Effect of Saponins in Pulsatillae Radix on Proliferation and Apoptosis of NCI-H460 Lung Cancer Cells and Screening of Its Differentially Expressed Proteins

Zi-yi GUAN; Lan-ying CHEN; Ying-ying LUO; Ya-ru CUI; Bin-yao SHOU; Li-fen ZHOU

doi:10.13422/j.cnki.syfjx.20191451

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Effect of Saponins in Pulsatillae Radix on Proliferation and Apoptosis of NCI-H460 Lung Cancer Cells and Screening of Its Differentially Expressed Proteins

Pharmacology | 更新时间：2021-02-09

- Effect of Saponins in Pulsatillae Radix on Proliferation and Apoptosis of NCI-H460 Lung Cancer Cells and Screening of Its Differentially Expressed Proteins
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 18, Pages: 49-56(2019)
- 作者机构：
  
  1.江西中医药大学　中药固体制剂制造技术国家工程研究中心，南昌　 330006；
  2.江西中医药大学　药学院，南昌　 330004
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20191451
  CLC： R22; R24; R73; R28; C37; TS193; R96
- Published：20 September 2019，
  
  Published Online：02 April 2019，
  
  Received：31 October 2018，
- 稿件说明：
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Zi-yi GUAN, Lan-ying CHEN, Ying-ying LUO, et al. Effect of Saponins in Pulsatillae Radix on Proliferation and Apoptosis of NCI-H460 Lung Cancer Cells and Screening of Its Differentially Expressed Proteins. [J]. Chinese Journal of Experimental Traditional Medical Formulae 25(18):49-56(2019)
DOI：

Zi-yi GUAN, Lan-ying CHEN, Ying-ying LUO, et al. Effect of Saponins in Pulsatillae Radix on Proliferation and Apoptosis of NCI-H460 Lung Cancer Cells and Screening of Its Differentially Expressed Proteins. [J]. Chinese Journal of Experimental Traditional Medical Formulae 25(18):49-56(2019) DOI： 10.13422/j.cnki.syfjx.20191451.

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。

Abstract

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 NCI-H460 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.

关键词

白头翁皂苷抗肿瘤凋亡蛋白质组学质谱检测Caspase途径丝裂原活化蛋白激酶

Keywords

saponins in Pulsatillae Radixanti-tumorapoptosisproteomicsmass spectrometric detectionCaspase pathwaymitogen-activated protein kinase

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