重楼皂苷A直接靶向ETS-1抑制胃癌细胞增殖并诱导凋亡的作用机制  

Mechanism of polyphyllin A in inhibition of proliferation and induction of apoptosis of gastric cancer cells by directly targeting ETS-1

作　　者：张亮 熊艺琏 任宇亮 刘雪文 司渊 刘莹 ZHANG Liang;XIONG Yi-lian;REN Yu-liang;LIU Xue-wen;SI Yuan;LIU Ying(School of Basic Medical Sciences,Hubei University of Medicine,Shiyan 442000,China;Hubei Key Laboratory of Wudang Local Chinese Medicine Research,Hubei University of Medicine,Shiyan 442000,China;Hubei Key Laboratory of Embryonic Stem Cell Research,Hubei University of Medicine,Shiyan 442000,China)

机构地区：[1]湖北医药学院基础医学院,湖北十堰442000 [2]湖北医药学院武当特色中药研究湖北省重点实验室,湖北十堰442000 [3]湖北医药学院胚胎干细胞研究湖北省重点实验室,湖北十堰442000

出　　处：《中国中药杂志》2022年第6期1650-1657,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82072928);十堰市科技局引导性项目(21Y08,21Y09);大学生创新创业训练项目(S202110929002,202110929004);湖北省科技厅创新群体项目(2021CFA009)。

摘　　要：该文旨在探讨重楼皂苷A(polyphyllin A,PPA)抑制胃癌(gastric cancer,GC)的分子机制。选用SGC7901和MGC803细胞株作为细胞模型。用不同浓度的PPA处理细胞后,通过MTT法、实时无标记细胞分析技术(real time cellular analysis,RTCA)和克隆形成实验检测PPA对GC细胞增殖能力的影响;流式细胞术检测细胞内活性氧(reactive oxygen species,ROS)变化水平;JC-1法检测线粒体膜电位;蛋白免疫印迹法检测凋亡相关蛋白caspase-9、caspase-3和PARP的表达及ETS-1、CIP2A和Akt信号通路蛋白的表达及磷酸化水平;通过分子对接分析PPA与ETS-1的作用位点;采用靶点稳定性药物亲和反应实验检测PPA与ETS-1的直接结合作用。PPA在低微摩尔浓度剂量下能够显著抑制GC细胞的增殖及克隆形成。PPA处理组的ROS明显增加,线粒体膜电位明显降低,PPA能下调caspase-9、caspase-3蛋白前体的表达,促进PARP切割,表明PPA诱导GC细胞发生线粒体途径的凋亡。PPA降低CIP2A的表达水平及下游Akt的磷酸化水平;分子对接模拟发现PPA与CIP2A的转录因子ETS-1的ETS domain发生结合,与其中的Pro319、Asp317形成氢键;靶点稳定性的药物结合实验进一步确证了PPA能显著阻止蛋白酶pronase对ETS-1的水解作用,表明PPA与ETS-1具有直接结合作用。PPA通过靶向ETS-1下调ETS-1/CIP2A/Akt信号通路抑制GC细胞增殖并诱导凋亡。The present study investigated the mechanism of polyphyllin A(PPA)in inhibiting gastric cancer(GC)cells.GC cells(SGC7901 and MGC803 cell lines)were treated with PPA at different concentrations.The effect of PPA on the proliferation of GC cells was detected by MTT assay,real-time cell analysis(RTCA)assay,and clone-forming assay,respectively.Reactive oxygen species(ROS)of GC cells was detected by flow cytometry.The change of mitochondrial membrane potential was detected by JC-1 assay.The expression and phosphorylation levels of apoptosis-related proteins(caspase-9,caspase-3,and PARP)and proteins related to the signaling pathway(ETS-1,CIP2 A,and Akt)were detected by Western blot.The binding sites of PPA to ETS-1 were analyzed by molecular docking.The affinity of PPA and ETS-1 was detected by drug affinity responsive target stability(DARTS)assay.PPA had a significant inhibitory effect on the proliferation and colony formation of GC cells at a low concentration.The PPA groups showed increased ROS and decreased mitochondrial membrane potential.PPA down-regulated the precursor expression of caspase-9 and caspase-3 and promoted the cleavage of PARP,suggesting that PPA induced the apoptosis of GC cells through the mitochondrial pathway.PPA significantly reduced expression levels of CIP2 A and the phosphorylation of downstream Akt.Molecular docking showed that PPA bound to the ETS domain of ETS-1,the transcription factor of CIP2 A,and formed hydrogen bonds with Pro319 and Asp317.DARTS assay further confirmed that PPA significantly prevented the hydrolysis of ETS-1 by pronase,which was inductive of the direct binding effect of PPA and ETS-1.PPA inhibits the proliferation and induces the apoptosis of GC cells by directly targeting ETS-1 to down-regulate the ETS-1/CIP2 A/Akt signaling pathway.

关 键 词：polyphyllin A 胃癌 ETS-1 增殖 凋亡 

分 类 号：R285[医药卫生—中药学]