重楼单体PP-11对人乳腺癌MDA-MB-231细胞增殖的抑制作用及其机制  

Inhibitory effect of PP-11,a monomer isolated from Paris polyphylla,on proliferation of human breast cancer MDA-MB-231 cells and its mecha⁃nism

作　　者：朱金艳 叶静怡 刘志龙[3] 李强[3] 赵娜 吴志慧 蒋建伟 林晨 汤杰 ZHU Jin-yan;YE Jing-yi;LIU Zhi-long;LI Qiang;ZHAO Na;WU Zhi-hui;JIANG Jian-wei;LIN Chen;TANG Jie(Department of Microbiology and Immunology,School of Medicine,Jinan University,Guangzhou 510632,China;Department of Biochemistry and Molecular Biology,School of Medicine,Jinan University,Guangzhou 510632,China;Department of General Surgery,The First Affiliated Hospital,Jinan University,Guangzhou 510632,China;Department of Oncology,Liyang People's Hospital,Liyang 213300,China.E-mail:[e-mail])

机构地区：[1]暨南大学基础医学院微生物和免疫学系,广东广州510632 [2]暨南大学基础医学院生物化学与分子生物学系,广东广州510632 [3]暨南大学附属第一医院肝胆外科,广东广州510632 [4]江苏省溧阳市人民医院肿瘤科,江苏溧阳213300

出　　处：《中国病理生理杂志》2021年第2期246-254,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82074064);广东省医学科学技术研究基金资助项目(No.A2019535);广东省中医药局资助项目(No.20201078);暨南大学附属第一医院科研培育专项基金资助项目(No.2019315)。

摘　　要：目的:探讨中药重楼(Paris polyphylla,PP)活性单体PP-11体外抑制人乳腺癌MDA-MB-231细胞增殖的作用及其机制。方法:采用不同浓度的PP-11作用于MDA-MB-231细胞,采用MTT法和集落形成实验检测细胞增殖情况;Hoechst 33258染色及Annexin V-FITC/PI双染流式细胞术检测细胞凋亡;JC-1染色检测线粒体膜电位改变;Western blot法检测细胞凋亡及自噬相关蛋白表达情况。再采用总caspase抑制剂Z-VAD-FMK、p38抑制剂SB203580和自噬抑制剂氯喹(chloroquine,CQ)进行阻断实验。结果:MTT测定结果显示,PP-11以剂量和时间依赖方式显著抑制MDA-MB-231细胞活力,其作用24、48和72 h后的IC_(50)分别为5.64、4.58和3.06μmol/L。集落形成实验结果提示,PP-11显著抑制MDA-MB-231细胞集落形成。Hoechst 33258染色观察到PP-11组MDA-MB-231细胞出现典型的细胞凋亡形态。Annexin V-FITC/PI双染流式细胞术结果显示,随着PP-11浓度的增加,MDA-MB-231细胞凋亡率逐渐升高。JC-1染色结果显示,PP-11处理的MDA-MB-231细胞线粒体膜电位下降。Western blot检测结果显示,PP-11处理的MDA-MB-231细胞Bcl-2家族蛋白中抗凋亡蛋白Bcl-2和Bcl-xL的表达显著减少,促凋亡蛋白Bim和Bok表达增加,p-p38和p-p53蛋白水平升高,p-ERK蛋白水平降低,cleaved caspase-9、cleaved caspase-3及cleaved PARP的蛋白水平显著升高;PP-11降低MDA-MB-231细胞p-STAT3蛋白水平及其下游蛋白c-Myc、cyclin D和Mcl-1的表达。总caspase抑制剂Z-VAD-FMK和p38 MAPK抑制剂SB203580均可减弱PP-11诱导的细胞凋亡。随着PP-11作用浓度的增加,细胞自噬相关蛋白LC3-Ⅱ表达增加,p62/SQSTM1表达下降;采用PP-11联合自噬阻断剂CQ,可逆转PP-11诱导的cleaved PARP表达,并提高细胞活力(P<0.05)。结论:重楼单体PP-11可显著抑制人乳腺癌MDA-MB-231细胞增殖。PP-11通过激活p38 MAPK信号通路、抑制ERK和JAK-Stat3信号通路诱导MDA-MB-231细胞发生线粒体相关的凋亡,并促进细胞自噬。AIM:To explore the anti-proliferation effect of PP-11,an active monomer isolated from Paris poly⁃phylla,on human breast cancer MDA-MB-231 cells and its molecular mechanism.METHODS:The MDA-MB-231 cells were incubated with different concentrations of PP-11.MTT assay and colony formation assay were used to detect the cell proliferation.The morphological changes of apoptotic cells were observed by Hoechst 33258 staining.The apoptosis was analyzed by flow cytometry with annexin V-FITC/PI double staining.JC-1 staining was used to detect the changes of mitochondrial membrane potential.The expression of apoptosis-related proteins and autophagy-related proteins was determined by Western blot.The total caspase inhibitor Z-VAD-FMK,p38 MAPK inhibitor SB203580,and autophagy inhibitor chloroquine(CQ)were used for blocking experiments.RESULTS:Treatment with PP-11 inhibited the proliferation of MDAMB-231 cells in time-and dose-dependent manners,and the IC_(50) values of PP-11 for 24 h,48 h and 72 h were 5.64,4.58 and 3.06μmol/L,respectively.PP-11 significantly inhibited the colony formation of MDA-MB-231 cells.The MDAMB-231 cells in PP-11 group showed obvious apoptotic morphological changes after Hoechst 33258 staining.The results of flow cytometry with annexin V-FITC/PI double staining showed that the percentage of apoptotic cells was increased gradually with the increase in PP-11 concentration.JC-1 staining assay showed that the mitochondrial membrane potential was decreased in PP-11 treatment group compared with control group.Western blot analysis showed that PP-11 significantly reduced the expression of anti-apoptosis proteins Bcl-2 and Bcl-xL,and significantly promoted the increases in pro-apoptotic proteins Bim and Bok.The protein levels of p-p38,p-p53,cleaved caspase-9,cleaved caspase-3 and cleaved PARP in PP-11 treatment group were significantly increased,and the protein level of p-ERK was significantly decreased.PP-11 inhibited the protein levels of p-Stat3 and downstream proteins c-Myc,cyclin D and Mcl-1 in the cell

关 键 词：重楼 乳腺癌 细胞增殖 细胞凋亡 自噬 

分 类 号：R737.9[医药卫生—肿瘤] R730.23[医药卫生—临床医学]