肉桂醛氧氟沙星酰腙诱导人肝癌SMMC-7721细胞凋亡的作用  

Cinnamaldehyde ofloxacin-3-ylhydrazone induces apoptosis of human hepatocarcinoma SMMC-7721 cells

作　　者：任争[1,2] 康玉华[2] 石贞玉[1] 皇甫超申[1] 胡国强[3] 刘彬[1] 

机构地区：[1]河南大学护理学院神经生物学研究所,河南开封475004 [2]河南大学淮河临床学院,河南开封475004 [3]河南大学化学生物学研究所,河南开封475004

出　　处：《药学学报》2010年第9期1109-1115,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(20872028);河南省教育厅自然科学研究计划项目(2009B310001;2010B310002);开封市科技发展计划项目(080312);河南大学科技攻关项目(07YBGG004)

摘　　要：为观察喹诺酮类化合物肉桂醛氧氟沙星酰腙化合物诱导人肝癌SMMC-7721细胞凋亡的作用,用不同浓度的N-(3-苯亚丙烯基)-6-氟-1,8-(2,1-丙氧基)-7-(4-甲基哌嗪-1-基)-喹啉-4(1H)-酮-3-甲酰肼(FQ16)与SMMC-7721细胞体外培养。MTT法检测FQ16对SMMC-7721细胞的增殖抑制作用;Hoechst33258/PI双染荧光染色法、TUNEL法及琼脂糖凝胶电泳检测细胞凋亡变化;以pBR322DNA为底物,采用琼脂糖凝胶电泳法测定FQ16对人DNA拓扑异构酶II活性的影响;高内涵活细胞成像系统测定细胞线粒体膜电位(△ψm)变化;RT-PCR方法观察Bcl-2、BaxmRNA的表达变化;Westernblotting方法测定caspase-9、caspase-8、caspase-3、p53、Bcl-2、Bax蛋白表达。结果显示,FQ16在0.625～10μmol·L?1的浓度范围能抑制细胞增殖,呈时间、浓度依赖性;各组FQ16作用24h后,细胞凋亡率显著高于对照组(P<0.05);琼脂糖凝胶电泳可见凋亡细胞典型的梯状DNA条带,并伴有线粒体膜电位降低。与对照组比较,FQ16能抑制DNA拓扑异构酶II的活性,使细胞BaxmRNA表达增高,Bcl-2mRNA表达水平下降,p53、Bax、caspase-9、caspase-3蛋白表达量增加,其中caspase-9、caspase-3活性裂解片段显著增加,caspase-8无变化,而Bcl-2蛋白表达水平下降。结果提示,肉桂醛氧氟沙星酰腙能够抑制DNA拓扑异构酶II活性,造成DNA损伤并激活线粒体凋亡通路,诱导人肝癌细胞凋亡。This study is to observe the effect of N-（3-phenylallylidene）-6-fluoro-1, 8-（2, 1-propoxy）-7-（4methylpiperazin-1-yl）-quinolin-4（1H）-one-3-carbonyl hyarazine （FQ16） on apoptosis of hepatocarcinoma SMMC-7721 cells in vitro. With different concentrations of FQ16 at different times used to treat SMMC-7721 cells in vitro, the proliferation of the cells and the inhibition effect of FQ16 on the cell proliferation were examined by MTT assay. Cell apoptosis was determined by Hoechst 33258/PI fluorescence staining, TUNEL and agarose gel electrophoresis method. The effect of FQ16 on topoisomerase II activity was measured by agarose gel electrophoresis using Plasmid pBR322 DNA as the substrate. Mitochondrial membrane potential （MMP, ψm） was measured by high content screening image system. The reverse transcriptase-polymerase chain reaction （RT-PCR） was used to detect the expression changes of Bcl-2 mRNA and Bax mRNA. The caspase-9, caspase-8, caspase-3, p53, Bcl-2 and Bax protein expressions were detected by Western blotting analysis. The results showed that the cell proliferation was inhibited by FQ16 at 0.625 ？ 10 μmol·L？1 in a time-dose dependent manner. Treatment of SMMC-7721 cells with different concentrations of FQ16 for 24 h increased the percentage of the apoptosis cells obviously （P 0.05）, the typical ladder DNA in apoptotic cells and a concomitant dissipation of the mitochondrial membrane potential. Compared with control group, FQ16 influenced obviously DNA topoisomerase II activity, stimulated DNA cleavage and inhibited DNA reunion mediated by topoisomerase II. In addition, FQ16 （3 ？ 7.39 μmol·L？1） increased mRNA expression of Bax and protein expression of p53, Bax, caspase-9, caspase-3, separately, and induced cytosolic accumulation of activities caspase-9 and caspase-3, whereas the mRNA and protein expression of Bcl-2 decreased with no change of caspase-8. Therefore it can be concluded that the effects of inhibited topoisomerase II and mitochondrial dependent path

关 键 词：喹诺酮衍生物 细胞凋亡 拓扑异构酶Ⅱ 线粒体膜电位 

分 类 号：R963[医药卫生—微生物与生化药学]