利用定量蛋白组学的方法研究甲异靛诱导K562细胞凋亡的机制
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摘要
目的:利用串联质谱标签系统(tandem mass tag,TMT)标记定量蛋白组学的方法,检测白血病细胞系K562细胞经甲异靛(meisoindigo)处理后蛋白表达的改变,探讨甲异靛诱导白血病细胞凋亡的作用机制。方法:用CCK8法检测甲异靛对K562细胞的半抑制浓度(inhibitory concentration 50,IC50),流式细胞术检测不同浓度甲异靛诱导K562细胞的凋亡水平。K562细胞经终浓度为0. 2%DMSO(对照组)和20μmol/L甲异靛(实验组)处理2 h后,提取总蛋白,TMT标记,高效液相色谱分级和质谱定量蛋白组学技术鉴定肽段和丰度信息,并进行3次技术重复。用Mascot软件鉴定蛋白,用GO(gene ontology)注释、富集和聚类分析方法分析差异表达蛋白。结果:在K562细胞系中,甲异靛以剂量依赖的方式诱导K562细胞凋亡(r=0. 98);蛋白质组学分析结果显示,共鉴定出蛋白质5 544个,其中有定量数据的蛋白质4 792个。与对照组相比,在实验组中表达差异1. 5倍以上的蛋白有8个,其中4个蛋白表达上调,4个蛋白表达下调,差异变化的蛋白主要与活性氧代谢相关。结论:应用定量蛋白质组学分析表明,包括DDIT4等蛋白的表达在甲异靛处理K562细胞系早期发生明显改变,提示活性氧代谢过程的改变可能在甲异靛促进凋亡的机制中发挥了重要作用。
Objective: To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags( TMT)-based proteomics technology,and to explore the mechanism for meisoindigo-inducing apoptosis. Methods: The half inhibitory concentration( IC50) of mesoindigo on K562 cells was determined by CCK8. The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO. Total proteins w ere extracted from the cells treated with 0. 2% DMSO( control) or 20 μmol/L meisoindigo( Test) for 2 hours. Then,the TMT-labeling HPLC-MS/MS was used to identify and quantify the peptides and their abundance,all the tests w ere repeated for 3 times. The Mascot software was used to identify the proteins; the GO annotations,enrichment and cluster analysis w ere used to analyze the differentially expressed proteins. Results:Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner( r = 0. 98),5 544 proteins w ere identified,4792 of which w ere quantified. The protein with expression difference > 1. 5-folds in Test group accoanted for 8,out of which the expression of 4 proteins w ere up-regulated and 4 w ere dow n-regulated. The differentially expressed proteins mainly associated with reactive oxygen species( ROS). Conclusion: Several proteins including DDIT4 w ere found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology. The ROS metabolic process may play important roles in meisoindigo-inducing apoptosis of K562 cells.
Objective: To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags( TMT)-based proteomics technology,and to explore the mechanism for meisoindigo-inducing apoptosis. Methods: The half inhibitory concentration( IC50) of mesoindigo on K562 cells was determined by CCK8. The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO. Total proteins w ere extracted from the cells treated with 0. 2% DMSO( control) or 20 μmol/L meisoindigo( Test) for 2 hours. Then,the TMT-labeling HPLC-MS/MS was used to identify and quantify the peptides and their abundance,all the tests w ere repeated for 3 times. The Mascot software was used to identify the proteins; the GO annotations,enrichment and cluster analysis w ere used to analyze the differentially expressed proteins. Results:Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner( r = 0. 98),5 544 proteins w ere identified,4792 of which w ere quantified. The protein with expression difference > 1. 5-folds in Test group accoanted for 8,out of which the expression of 4 proteins w ere up-regulated and 4 w ere dow n-regulated. The differentially expressed proteins mainly associated with reactive oxygen species( ROS). Conclusion: Several proteins including DDIT4 w ere found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology. The ROS metabolic process may play important roles in meisoindigo-inducing apoptosis of K562 cells.
