科罗索酸诱导细胞内ROS及Ca^2+超载抗人结肠癌的研究  

Corosolic acid induced intracellular ROS and Ca^2+overload against human colon cancer

作　　者：鲍晓文 张铭雅 王宇[1] 朱志铭 马博[1] BAO Xiaowen;ZHANG Mingya;WANG Yu;ZHU Zhiming;MA Bo(College of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 211800, China)

机构地区：[1]南京工业大学药学院,江苏南京211800

出　　处：《南京工业大学学报（自然科学版）》2020年第5期663-670,共8页Journal of Nanjing Tech University(Natural Science Edition)

基　　金：国家自然科学基金(81773802);江苏省先进生物制造创新计划(XTD1819)。

摘　　要：为了研究科罗索酸对人结肠癌SW480细胞的抗增殖、促凋亡作用及其机制,分别采用不同浓度的科罗索酸干预SW480细胞24和48 h后,用四甲基偶氮唑蓝(MTT)法检测细胞活力并计算半数抑制浓度(IC 50);使用4,6-联-脒-2-苯基吲哚(DAPI)染色以及Annexin V-FITC/PI双染检测科罗索酸诱导细胞凋亡的作用;通过单克隆实验考察科罗索酸对SW480细胞增殖能力的影响;采用2′,7′二氯荧光素二乙酸酯(DCFH-DA)法和二氢乙啶(DHE)超氧化合物阴离子荧光探针法检测科罗索酸对SW480细胞内的活性氧(ROS)浓度的影响;荧光探针(JC-1)检测细胞线粒体膜电位;Ca^2+荧光探针Fluo-4AM检测细胞内Ca^2+浓度;蛋白质免疫印迹法检测科罗索酸对线粒体凋亡通路相关蛋白Cleaved Caspase-3、Bax、Bcl-2和Cleaved PARP表达的影响。结果表明:科罗索酸干预细胞24和48 h的半数抑制浓度分别为12.13和7.79μmol/L;低于半数抑制浓度的科罗索酸可以显著抑制SW480细胞的增殖;与空白对照组相比,科罗索酸(3.75、7.5和15μmol/L)干预SW480细胞后,其细胞凋亡率分别为20.05%、20.95%和31.60%,线粒体膜电位显著下降;同时,Cleaved Caspase 3、Bax和Cleaved PARP蛋白表达水平显著上升,Bcl 2表达水平显著降低,科罗索酸激活线粒体凋亡通路诱导SW480细胞凋亡。科罗索酸可以明显抑制SW480细胞的增殖并激活线粒体凋亡通路发挥抗肿瘤作用,其机制与上调细胞内活性氧浓度和Ca^2+超载有关。To study the anti-proliferation and pro-apoptotic effect and mechanism of corosolic acid(CA)on human colon cancer cell SW480,a series concentration of CA were used to intervene human colon cancer SW480 cells for 24 and 48 h.The cell viability was detected by the tetramethyl azozo blue(MTT)method,and the half-inhibitory concentration(IC50)was calculated.The 4,6-bialamidine-2-phenylindole(DAPI)staining and Annexin V-FITC/PI double staining method were used to detect the effect of CA induced apoptosis.The colony formation assay was employed to evaluate the ability of CA on tumor cell proliferation.Intracellular reactive oxygen species(ROS)concerntration were determined by the fluorescent probe 2′,7′-dichlorofluorescein diacetate(DCFH-DA)and dihydroethidium(DHE)staining.Mitochondrial membrane potential was determined by the JC-1 probe method.Intracellular Ca^2+concerntration was detected by Ca^2+fluorescent probe Fluo-4AM.Western blotting was used to detect the effect of CA on the expression of mitochondrial apoptosis pathway-related proteins Cleaved Caspase-3,Bax,Bcl-2 and Cleaved PARP.Results showed that the IC50 values of CA in SW480 cells at 24 and 48 h were 12.13 and 7.79μmol/L,respectively,and CA at lower levels could significantly inhibit human colon cancer SW480 cells.Compared with control group,the apoptosis rate of SW480 cells after CA(3.75,7.5 and 15μmol/L)was 20.05%,20.95%and 31.60%,respectively.The mitochondrial membrane potential was significantly decreased.At the same time,Cleaved Caspase-3,Bax and Cleaved PARP protein expression increased significantly,and Bcl-2 protein expression decreased significantly,which indicated CA activated mitochondrial apoptosis pathway to induce apoptosis in SW480 cells.CA could significantly inhibit the proliferation of SW480 cells and activate the mitochondrial apoptosis pathway to play an anti-tumor effect.The mechanism could be related to the increase of intracellular ROS concentration and Ca^2+overload.

关 键 词：科罗索酸 结肠癌 细胞凋亡 活性氧 细胞内CA^2+ 

分 类 号：R9[医药卫生—药学] R73