迭鞘石斛乙醚提取物对肺癌细胞抑制机制的定量蛋白质组学研究
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摘要
迭鞘石斛Dendrobium denneanum作为名贵中药材具有悠久的使用历史,研究发现其乙醚提取物相比传统的醇或水提取物对肺癌细胞具有更强的抑制作用,具备更好的开发前景。该研究采用基于SILAC(stable isotope labeling with amino acids incell cultures)的定量蛋白质组学方法,从蛋白质组层面对迭鞘石斛乙醚提取物抑制人肺癌细胞A549的作用机制进行了系统研究,共检测到A549细胞的4 855种蛋白。数据分析显示迭鞘石斛乙醚提取物导致A549细胞中237种蛋白的表达出现明显差异,其中193种蛋白含量出现明显的上调,44种蛋白含量出现明显的下调。差异表达蛋白主要与细胞内生物大分子的合成运输及代谢、分子伴侣、DNA修复、氧化还原酶、细胞黏附、细胞周期、细胞凋亡与自噬等功能相关。结果显示,迭鞘石斛乙醚提取物会严重紊乱肺癌细胞内蛋白等物质的代谢过程,诱发内质网应激反应,同时使细胞的自我修复机制出现异常,导致细胞的黏附与增殖功能受损。另一方面,乙醚提取物会引起肺癌细胞内ROS水平的剧烈升高,同时使细胞内的氧化还原系统发生紊乱。在上述多重因素的影响下,肺癌细胞启动细胞凋亡和细胞自噬等多种进程,加速细胞的死亡。该研究结果从蛋白质组层面系统阐释了迭鞘石斛乙醚提取物抑制肺癌细胞的作用机制,为迭鞘石斛乙醚提取物的抑癌作用研究提供了理论基础,有利于从迭鞘石斛乙醚提取物中开发出新型的抗癌药物。
Dendrobium denneanum have been used for a long time as rare medicinal herbs in traditional Chinese medicine. Our previous works found that ether extract of D. denneanum had higher anticancer activities than alcohol or water extract,thus with better development prospects. Quantitative proteomics based on SILAC technique was used to investigate the anticancer mechanism of D. denneanum on lung tumor cell line A549,and 4 855 proteins were detected in A549 cells. Quantitative proteomics experiments found that 193 proteins of A549 cells were up-regulated,and 44 proteins were down-regulated by ether extract of D. denneanum. Those proteins are associated with synthesis,transport and metabolism of biological macromolecules,chaperone,DNA repair,oxidoreductase,cell adhesion,cell cycle,apoptosis and autophagy. Through the function analysis of differentially expressed proteins,it was inferred that ether extract of D. denneanum caused cell protein metabolism disorder,endoplasmic reticulum stress response,abnormal self-repair mechanism of cells,damage of cell adhesion and proliferation; besides,it caused a dramatic increase in ROS level in A549 cells,and upset the balance of intracellular oxidation reduction system. Affected by the above factors,lung cancer cells initiated apoptosis and autophagy,which accelerated cell death. This research explains the anticancer mechanism of D. denneanum from the perspective of quantitative proteomics,and lays a foundation for future research and development of new anticancer drugs based on ether extract of D. denneanum.
Dendrobium denneanum have been used for a long time as rare medicinal herbs in traditional Chinese medicine. Our previous works found that ether extract of D. denneanum had higher anticancer activities than alcohol or water extract,thus with better development prospects. Quantitative proteomics based on SILAC technique was used to investigate the anticancer mechanism of D. denneanum on lung tumor cell line A549,and 4 855 proteins were detected in A549 cells. Quantitative proteomics experiments found that 193 proteins of A549 cells were up-regulated,and 44 proteins were down-regulated by ether extract of D. denneanum. Those proteins are associated with synthesis,transport and metabolism of biological macromolecules,chaperone,DNA repair,oxidoreductase,cell adhesion,cell cycle,apoptosis and autophagy. Through the function analysis of differentially expressed proteins,it was inferred that ether extract of D. denneanum caused cell protein metabolism disorder,endoplasmic reticulum stress response,abnormal self-repair mechanism of cells,damage of cell adhesion and proliferation; besides,it caused a dramatic increase in ROS level in A549 cells,and upset the balance of intracellular oxidation reduction system. Affected by the above factors,lung cancer cells initiated apoptosis and autophagy,which accelerated cell death. This research explains the anticancer mechanism of D. denneanum from the perspective of quantitative proteomics,and lays a foundation for future research and development of new anticancer drugs based on ether extract of D. denneanum.
引文
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[24] Ciccia A,Elledge S J. The DNA damage response:making it safeto play with knives[J]. Mol Cell,2010,40(2):179.
[25] Hassan M,Watari H,Abu Almaaty A,et al. Apoptosis and mo-lecular targeting therapy in cancer[J]. Biomed Res Int,2014,2014:150845.
[26] White E,Mehnert J M,Chan C S. Autophagy,metabolism,andcancer[J]. Clin Cancer Res,2015,21(22):5037.
[27]陈易斯,宋福永.选择性自噬接头蛋白p62/sequestosome 1的研究进展[J].中国药理学与毒理学杂志,2016,30(3):258.
[28] Milroy L G,Ottmann C. The renaissance of Ras[J]. ACS ChemBiol,2014,9(11):2447.