引文
1 Xiao Z,Hao Y,Liu B,et al.Indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in China.Leuk lymphoma,2002;43(9):1763-1768.
2 Kim SU,Song KS,Jung DS,et al.Production of indoxyl derivatives in indole-supplemented tissue cultures of Polygonum tinctorium.Planta Med,1996;62(1):54-56.
3 Duensing S,Duensing A,Lee DC,et al.Cyclin-dependent kinase inhibitor indirubin-3'-oxime selectively inhibits human papillomavirus type 16 E7-induced numerical centrosome anomalies.Oncogene,2004;23(50):8206-8215.
4 Blazevic T,Heiss EH,Atanasov AG,et al.Indirubin and indirubin derivatives for counteracting proliferative diseases.Evid Based Complement Alternat Med,2015;2015:654098.
5陶忠华,尹宏文.治疗慢性粒细胞白血病新药甲异靛.中国新药杂志,1993;2(01):10-13.
6竺晓凡,钱林生,刘鸿,等.甲异靛治疗慢性粒细胞白血病远期疗效观察.中华血液学杂志,1998;19(03):45-46.
7左明新,李燕,周建华,等.甲异靛对K562和HL-60细胞Wnt信号通路的影响.中国实验血液学杂志,2010;18(03):579-582.
8 Xiao Z,Wang Y,Lu L,et al.Anti-angiogenesis effects of meisoindigo on chronic myelogenous leukemia in vitro.Leuk Res,2006;30(1):54-59.
9 Schubert OT,Rost HL,Collins BC,et al.Quantitative proteomics:Challenges and opportunities in basic and applied research.Na Protoc,2017;12(7):1289-1294.
10 Sawyers CL.Chronic myeloid leukemia.N Engl J Med,1999;340(17):1330-1340.
11 Goss VL,Lee KA,Moritz A,et al.A common phosphotyrosine signature for the Bcr-Abl kinase.Blood,2006;107(12):4888-4897.
12 Groarke JD,Cheng S,Moslehi J.Cancer-drug discovery and cardiovascular surveillance.N Engl J Med,2013;369(19):1779-1781.
13 Thompson A,Schfer J,Kuhn K,et al.Tandem mass tags:A nove quantification strategy for comparative analysis of complex protein mixtures by ms/ms.Anal Chem,2003;75(8):1895-1904.
14刘兵城,肖志坚.甲异靛对K562细胞bcr-abl信号通路的影响以及诱导细胞凋亡机制研究.中华血液学杂志,2008;29(12):815-818.
15吴英理,邬维礼,孙关林.甲异靛对K562细胞的作用及其机制的初步研究.中华血液学杂志,2004;25(01):46-48.
16 Ellisen LW,Ramsayer KD,Johannessen CM,et al.Redd1,a developmentally regulated transcriptional target of p63 and p53,links p63 to regulation of reactive oxygen species.Mol Cell,2002;10(5):995-1005.
17 Schwarzer R,Tondera D,Arnold W,et al.Redd1 integrates hypoxia-mediated survival signaling downstream of phosphatidylinositol 3-kinase.Oncogene,2005;24(7):1138-1149.
18 Cheng X,Kim JY,Ghafoory S,et al.Methylisoindigo preferentially kills cancer stem cells by interfering cell metabolism via inhibition of LKB1 and activation of AMPK in PDACs.Mol Oncol,2016;10(6):806-824.
19 Lee CC,Lin CP,Lee YL,et al.Meisoindigo is a promising agent with in vitro and in vivo activity against human acute myeloid leukemia.Leuk Lymphoma,2010;51(5):897-905.
20 Mingxin Z,Yan L,Hongbo W,et al.The antitumor activity of meisoindigo against human colorectal cancer HT-29 cells in vitro and in vivo.J Chemother,2008;20(6):728-733.
