寨卡提取物体外对人结肠癌细胞凋亡的诱导作用及其作用机制研究

Induction of the Extract from Thlaspi arvense Linn.on Apoptosis in Human Colorectal Carcinoma LoVo Cells and its Mechanisms in vitro

目的研究寨卡提取物诱导人结肠癌LoVo细胞凋亡的作用,并对其可能的作用机制进行探讨。方法以寨卡提取物作用于人结肠癌LoVo细胞,采用MTT法分析寨卡提取物对LoVo肿瘤细胞的抑制生长作用;采用Annexin V-FITC荧光染色实验,考察细胞在药物作用下的质膜变化,对提取物诱导LoVo细胞凋亡进行检测;采用比色法测定寨卡提取物对人大肠癌LoVo肿瘤细胞内还原型谷胱甘肽(GSH)的耗竭作用和对谷胱甘肽-S转移酶(GST)的诱导作用;同时利用DCFH-DA探针,检测LoVo肿瘤细胞给药后的活性氧水平;应用W estern蛋白印迹法检测Caspase-3和Caspase-9蛋白的表达。结果寨卡提取物具有明显的体外抗肿瘤活性,并呈剂量依赖关系,IC50为550μg.m l-1。该药物可通过改变细胞质膜,耗竭细胞内谷胱甘肽含量,增加谷胱甘肽-S转移酶含量,上调肿瘤细胞内活性氧水平等途径,提高Caspase-3和Caspase-9蛋白的表达,诱导LoVo细胞发生凋亡。结论寨卡提取物可诱导LoVo细胞凋亡,其作用机制可能通过耗竭细胞内GSH的含量,以ROS为介导直接作用于细胞线粒体,诱导凋亡的产生。寨卡提取物对LoVo细胞凋亡的诱导作用还可能与其增加Caspase-3和Caspase-9蛋白表达有关。

Objective To study the induction of the extract from Thlaspi arvense Linn.（ZAF） on apoptosis in human colorectal carcinoma LoVo cells and its mechanisms in vitro.Methods MTT assay was employed to evaluate the survival of LoVo cells.The induction of apoptosis was detected by Annexin V-FITC staining.The depletion of glutathione（GSH） and the induction of glutathione-S-transferase（GST） effects of ZAF in LoVo cells were dectected.The level of reactive oxygen species（ROS） in LoVo cells was analyzed with DCFH-DA as probe.The protein expressions of Caspase-3 and Caspase-9 in LoVo cells were assayed by Western blot.Results ZAF effectively inhibited LoVo cell viability in vitro and induced LoVo cells apoptosis in a dose-dependent manner,and IC50 was 550μg·ml-1.ZAF could obviously deplet the level of GSH level,increase activity of GST,and accumulate ROS in LoVo cells.Over-expression of Caspase-3 and Caspase-9 mediated by ZAF in a concentration-dependent manner were observed.Conclusion ZAF can induce apoptosis of LoVo cells,which is relative to the changes of the cells redox states and upregulating Caspase-3 and Caspase-9 expression.