环巴胺诱导大肠癌Caco-2细胞凋亡效应和线粒体蛋白质组学研究

J4 ›› 2011, Vol. 37 ›› Issue (3): 407-412.

环巴胺诱导大肠癌Caco-2细胞凋亡效应和线粒体蛋白质组学研究

代恩勇¹, 张桂珍², 卢振霞¹, 杜珍武², 白鹭鹭¹, 孟令俊¹

（1.吉林大学中日联谊医院肿瘤血液科|吉林长春 130033；2.吉林大学中日联谊医院中心研究室| |吉林长春 130033)

收稿日期:2011-04-01 出版日期:2011-05-28 发布日期:2011-05-28
通讯作者: 卢振霞 E-mail:E-mail: lzx2010@yahoo.com.cn
作者简介:代恩勇(1977-),男,吉林省吉林市人,主治医师|在读医学博士|主要从事大肠癌基因诊断及治疗研究。
基金资助:
国家自然科学基金面上项目资助课题(30870355)；吉林省科技厅科技发展计划项目资助课题（200705316）

Effect of cyclopamine on apoptosis of colorectal cancer  Caco-2 cells and study on proteomics of |mitochondria

DAI En-Yong¹, ZHANG Gui-Zhen², LEI Zhen-Xia¹, DU Zhen-Wu², BAI Lu-Lu¹, MENG Lian-Dun¹

(1．Department of Oncology and Hematology,China-Japan Union Hospital,Jilin University,Changchun 130033,China;2.Central Laboratory,China-Japan Union Hospital,Jilin University,Changchun 130033,China)

Received:2011-04-01 Online:2011-05-28 Published:2011-05-28

摘要/Abstract

摘要：

目的：探讨环巴胺对大肠癌Caco-2细胞的促凋亡效应，在线粒体蛋白水平阐明其作用机制。方法: 取对数生长期大肠癌Caco-2细胞分为环巴胺处理组及阴性对照组，以5、10、20和40　μmol·L^-1环巴胺处理Caco-2细胞，分别应用MTS法及流式细胞术测定Caco-2细胞生长抑制率及凋亡率，采用nano LC-ESI-MS方法检测环巴胺处理组与阴性对照组细胞线粒体蛋白质组学差异。结果:环巴胺处理组Caco-2细胞分别经10、20和40 μmol·L^-1环巴胺作用24、48和72 h后，生长抑制率(分别为23.1%±1.8%，46.2±0.9%，53.4±2.3%；45.1%±2.8%，73.0%±2.5%，81.2%±1.6%；59.7±2.3%，87.5±1.4%，91±1.06%)明显高于阴性对照组(1.8%±0.2%,2.5%±0.1%,3.7%±0.3%)（P<0.01）；经5、10、20和40 μmol·L^-1环巴胺作用于Caco-2细胞48 h后，细胞凋亡率分别为24.1%±1.3%、31.7%±1.6%、50.5%±2.3%和64.0%±1.9%，明显高于阴性对照组(14.4%±0.7%)（P<0.01）。蛋白质组学差异分析，环巴胺作用后，Caco-2细胞线粒体共有32种蛋白表达下调， 25种蛋白表达上升，这些蛋白功能主要涉及细胞凋亡、细胞骨架、核酸、核糖体及蛋白质代谢等，其中与凋亡关系密切的蛋白包括ATF6、Clusterin、OPA1、RGD1565411和Cofilin。结论:环巴胺通过阻断Hedgehog(HH)通路，抑制大肠癌Caco-2细胞增殖，其机制与促进细胞凋亡有关;环巴胺可明显改变Caco-2细胞线粒体蛋白表达，其差异与凋亡关系密切。

关键词: 环巴胺；Hedgehog通路；大肠肿瘤；蛋白质组学

Abstract:

Objective:To investigate the effect of cyclopamine on proliferation,apoptosis of colorectal cancer Caco-2 cells and expounds its mechanism in the mitochondria using proteomic methods.Methods The colorectal cancer Caco-2 cells in logarithmic growth phase were divided into cyclopamine treatment groups and negative control groups. The Caco-2 cells were treated with 5,10,20 and 40 μmol·L^-1 cyclopamine,MTS assay and flow cytometric analysis were applied to detect the inhibitory rate of growth and the apoptotic rate of Caco-2 cells. Nano-LC-ESI-MS method was used to detect the protein expression differences in mitochondria between various groups.Results The growth inhibition rate of Caco-2 cells in treatment groups by 10,20,40 μmol·L^-1 cyclopamine after 24,48 and 72 h were 23.1%±1.8%,46.2%±0.9%,53.4%±2.3%,45.1%±2.8%,73.0%±2.5%,81.2%±1.6%,59.7%±2.3%,87.5%±1.4% and 91.0%±1.06%,they were obviously higher than those in negative control groups(1.8%±0.2%,2.5%±0.1%,and 3.7%±0.3%)（P<0.01）.The apoptotic rates of Caco-2 cell in treatment groups by 5,10,20, 40 μmol·L^-1 cyclopamine after 48 h were 24.1%±1.3%，31.7%±1.6%，50.5%±2.3%, and 64.0%±1.9%，they wer higher than that in negative control groups (14.5%±0.7%)(P<0.01). Proteomics difference analysis showed that Caco-2 cell mitochondrial proteins obviously changed after treated with cyclopamine,the expressions of 32 proteins were down-regulated and the expressions of 25 proteins were up-regulated. Function analysis showed that these proteins involved in apoptosis,cytoskeleton,nucleic acid,ribosomes and protein metabolism,etc,among them Clusterin,OPA1,ATF6,RGD1565411 and Cofilin had close relation with apoptosis.Conclusion Cyclopamine can inhibit the proliferation of colorectal cancer Caco-2 cells by blocking HH pathways,its mechanism involve in promoting apoptosis.Cyclopamine could change the protein expression of Caco-2 cell mitochondria obviously,the differences are related to apoptosis closely.

Key words: cyclopamine;colorectal neoplasms;Hedgehog signaling pathway;proteomics

中图分类号:

R735.3

代恩勇, 张桂珍, 卢振霞, 杜珍武, 白鹭鹭, 孟令俊. 环巴胺诱导大肠癌Caco-2细胞凋亡效应和线粒体蛋白质组学研究[J]. J4, 2011, 37(3): 407-412.

DAI En-Yong, ZHANG Gui-Zhen, LEI Zhen-Xia, DU Zhen-Wu, BAI Lu-Lu, MENG Lian-Dun. Effect of cyclopamine on apoptosis of colorectal cancer  Caco-2 cells and study on proteomics of |mitochondria[J]. J4, 2011, 37(3): 407-412.

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