白头翁皂苷对NCI-H460细胞增殖、凋亡的影响及其差异表达蛋白筛选
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摘要
目的:采用nano LC-LTQ-Orbitrap-MS/MS技术筛选白头翁皂苷抑制人大细胞肺癌细胞NCI-H460增殖及诱导凋亡作用的差异表达蛋白,初步推测其作用机制。方法:通过体外培养NCI-H460,人卵巢腺癌细胞SK-OV-3,人胃腺癌细胞SGC-7901,利用噻唑蓝(MTT)比色法检测白头翁皂苷的抗肿瘤活性;磷脂酰丝氨酸外翻分析-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)染色流式细胞术及4',6-二脒基-2-苯基吲哚(DAPI)染色检测对细胞凋亡的影响;运用nano LC-LTQ-Orbitrap-MS/MS技术对白头翁皂苷干预后NCI-H460细胞全蛋白进行分析与鉴定;利用Thermo Proteome Discoverer 1. 4软件进行蛋白质鉴定,Sieve 2. 1软件对所有样本蛋白进行相对定量分析,筛选并鉴定差异表达蛋白后,通过DAVID Bioinformatics Resources 6. 8和京都基因及基因组百科全书(KEGG)数据库对差异表达蛋白进行生物信息学分析。结果:白头翁皂苷对NCI-H460,SK-OV-3及SGC-7901细胞的增殖均有一定的抑制作用,且能诱导NCI-H460细胞的凋亡。白头翁皂苷主要通过调节核糖体的生物功能、糖酵解/糖异生、碳代谢、蛋白质的生物合成及mRNA的代谢等生物进程来影响NCI-H460细胞的增殖及凋亡,可能通过干预丝裂原活化蛋白激酶(MAPK)信号通路和调控Caspase途径来诱导NCI-H460细胞的凋亡。结论:白头翁皂苷具有抑制NCI-H460细胞增殖及诱导其凋亡的作用,其作用机制可能与干预MAPK信号通路和调控Caspase途径等有关,为研究白头翁皂苷抗肿瘤的分子机制提供了新思路。
Objective: To screen the differentially expressed proteins of saponins in Pulsatillae Radix inhibiting the proliferation and induce apoptosis on NCI-H460 tumor cells based on proteome technology using nano LC-LTQ-Orbitrap-MS/MS,and preliminarily speculate the potential mechanism. Method: NCI-H460,SK-OV-3 and SGC-7901 tumor cells were cultured in vitro. Methylthiazoletetrazolium( MTT) assay was used to detect the inhibitory rate of saponins in Pulsatillae Radix on three tumor cell lines. Effect of saponins in Pulsatillae Radix on apoptosis was analyzed by Annexin V-fluorescein isothiocyanate( FITC)/propidium iodide( PI) staining flow cytometry and 4',6-diamidino-2-phenylindole( DAPI) staining. Apoptosis was analyzed using flow cytometry and DAPI stain. Nano LC-LTQ-Orbitrap-MS/MS was used to investigate the changes in the protein profiles on NCIH460 cells treated with saponins in Pulsatillae Radix. Proteins exhibiting differential expression were analyzed by DAVID Bioinformatics Resources 6. 8 and Kyoto encyclopedia of genes and genomes( KEGG) database. The differentially expressed proteins were verified by Western blot. Result: Saponins in Pulsatillae Radix could inhibit the proliferation of NCI-H460,SK-OV-3 and SGC-7901 tumor cells and induce apoptosis of NCI-H460 tumor cells.Effect of Saponins in Pulsatillae Radix on the proliferation and apoptosis of NCI-H460 tumor cells was mainly related to the regulation of biological function of ribosome, glycolysis/gluconeogenesis and other biological processes. It was possible to induce apoptosis of NCI-H460 tumor cells by interfering mitogen-activated protein kinase( MAPK) signaling pathway and regulating the Caspase pathway. Conclusion: Saponins in Pulsatillae Radix can inhibit the proliferation and induce the apoptosis of NCI-H460 tumor cells,the mechanism may be related to the intervention of MAPK signaling pathway and the regulation of Caspase pathway. These findings are helpful to elucidate the molecular mechanism of the anti-tumor effect of saponins in Pulsatillae Radix.
