竹节香附素A对人结肠癌细胞HCT-116增殖、凋亡、迁移及侵袭活性的影响  

Effects of Raddeanin A on Proliferation,Apoptosis,Migration and Invasion of Human Carcinoma Colon Cell Line HCT-116

作　　者：王宇[1] 姜修博 韩豆 朱志铭 宋长琴 赵昂[1] 张琪[1] 刘昌辉[2] 马博[1] WANG Yu;JIANG Xiubo;HAN Dou;ZHU Zhiming;SONG Changqin;ZHAO Ang;ZHANG Qi;LIU Changhui;MA Bo;(School of Pharmaceutical Sciences, Nanjing Tech University;Institute of Clinical Medicine, Guangzhou University of Chinese Medicine)

机构地区：[1]南京工业大学药学院,药动药效研究与评价中心,江苏南京210009 [2]广州中医药大学临床药理研究所,广东广州510405

出　　处：《中药新药与临床药理》2018年第2期123-130,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金青年项目(81302835); 2015年江苏高校优秀科技创新团队计划项目(苏教科[2015]4号); 江苏省大学生创新创业训练计划项目(省级重点)(201710291028Z)

摘　　要：目的研究竹节香附素A(Raddeanin A,RA)对人结肠癌细胞HCT-116增殖、凋亡、迁移及侵袭活性的影响,并初步探讨其作用机制。方法采用不同浓度的RA(0.125~50.0μmol·L^(-1))处理HCT-116细胞24,48 h,四甲基偶氮唑蓝(MTT)法检测细胞的增殖能力。RA(1.0,2.0,4.0μmol·L^(-1))干预后,采用Annexin V/PI双染法检测细胞凋亡率;JC-1染色法检测线粒体膜电位变化;DCFH-DA法检测细胞内活性氧簇(ROS)水平;采用Western Blot法检测细胞中Cleaved caspase-3蛋白表达。选择低于凋亡浓度的RA(0.25,0.5,1.0μmol·L^(-1))作用于HCT-116细胞,采用细胞划痕实验和Transwell穿膜实验观察不同浓度RA对HCT-116细胞迁移的影响;采用铺有Matrigel胶的Transwell实验检测RA对HCT-116细胞侵袭能力的影响;采用RT-PCR法和Western Blot法检测RA对HCT-116细胞中MMP-2、MMP-9 m RNA及蛋白表达的影响。结果 RA干预24,48 h后的IC50值分别为5.967μmol·L^(-1)和4.797μmol·L^(-1),且呈现浓度与时间依赖性。与空白对照组比较,RA1.0,2.0,4.0μmol·L^(-1)浓度组凋亡、坏死的HCT-116细胞明显增多(P<0.05,P<0.01);线粒体膜电位坍塌率分别为70.2%,74.4%和91.6%,差异均有统计学意义(P<0.01);细胞内ROS含量均显著增加(P<0.01),且呈现浓度依赖性;Cleaved caspase-3蛋白的表达水平随RA浓度的增加而显著提高(P<0.01)。RA干预后与空白对照组比较,0.5,1.0μmol·L^(-1)浓度组的细胞愈合率显著降低(P<0.01);RA 0.25,0.5,1.0μmol·L^(-1)浓度组均能显著抑制HCT-116细胞的迁徙能力(P<0.01),均能显著抑制HCT-116细胞体外侵袭能力(P<0.05,P<0.01);RA 0.5,1.0μmol·L^(-1)浓度组MMP-2、MMP-9 m RNA及蛋白相对表达水平均显著降低(P<0.05,P<0.01)。结论 RA既能够有效抑制人结肠癌HCT-116细胞增殖,又能诱导该细胞的凋亡,可能与诱导细胞内ROS产生,导致线粒体膜电位坍塌,激活caspase信号通路有关。低于凋亡浓度的RA(0.25,0.5,1.0μmol·L^(-1))能明显抑制HCT-116细胞的迁移�Objective To investigate roles of Raddeanin A（RA） on proliferation, apoptosis, migration and invasion of human carcinoma colon HCT-116 cells, and the mechanism of its role on migration and invasion.Methods HCT-116 cells were treated with RA at a range of 0.125 to 50.0 μmol·L^（-1） for 24 h and 48 h respectively.Cell proliferation was assessed by MTT（Methyl Thiazolyl Tetrazolium） assay. The apoptosis rate of HCT-116 cells was calculated by Annexin V/PI double staining. Meanwhile,the level of electric potential of mitochondrion wasdetected by JC-1;and the level of ROS（reactive oxygen species） in HCT-116 cell was assessed by DCFH-DA.Western Blot was carried out to analyze the expression of cleaved caspase-3. Furthermore,cell migration at different concentrations of RA（0.25,0.5,1.0 μmol·L^（-1）） on HCT-116 cells was assayed by the scratch wound healing and Transwell chamber assay. And Matrigel cell invasion assay was used to detect the effect of RA on the invasion of HCT-116 cells,real-time quantitative PCR and Western Blot were used to detect the m RNA and protein levels of matrix metalloproteinase-2（MMP-2） and matrix metalloproteinase-9（MMP-9） in the cells. Results IC_（50） values were 5.967 μmol·L^（-1） and 4.797 μmol·L^（-1） after cells were treated with RA for 24 h and 48 h,which showed dose-and time-dependent manner. Compared with the control,numbers of apoptotic and necrotic HCT-116 cells were significantly increased（P〈0.05,P〈0.01） in RA（1.0,2.0,4.0 μmol·L^（-1）） groups,and the collapse levels ofmitochondrial membrane potential were 70.2%, 74.4%, which were both statistically significant（P〈0.01）compared with the control（91.6%）. Meanwhile, the level of intracellular ROS and the expression of cleavedcaspase-3 were significantly increased（P〈0.01） in a dose-dependent manner. Compared with the control,thescratch wound healing of the cells were significantly decreased after treating with RA（0.5,1.0 μmol·L^（-1））（P〈0.01）;an

关 键 词：竹节香附素A 结肠癌细胞HCT-116 细胞凋亡 细胞增殖 细胞迁移 细胞侵袭 

分 类 号：R285.5[医药卫生—中药学]