[2] Ferlay J,Shin H R,Bray F,et al. Estimates of worldwide bur-den of cancer in 2008:GLOBOCAN 2008[J]. Int J Cancer,2010,127(12):2893.
[3]刘思远,李志鹏,彭召云,等.花青素对肺癌细胞相关炎性因子信号转导通路作用的研究进展[J].中国实验方剂学杂志,2017,23(14):219.
[4]董洁,袁将,王加利,等.维药阿纳其根抗肿瘤活性部位筛选及其化学成分的分离鉴定[J].中国中药杂志,2017,42(20):3932
[5]王佳娜,张艳,宋丽艳,等.刺齿凤尾蕨化学成分及其体外抗肿瘤活性研究[J].中国中药杂志,2017,42(21):4159
[6] Lam Y,Ng T B,Yao R M,et al. Evaluation of chemical constit-uents and important mechanism of pharmacological biology indendrobium plants[J]. Evid Based Complement Alternat Med,2015,2015:841752.
[7] Tsai A C,Pan S L,Lai C Y,et al. The inhibition of angiogene-sis and tumor growth by denbinobin is associated with the bloc-king of insulin-like growth factor-1 receptor signaling[J]. J NutrBiochem,2011,22(7):625.
[8] Song J I,Kang Y J,Yong H Y,et al. Denbinobin,a phenan-threne from dendrobium nobile,inhibits invasion and induces ap-optosis in SNU-484 human gastric cancer cells[J]. Oncol Rep,2012,27(3):813.
[9] Chen P H,Peng C Y,Pai H C,et al. Denbinobin suppressesbreast cancer metastasis through the inhibition of Src-mediatedsignaling pathways[J]. J Nutr Biochem,2011,22(8):732.
[10] Fang H,Hu X,Wang M,et al. Anti-osmotic and antioxidantactivities of gigantol from Dendrobium aurantiacum var. dennea-num against cataractogenesis in galactosemic rats[J]. J Ethno-pharmacol,2015,172:238.
[11]罗傲雪,宋关斌,范益军,等.迭鞘石斛抗肿瘤作用动物实验研究[J].四川大学学报:自然科学版,2005,42(6):1281.
[12]罗傲雪,宋关斌,淳泽,等.迭鞘石斛抗肿瘤作用研究[J].应用与环境生物学报,2007(2):184.
[13] Prasad R,Koch B. Antitumor activity of ethanolic extract of Den-drobium formosum in T-cell lymphoma:an in vitro and in vivostudy[J]. Biomed Res Int,2014,2014:753451.
[14] Wang Q,Sun P,Li G,et al. Inhibitory effects of Dendrobiumcandidum Wall ex Lindl. on azoxymethane-and dextran sulfatesodium-induced colon carcinogenesis in C57BL/6 mice[J]. On-col Lett,2014,7(2):493.
[15]张华,李莹,刘静,等.基于定量蛋白质组学分析迭鞘石斛对肺癌细胞的抑制作用及机理[J].应用与环境生物学报,2017,23(1):100.
[16]李莹,静刘,张雪琴,等.石斛不同极性成分的提取及其对肺癌细胞的抑制作用[J].应用于环境生物学报,2018,24(1):13.
[17] Brossier N M,Prechtl A M,Longo J F,et al. Classic Ras pro-teins promote proliferation and survival via distinct phosphopro-teome alterations in neurofibromin-null malignant peripheralnerve sheath tumor cells[J]. J Neuropathol Exp Neurol,2015,74(6):568.
[18] Vasan N,Boyer J L,Herbst R S. A RAS renaissance:emergingtargeted therapies for KRAS-mutated non-small cell lung cancer[J]. Clin Cancer Res,2014,20(15):3921.
[19] Prasad R,Rana N K,Koch B. Dendrobium chrysanthum ethanol-ic extract induces apoptosis via p53 up-regulation in He La cellsand inhibits tumor progression in mice[J]. J Complement IntegrMed,2017,doi:10. 1515/jcim-2016-0070.
[20]许婉琦,王奕博,孙志蓉.石斛属植物抗肿瘤研究情况分析[J].中国现代应用药学,2017,34(1):130.
[21] Oakes S A,Papa F R. The role of endoplasmic reticulum stress inhuman pathology[J]. Annu Rev Pathol,2015,10:173.
[22] Dandekar A,Mendez R,Zhang K. Cross talk between ER stress,oxidative stress,and inflammation in health and disease[J].Methods Mol Biol,2015,1292:205.
[23] ShaltielⅠA,Krenning L,Bruinsma W,et al. The same,onlydifferent-DNA damage checkpoints and their reversal throughoutthe cell cycle[J]. J Cell Sci,2015,128(4):607.
[24] Ciccia A,Elledge S J. The DNA damage response:making it safeto play with knives[J]. Mol Cell,2010,40(2):179.
[25] Hassan M,Watari H,Abu Almaaty A,et al. Apoptosis and mo-lecular targeting therapy in cancer[J]. Biomed Res Int,2014,2014:150845.
[26] White E,Mehnert J M,Chan C S. Autophagy,metabolism,andcancer[J]. Clin Cancer Res,2015,21(22):5037.
[27]陈易斯,宋福永.选择性自噬接头蛋白p62/sequestosome 1的研究进展[J].中国药理学与毒理学杂志,2016,30(3):258.
[28] Milroy L G,Ottmann C. The renaissance of Ras[J]. ACS ChemBiol,2014,9(11):2447.