21王一,连小云,张玎,等.甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究.现代肿瘤医学,2011;19(07):1312-1315.
2 Kim SU,Song KS,Jung DS,et al.Production of indoxyl derivatives in indole-supplemented tissue cultures of Polygonum tinctorium.Planta Med,1996;62(1):54-56.
3 Duensing S,Duensing A,Lee DC,et al.Cyclin-dependent kinase inhibitor indirubin-3'-oxime selectively inhibits human papillomavirus type 16 E7-induced numerical centrosome anomalies.Oncogene,2004;23(50):8206-8215.
4 Blazevic T,Heiss EH,Atanasov AG,et al.Indirubin and indirubin derivatives for counteracting proliferative diseases.Evid Based Complement Alternat Med,2015;2015:654098.
5陶忠华,尹宏文.治疗慢性粒细胞白血病新药甲异靛.中国新药杂志,1993;2(01):10-13.
6竺晓凡,钱林生,刘鸿,等.甲异靛治疗慢性粒细胞白血病远期疗效观察.中华血液学杂志,1998;19(03):45-46.
7左明新,李燕,周建华,等.甲异靛对K562和HL-60细胞Wnt信号通路的影响.中国实验血液学杂志,2010;18(03):579-582.
8 Xiao Z,Wang Y,Lu L,et al.Anti-angiogenesis effects of meisoindigo on chronic myelogenous leukemia in vitro.Leuk Res,2006;30(1):54-59.
9 Schubert OT,Rost HL,Collins BC,et al.Quantitative proteomics:Challenges and opportunities in basic and applied research.Na Protoc,2017;12(7):1289-1294.
10 Sawyers CL.Chronic myeloid leukemia.N Engl J Med,1999;340(17):1330-1340.
11 Goss VL,Lee KA,Moritz A,et al.A common phosphotyrosine signature for the Bcr-Abl kinase.Blood,2006;107(12):4888-4897.
12 Groarke JD,Cheng S,Moslehi J.Cancer-drug discovery and cardiovascular surveillance.N Engl J Med,2013;369(19):1779-1781.
13 Thompson A,Schfer J,Kuhn K,et al.Tandem mass tags:A nove quantification strategy for comparative analysis of complex protein mixtures by ms/ms.Anal Chem,2003;75(8):1895-1904.
14刘兵城,肖志坚.甲异靛对K562细胞bcr-abl信号通路的影响以及诱导细胞凋亡机制研究.中华血液学杂志,2008;29(12):815-818.
15吴英理,邬维礼,孙关林.甲异靛对K562细胞的作用及其机制的初步研究.中华血液学杂志,2004;25(01):46-48.
16 Ellisen LW,Ramsayer KD,Johannessen CM,et al.Redd1,a developmentally regulated transcriptional target of p63 and p53,links p63 to regulation of reactive oxygen species.Mol Cell,2002;10(5):995-1005.
17 Schwarzer R,Tondera D,Arnold W,et al.Redd1 integrates hypoxia-mediated survival signaling downstream of phosphatidylinositol 3-kinase.Oncogene,2005;24(7):1138-1149.
18 Cheng X,Kim JY,Ghafoory S,et al.Methylisoindigo preferentially kills cancer stem cells by interfering cell metabolism via inhibition of LKB1 and activation of AMPK in PDACs.Mol Oncol,2016;10(6):806-824.
19 Lee CC,Lin CP,Lee YL,et al.Meisoindigo is a promising agent with in vitro and in vivo activity against human acute myeloid leukemia.Leuk Lymphoma,2010;51(5):897-905.
20 Mingxin Z,Yan L,Hongbo W,et al.The antitumor activity of meisoindigo against human colorectal cancer HT-29 cells in vitro and in vivo.J Chemother,2008;20(6):728-733.
21王一,连小云,张玎,等.甲异靛对人乳腺癌MCF-7细胞增殖和凋亡影响的实验研究.现代肿瘤医学,2011;19(07):1312-1315.