槐定碱抑制人胶质瘤U87MG细胞株增殖及其作用机制研究

Sophoridine inhibits proliferation of human glioma U87MG cell line and its mechanism

目的探讨槐定碱抑制人胶质瘤U87恶性胶质瘤(U87MG)细胞株增殖作用及其相关机制。方法将各个浓度的槐定碱加入到人胶质瘤U87MG细胞株中,应用四唑氮盐比色法测定槐定碱对肿瘤细胞生长的抑制作用,流式细胞仪测定槐定碱对U87MG细胞凋亡和细胞周期的变化,生化检验测定肿瘤细胞内活性氧簇(ROS)和谷胱甘肽(GSH)含量,实时荧光定量聚合酶链式反应和蛋白免疫印迹法分别检测肿瘤相关基因mRNA表达和蛋白表达的变化,荧光素酶报告基因法检测肿瘤细胞中泛素-蛋白酶体、叉头盒蛋白M1(FoxM1)、核转录因子kappa B(NF-kb)、激活子蛋白-1(AP-1)的活性的变化。结果槐定碱能够显著抑制细胞增殖,阻滞细胞周期在G2/M期;诱导细胞凋亡,引起ROS产生和GSH含量减少;促使p27、周期蛋白依赖性激酶2、Survivin、Livin、B淋巴细胞瘤-2、E2启动子结合细胞因子1等基因表达下调,同时上调半胱氨酸天冬氨酸蛋白酶-3/8、p53、线粒体来源的第2个半胱氨酸蛋白酶激活剂、c-Jun氨基末端激酶和p38-丝裂原活化蛋白激酶等基因的表达;显著抑制泛素-蛋白酶体活性和FoxM1、NF-kb、AP-1的转录活性。结论槐定碱可能通过诱导细胞凋亡和ROS积聚,激活线粒体信号通路来发挥抗肿瘤活性。槐定碱能够作为一种新的药物来治疗胶质瘤。

Objective The effect and mechanism of sophoridine on proliferation inhibition in human U87 malignant glioma （U87MG） cell line were investigated. Methods Sophoridine at different concentrations were added into human neuroglioma U87 line cells. 3-（4,5-dimethylthiazol-zyl ）-5-（ 3- carboxyrocthoxyphenyl ） -2- （ 4-sulfophenyl ） - 2H-tetrazoliuzolium, inner salt assay was employed to detect the inhibitory effect of sophoridine in tumor cells, flow cytometry to detect apoptosis and cell cycle, and biochemical assay to determinate intracellular reactive oxygen species （ROS） and glutathione （GSH） contents, real time-polymerase chain reaction to detect the changes in tumor- related gene mRNA expression, Western Blot to detect the changes in tumor-related gene protein expression, and luciferase report gene assay to detect the activity of ubiquifin-proteasome, forkhead box M1 （ FoxM1 ）, nuclear transcription factor kappa B （NF-κb） and activator protein 1 （AP-1） in tumor cells.Results Sophoridine significantly inhibited the cell proliferation, G2/M phase arrest, induced cell apoptosis and caused ROS generation and GSH content reduction. Sophoridine also triggered significantly the down-regulating of the expression of p27, eyclin-dependent kinases2, Survivin, Livin, B-cell lymphoma-2, E2-promoter binding factor and the transcriptional activity of FoxM1, NF-κb and AP-1, meanwhile, up-regulating of the expression of cysteinyl aspartate-specific protease-3/8, p53, second mitochondria derived activator of caspase, c-Jun N-terminal kinase and p38-mitogen- activated protein kinase. Sophoridine significantly inhibited the activity of ubiquitin-proteasome in tumor cells. Conclusion Sophoridine shows obvious anti-cancer activity on glioma cells by inducing cell apoptosis, ROS accumulation, and activating mitochondrial signal pathways. Sophoridine can be used as a new drug for the treatment of glioma.