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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。
To observe the effect of the quinolone compound ofloxacin hydrazone on the apoptosis of human hepatocarcinoma SMMC-7721 cells, the effect of different concentrations of N- (3-benzilidene) -6-fluoro-1,8- 2-propoxy) -7- (4-methylpiperazin-1-yl) -quinoline-4 (1H) -one-3-carboxylic acid hydrazide (FQ16) and SMMC-7721 cells were cultured in vitro. MTT assay was used to detect the inhibitory effect of FQ16 on proliferation of SMMC-7721 cells; Hoechst33258 / PI double staining staining, TUNEL assay and agarose gel electrophoresis were used to detect the changes of apoptosis; pBR322DNA was used as substrate, and agarose gel electrophoresis The effect of FQ16 on the activity of human DNA topoisomerase II was assayed. The changes of △ mitochondrial membrane potential (△ ψm) were measured by high-content living cell imaging system. The expressions of Bcl-2 and Bax mRNA were observed by RT- 9, caspase-8, caspase-3, p53, Bcl-2, Bax protein expression. The results showed that FQ16 could inhibit cell proliferation in a concentration range of 0.625 ~ 10μmol·L-1 in a time-and concentration-dependent manner. The apoptosis rate of FQ16 was significantly higher than that of the control group (P <0.05) Glucose gel electrophoresis shows a typical DNA ladder apoptotic cells, accompanied by decreased mitochondrial membrane potential. Compared with the control group, FQ16 could inhibit the activity of DNA topoisomerase II, increase the expression of Bax mRNA and the expression of Bcl-2 mRNA, and increase the expression of p53, Bax, caspase-9 and caspase-3, , Caspase-3 active fragment increased significantly, caspase-8 unchanged, and decreased Bcl-2 protein expression. The results suggest that the cinnamic aldehyde ofloxacin hydrazone can inhibit the activity of DNA topoisomerase II, cause DNA damage and activate the mitochondrial apoptosis pathway, and induce the apoptosis of human hepatoma cells.