang Phytomedicine Resource and Utilization(Ministry of Education),School of Pharmacy,Shihezi University,Shihezi 832061;Featured Laboratory for Biosynthesis and Target Discovery of Active Components of Traditional Chinese Medicine,School of Integrated Traditional Chinese and Western Medicine,Binzhou Medical University,Yantai 264003;Yantai Key Laboratory of Pharmacology of Traditional Chinese Medicine in Tumor Metabolism,School of Integrated Traditional Chinese and Western Medicine,Binzhou Medical University,Yantai 264003,China)
出处
《中国药科大学学报》
CAS
CSCD
北大核心
2023年第6期757-768,共12页
Journal of China Pharmaceutical University
基金
国家自然科学基金资助项目(No.81872162)。
