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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 Topoisomerase is an important target of many tumor chemotherapies discovered in recent years and is closely related to the occurrence, proliferation and development of tumor cells. This study analyzed the antitumor activity of two novel topoisomerase inhibitors in vitro and explored the mechanism of action. Methods MTT assay was used to detect the inhibitory effect of two test substances on the proliferation of human tumor cells. The effect of inhibitors on Topo Ⅰ catalytic activity was detected by the pBR322 helicase reaction. The protein expression of Topo Ⅰ and Topo Ⅱ were detected by Western blotting. Hoechst 33258 Fluorescence staining, AO fluorescence staining, comet assay and flow cytometry were used to observe the effects of two test substances on the apoptosis and cell cycle of HEp-2 cells. Results The results of MTT assay showed that the tested compounds 1 and 2 showed significant antitumor activity against nine kinds of human tumor cell lines in a concentration-dependent manner (P <0.05). The test substances 1 and 2 inhibited Topo-mediated Of the plasmid DNA supercoiled, and the two kinds of test substance can down-regulate Topo I, II protein expression in the cells, P <0.05; Hoechst 33258 fluorescence staining showed that the test substance can induce the apoptosis of HEp-2 cells; AO staining The same results were also obtained. Two kinds of test substances were detected by comet assay, which could lead to DNA damage. The result of flow cytometry showed that the apoptosis rate of treated group was higher than that of control group (P <0.05) 1 inhibited the cell cycle of HEp-2 cells in S phase and G2 / M phase (P <0.01), while in test cell 2, it blocked in G1 phase (72h) (P <0.05). Western blotting showed that HEp- The expression of pro-apoptotic proteins Bid, Bax and JNK increased (P <0.01), the expression of anti-apoptotic protein Bcl-2 decreased (P <0.01) and the expressions of Cyclin B1 and Cyclin D1 decreased (P <0.05). CONCLUSION: Compounds 1 and 2 can inhibit the growth of HEp-2 cells, which may be related to the following mechanisms: By inhibiting the catalytic activity of Topo I and down-regulating the expression of Topo I and Topo II, regulating the expression of apoptosis-related genes and inducing their apoptosis Death; resulting in tumor cell cycle disorders and promote apoptosis.