新型拓扑异构酶抑制剂抗肿瘤活性及其机制研究  

Antitumor effect of two novel topoisomerase inhibitors and the molecular mechanisms

作　　者：陈列松[1] 虞莹[2] 王攀[2] 王文娟[2] 梁中琴[2] 

机构地区：[1]苏州大学附属第二医院放疗科,江苏苏州215004 [2]苏州大学药学院肿瘤药理学系,江苏苏州215009

出　　处：《中华肿瘤防治杂志》2016年第9期553-562,共10页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81102466;81373430)

摘　　要：目的拓扑异构酶是近年来发现的多种肿瘤化疗的重要靶点,与肿瘤细胞的发生、增殖和发展密切相关。本研究分析两种新型拓扑酶抑制剂的体外抗肿瘤活性,并初步探讨其作用机制。方法采用MTT法检测两种受试物对人肿瘤细胞增殖的抑制作用;质粒pBR322解旋反应检测抑制剂对TopoⅠ催化活性的影响,蛋白质印迹法检测细胞中TopoⅠ和TopoⅡ的蛋白表达变化;Hoechst 33258荧光染色、AO荧光染色、彗星实验和流式细胞术等方法观察两种受试物对HEp-2细胞凋亡和细胞周期的影响。结果 MTT法的结果显示,受试物1和2对9种人肿瘤细胞株均有显著的抗肿瘤活性(P<0.05),并呈现浓度依赖性;受试物1和2可抑制TopoⅠ介导的质粒DNA超螺旋的解旋,并且两种受试物都可下调细胞中TopoⅠ、Ⅱ的蛋白表达,P<0.05;Hoechst 33258荧光染色表明受试物可诱导HEp-2细胞凋亡,AO染色也得到相同的结果;彗星实验检测出两种受试物可导致DNA损伤;流式细胞仪检测的结果表明,处理组的细胞凋亡率高于对照组(P<0.05),并且,受试物1使HEp-2细胞周期在S期和G2/M期发生阻滞(P<0.01),而受试物2使其阻滞在G1期(72h),P<0.05;蛋白质印迹法结果显示促凋亡蛋白Bid、Bax和JNK表达增加(P<0.01),抗凋亡蛋白Bcl-2表达减少(P<0.01),细胞周期蛋白Cyclin B1和Cyclin D1表达减少(P<0.05)。结论受试物1和2可以抑制HEp-2细胞的生长,这可能与以下机制有关:通过抑制TopoⅠ的催化活性并下调细胞中TopoⅠ和TopoⅡ的表达;调控凋亡相关基因的表达,诱导其凋亡;造成肿瘤细胞周期紊乱,促进凋亡。OBJECTIVE Topoisomerases are considered as important therapeutic targets for a wide range of cancers, due to their association with the initiation, proliferation and survival of cancer ceils. We investigated the antitumor effect of two novel topoisomerase inhibitors and the molecular mechanisms. METHODS Cell inhibition rates of 9 human tumor cell lines treated with Compound 1 or Compound 2 were assessed with MTT assay. Topo I mediated-pBR322 DNA unwinding were measured using agarose gel electrophoresis. The expression of Topo Ⅰ/Ⅱ in HEp-2 cells was evaluated by western blot analysis. The activation of apoptotic effect and cell cycle arrest of HEp-2 cells were characterized by Hoechst 33258 staining, AO staining, comet assay and flow cytometry. RESULTS MTT assay indicated that Compound 1 and Compound 2 decreased the viability of 9 human tumor cell lines in dose-dependent manner（P〈0.05） ; Compound 1 and Compound 2 inhibited the Topo I-mediated relaxation of supercoiled pBR322 plasmid DNA effectively. Western blot analysis indicated that the compounds could decrease the Topo Ⅰ/Ⅱ protein expression（P〈0.05） ; Apoptosis was induced in HEp-2 cells by the compounds as detected by Hoechst 33258 staining and AO staining; DNA damage induced by the compounds was detected by comet assay（P;0.05） ; flow cytometry analysis indicated that the compounds increased cell apoptosis, S and G2/M cell arrest （induced by Compound 1, 72 h, P〈0. 01） and G1 cell arrest （induced by Compound 2, 72 h, P〈0. 05） ; Western blot analysis indicated that the compounds increase Bid, Pax and JNK expression（P〈0.01）, decrease Bcl-2（P〈0.01）, Cyclin B1 and Cyclin D1 expression（P〈0.05）. CONCLUSIONS Both Compound 1 and Compound 2 inhibit the proliferation of HE;2 cells. The mechanisms are probably related to the factors as follows： Inhibition of catalytic activity of Topo Ⅰ and down-regulation of Topo Ⅰ/Ⅱ protein expression in cells; Regulating transcription and expression of tumor related genes o

关 键 词：拓扑酶抑制剂 人肝癌HEp-2细胞 细胞凋亡 细胞周期 

分 类 号：R96[医药卫生—药理学]