开口箭乙醇提取物诱导肝癌细胞HepG2凋亡活性研究
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摘要
目的探讨开口箭根部乙醇提取物(ERT-95)对肝癌细胞HepG2生长的影响及其抗肿瘤作用的可能机制。方法采用MTT法绘制HepG2细胞生长曲线,检测ERT-95对HepG2细胞的生长抑制作用;采用光学显微镜观察HepG2细胞的凋亡形态变化;采用Hochest染色原理检测HepG2细胞凋亡情况;采用Western blot检测Cyt C蛋白的表达;采用caspase活性检测试剂盒检测caspase-3、caspase-9的活性变化;采用DNA ladder凝胶电泳技术检测DNA的裂解情况。结果 MTT结果显示,ERT-95对HepG2细胞的抑制作用呈剂量依赖性(IC50为52.57μg·mL-1);光学显微镜下,处理组细胞出现皱缩、变圆等形态变化;Hochest-33258染色后的处理组细胞呈现典型的凋亡结构;Western blot结果显示,随着药物浓度增加,Cyt C蛋白表达上调;caspase活性检测显示,caspase-3、caspase-9活性逐步升高;与对照组相比,处理组琼脂糖凝胶电泳图谱出现DNA条带。结论ERT-95有显著的体外促肿瘤细胞凋亡作用,初步研究其促肿瘤细胞凋亡机制是通过线粒体释放Cyt C,进一步活化caspase-9和caspase-3而激活内源性核酸内切酶,导致DNA裂解而实现的。
Objective To observe the antitumor effect of ethanol extract from the root of Tupistra chinensis Baker.(ERT-95) on the liver cancer cell line HepG2, and to explore its potential mechanisms. Methods The growth curves and the cytostatic effect of ERT-95 on the HepG2 cells were evaluated by MTT assay. The morphological changes in HepG2 cells were observed by optical microscopes; ERT-95 induced cell apoptosis death on HepG2 cells was measured by Hochest 33258 staining. The effect on expressions of Cytochrome C(Cyt C) protein in HepG2 cells were detected by Western blot. Caspase-3 and caspase-9 activities were measured with caspase activity assay kit. The infected cells were examined for DNA fragmentation by agarose gel electrophoresis. ResultsMTT assay showed that ERT-95 was able to inhibit the HepG2 cells in a dose-dependent manner. The concentration required for 50% inhibition(IC50) was 52.57 μg·mL- 1. The morphological changes due to apoptosis incluede shrinking of cells and r ound cells under an optical microscope; Hochest 33258 staining of the fi xed cells revealed typical apoptotic structures. By Western blot, expression of Cyt C proteins in the cell line HepG2 was up-regulated with various concentrations of ERT-95. The activity of caspase-3 and caspase-9 after the treatment with ERT-95 was promoted; DNA fragment was also found by agarose gel electrophoresis in the administration group, but not in the control group. Conclusion ERT-95 has signifi cant in vitro antitumor effect. Its anti-tumor mechanism is that mitochondria releases Cyt C which further activates caspase-9 and caspase-3, triggering the activation of endogenous endonucleases and causing DNA cleavage.
Objective To observe the antitumor effect of ethanol extract from the root of Tupistra chinensis Baker.(ERT-95) on the liver cancer cell line HepG2, and to explore its potential mechanisms. Methods The growth curves and the cytostatic effect of ERT-95 on the HepG2 cells were evaluated by MTT assay. The morphological changes in HepG2 cells were observed by optical microscopes; ERT-95 induced cell apoptosis death on HepG2 cells was measured by Hochest 33258 staining. The effect on expressions of Cytochrome C(Cyt C) protein in HepG2 cells were detected by Western blot. Caspase-3 and caspase-9 activities were measured with caspase activity assay kit. The infected cells were examined for DNA fragmentation by agarose gel electrophoresis. ResultsMTT assay showed that ERT-95 was able to inhibit the HepG2 cells in a dose-dependent manner. The concentration required for 50% inhibition(IC50) was 52.57 μg·mL- 1. The morphological changes due to apoptosis incluede shrinking of cells and r ound cells under an optical microscope; Hochest 33258 staining of the fi xed cells revealed typical apoptotic structures. By Western blot, expression of Cyt C proteins in the cell line HepG2 was up-regulated with various concentrations of ERT-95. The activity of caspase-3 and caspase-9 after the treatment with ERT-95 was promoted; DNA fragment was also found by agarose gel electrophoresis in the administration group, but not in the control group. Conclusion ERT-95 has signifi cant in vitro antitumor effect. Its anti-tumor mechanism is that mitochondria releases Cyt C which further activates caspase-9 and caspase-3, triggering the activation of endogenous endonucleases and causing DNA cleavage.
引文
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[2]何苗,龙春燕,宋银宏.去甲基化药物治疗肿瘤的研究进展[J].中国药房,2012,23(29):2764-2767.
[3]施汀兰,杨雪,冉长清.中药抗肿瘤作用研究概况[J].重庆中草药研究,2008,12(2):32-36.
[4]芦柏震,陈小娟.中药抗肿瘤转移浅谈[J].海峡药学,2010,22(2):6-9.
[5]丁安伟.中药研究的现状及展望[J].南京中医药大学学报,1997,13(4):195-197.
[6]张秀云,周凤琴.中药抗肿瘤研究进展[J].辽宁中医药大学学报,2012,14(11):142-144.
[7]陈勇,刘婧,谢臻.开口箭属植物研究概况[J].时珍国医国药,2004,15(12):8602-8611.
[8]杨春艳,刘朝奇,邹坤,等.开口箭皂苷体外抗肿瘤作用的初步实验研究[J].时珍国医国药,2009,20(10):2390-2392.
[9]亏晓哗,钟雪云.Caspases与细胞凋亡[J].暨南大学学报:医学版,2000,21(6):121-124.
[10]李宏,冯有胜.线粒体Cyt C的释放机制及其在细胞凋亡中的作用[J].生物信息学,2010,8(3):210-213.
[11]王叨.细胞色素C与细胞凋亡的研究进展[J].中国小儿血液,2004,9(4):181-183.
[12]方希敏.细胞色素C与细胞凋亡[J].国外医学:临床生物化学与检验学分册,2005,26(1):43-46.
[13]Adams JM,Cory S.Life-or-death decisions by the Bcl-2 protein family[J].Trends Biochem Sci,2001,26(1):61-66.
[14]朱正光,余传林,蔡晶,等.开口箭提取物的抗肿瘤作用研究[J].中药材,2006,29(13):277-279.
[15]汪鋆植,邹坤,徐宏伟.开口箭皂苷抗炎活性成分呢研究[J].时珍国医国药,2006,17(10):1970-1971.