Objective: To screen the differentially expressed proteins of saponins in Pulsatillae Radix inhibiting the proliferation and induce apoptosis on NCI-H460 tumor cells based on proteome technology using nano LC-LTQ-Orbitrap-MS/MS,and preliminarily speculate the potential mechanism. Method: NCI-H460,SK-OV-3 and SGC-7901 tumor cells were cultured in vitro. Methylthiazoletetrazolium( MTT) assay was used to detect the inhibitory rate of saponins in Pulsatillae Radix on three tumor cell lines. Effect of saponins in Pulsatillae Radix on apoptosis was analyzed by Annexin V-fluorescein isothiocyanate( FITC)/propidium iodide( PI) staining flow cytometry and 4',6-diamidino-2-phenylindole( DAPI) staining. Apoptosis was analyzed using flow cytometry and DAPI stain. Nano LC-LTQ-Orbitrap-MS/MS was used to investigate the changes in the protein profiles on NCIH460 cells treated with saponins in Pulsatillae Radix. Proteins exhibiting differential expression were analyzed by DAVID Bioinformatics Resources 6. 8 and Kyoto encyclopedia of genes and genomes( KEGG) database. The differentially expressed proteins were verified by Western blot. Result: Saponins in Pulsatillae Radix could inhibit the proliferation of NCI-H460,SK-OV-3 and SGC-7901 tumor cells and induce apoptosis of NCI-H460 tumor cells.Effect of Saponins in Pulsatillae Radix on the proliferation and apoptosis of NCI-H460 tumor cells was mainly related to the regulation of biological function of ribosome, glycolysis/gluconeogenesis and other biological processes. It was possible to induce apoptosis of NCI-H460 tumor cells by interfering mitogen-activated protein kinase( MAPK) signaling pathway and regulating the Caspase pathway. Conclusion: Saponins in Pulsatillae Radix can inhibit the proliferation and induce the apoptosis of NCI-H460 tumor cells,the mechanism may be related to the intervention of MAPK signaling pathway and the regulation of Caspase pathway. These findings are helpful to elucidate the molecular mechanism of the anti-tumor effect of saponins in Pulsatillae Radix.
引文
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[3]石宝俊,李茜,张晓琦,等.中药白头翁地上部分的三萜皂苷成分[J].药学学报,2007,42(8):826-866.
[4]罗颖颖,陈兰英,崔亚茹,等.白头翁皂苷抑制人HT29结肠癌细胞增殖及诱导凋亡作用研究[J].中药药理与临床,2013,29(5):52-56.
[5]罗颖颖,陈兰英,简晖,等.白头翁皂苷调节Bel-7402人肝癌异体移植瘤裸鼠能量代谢的研究[J].中草药,2014,45(7):973-977.
[6] Santos R,Ursu O,Gaulton A,et al. A comprehensive map of molecular drug targets[J]. Nat Rev Drug Discov,2016,16(1):19-34.
[7] LU C L,QU X Y,JIANG J G. Proteomics and syndrome of Chinese medicine[J]. J Cell Mol Med,2010,14(12):2721-2728.
[8]林满遍,陈亮,吴水生.钩吻总碱对人结肠癌细胞增殖、凋亡的影响及其机制[J].中国实验方剂学杂志,2018,24(4):149-153.
[9]李红蓉,常丽萍,秘红英,等.巨噬细胞对内皮细胞凋亡的影响及通心络的干预作用[J].中国实验方剂学杂志,2017,23(13):121-127.
[10] Wis'niewski J R,Zougman A,Nagaraj N,et al. Universal sample preparation method for proteome analysis[J].Nat Methods,2009,6(5):359-362.
[11] HUANG D W,Sherman B T,Lempicki R A.Bioinformatics enrichment tools:paths toward the comprehensive functional analysis of large gene lists[J]. Nucleic Acids Res,2009,37(1):1-13.
[12]王征,张宁,孙艳妮,等.白头翁寒热药性的细胞评价[J].药物评价研究,2013,36(5):359-362.
[13] ZHAO L,Nicholson J K,LU A,et al. Targeting the human genome-microbiome axis for drug discovery:inspirations from global systems biology and traditional Chinese medicine[J]. J Proteome Res,2012,11(7):3509-3519.
[14] WANG C,Maass T,Krupp M,et al. A systems biology perspective on cholangiocellular carcinoma development:focus on MAPA-signaling and the extracellular environment[J]. J Hepatol,2009,50(6):1122-1131.
[15] Adams J M,Cory S. The Bcl-2 apoptotic switch in cancer development and therapy[J]. Oncogene,2007,26(9):1324-1337.
[16] Roderick H L,Cook S J. Ca2+signalling checkpoints in cancer:remodelling Ca2+for cancer cell proliferation and survival[J]. Nat Rev Cancer,2008,8(5):361-375.
[17] Stanley R F,Piszczatowski R T,Bartholdy B,et al. A myeloid tumor suppressor role for NOL3[J]. J Exp Med,2017,214(3):753-771.