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目的探讨氟喹诺酮类化合物三甲氧基苯甲醛环丙沙星席夫碱诱导人肝癌SMMC-7721细胞凋亡作用。方法用不同浓度的1-环丙基-6-氟-7-(哌嗪~(-1)-基)-3-[5-苄硫基-4-(3,4,5-三甲氧苯甲叉基氨基)~(-1),2,4-三唑-3-基]-喹啉(1-H)-4-酮(M27)与人肝癌SMMC-7721细胞、结肠癌HCT~(-1)16细胞株、白血病JURKET细胞株体外培养。MTT法检测M27及其前体化合物环丙沙星盐酸盐对肿瘤细胞的增殖抑制作用;Hoechst 33258荧光染色法、TUNEL法检测细胞凋亡变化;以p BR322 DNA为底物,琼脂糖电泳法检测M27对拓扑异构酶Ⅱ的活性的影响;高内涵活细胞成像系统测定线粒体跨膜电位(△ψm)变化;Western blotting法检测p53、Caspase-9、Caspase-3、Caspase-8、Bcl-2和Bax等凋亡相关蛋白的表达以及细胞色素C在线粒体内外分布的变化。结果 M27在10~60μmol·L~(-1)内抑制肿瘤细胞增殖,呈浓度、时间依赖性。作用于SMMC-7721细胞、HCT~(-1)16细胞、JURKET细胞24 h IC50值分别为37.97、46.07、44.33μmol·L~(-1),而环丙沙星盐酸盐对SMMC-7721细胞增殖抑制作用不显著。各组M27作用于SMMC-7721细胞24 h,细胞凋亡率显著高于对照组(P<0.05),并伴有细胞线粒体膜电位(Δψm)降低。与对照组比较,M27能够抑制DNA拓扑异构酶Ⅱ的活性,使细胞p53、Bax、Caspase-8、Caspase-9和Caspase-3蛋白表达及活性片段增加,Bcl-2的表达量降低;线粒体细胞色素C减少,胞浆细胞色素C增多。结论三甲氧基苯甲醛环丙沙星席夫碱能够抑制拓扑异构酶Ⅱ的活性,造成DNA损伤,诱导人肝癌SMMC-7721细胞凋亡。
Objective To investigate the apoptosis of SMMC-7721 cells induced by fluoroquinolone trimethoxybenzaldehyde and ciprofloxacin Schiff base. Methods Different concentrations of 1-cyclopropyl-6-fluoro-7- (piperazine-1-yl) -3- [5-benzylthio-4- (M27) and human hepatoma SMMC-7721 cells, colon cancer HCT ~ (-1), 2,4-triazol-3-yl] -1) 16 cell line, leukemia JURKET cell line in vitro. The inhibitory effect of M27 and its precursor compound ciprofloxacin hydrochloride on the proliferation of tumor cells was detected by MTT assay. The apoptosis of tumor cells was detected by Hoechst 33258 staining and TUNEL assay. The pBR322 DNA was used as substrate and agarose gel electrophoresis The effect of M27 on the activity of topoisomerase Ⅱ was examined. The mitochondrial transmembrane potential (△ ψm) was determined by high-content living cell imaging system. The expressions of p53, Caspase-9, Caspase-8, Bcl- 2, Bax and other apoptosis-related proteins and the change of mitochondrial cytochrome C distribution inside and outside the mitochondria. Results M27 inhibited tumor cell proliferation in a concentration-dependent and time-dependent manner in 10 ~ 60μmol·L -1. The IC50 values of HCT-16 cells and JURKET cells for 24 h were 37.97, 46.07 and 44.33 μmol·L -1, respectively, while the effect of ciprofloxacin hydrochloride on SMMC-7721 cells The inhibition of cell proliferation was not significant. The apoptosis rate of M27 in SMMC-7721 cells was significantly higher than that in control group (P <0.05) at 24 h, and accompanied with decreased mitochondrial membrane potential (Δψm). Compared with the control group, M27 could inhibit the activity of DNA topoisomerase Ⅱ, increase the expression and activity of p53, Bax, Caspase-8, Caspase-9 and Caspase-3 protein and decrease the expression of Bcl- Cytochrome C decreased cytoplasmic cytochrome C increased. Conclusion Trimethoxybenzaldehyde-ciprofloxacin Schiff base can inhibit the activity of topoisomerase Ⅱ, cause DNA damage and induce apoptosis in human hepatoma SMMC-7721 cells.