乙氧基血根碱靶向CIP2A抑制SGC7901/DDP细胞增殖及活化自噬的作用机制研究  

Ethoxysanguinarine directly targets CIP2A to inhibit proliferation and induce autophagy of SGC7901/DDP cells

作　　者：万芳 谭苗 向雨晨 刘雪文 彭鹏 刘莹 WAN Fang;TAN Miao;XIANG Yu-chen;LIU Xue-wen;PENG Peng;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 Laboralory of Embryonic Stem Cell Research,Hubei Universiy 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21期5890-5899,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82072928);湖北省自然科学基金创新群体项目(2021CFA009);十堰市科技局引导性项目(21Y08,21Y09);湖北医药学院研究生科技创新项目(YC2022032);2022年大学生创新创业训练计划项目(202210929007)。

摘　　要：该文旨在探讨乙氧基血根碱(ethoxysanguinarine, Eth)对顺铂(cisplatin, DDP)耐药的人胃癌细胞的影响及其机制。选用SGC7901和DDP耐药的人胃癌细胞系SGC7901/DDP作为细胞模型;Western blot检测多药耐药相关蛋白在SGC7901和SGC7901/DDP细胞的表达水平;MTT实验检测DDP对SGC7901细胞和SGC7901/DDP细胞增殖的抑制作用;用增加浓度的Eth处理SGC7901和SGC7901/DDP细胞后,通过MTT实验检测Eth对SGC7901和SGC7901/DDP细胞增殖的影响;通过台盼蓝拒染实验、克隆形成实验和共聚焦高内涵成像分析系统检测Eth对SGC7901/DDP细胞增殖能力的影响;DAPI染色、Anne-xin V-FITC/PI染色流式细胞术检测Eth对SGC7901/DDP细胞凋亡的影响;GFP-LC3转染实验检测Eth对SGC7901/DDP细胞自噬的影响;Western blot检测凋亡相关蛋白caspase-9、caspase-3和PARP的表达、自噬相关蛋白LC3-Ⅱ的表达、多药耐药蛋白P-gp的表达、mTORC1下游关键效应子(mTOR、P70S6K、4E-BP1)的蛋白及磷酸化水平、CIP2A和Akt的蛋白及磷酸化水平;通过RT-qPCR分析Eth处理24 h后,SGC7901/DDP细胞中CIP2A的mRNA水平;在SGC7901/DDP细胞中,瞬时转染pcDNA3.1-HA-CIP2A,过表达CIP2A,并用Eth处理,通过MTT实验检测对SGC7901/DDP细胞增殖的影响,通过Western blot检测相关蛋白的表达变化;通过分子对接分析Eth与CIP2A的作用位点;进一步通过靶点稳定性药物亲和反应试验检测Eth与CIP2A的结合稳定性;SwissADME预测Eth的药代动力学性质和类药活性。结果表明SGC7901/DDP细胞对Eth的敏感性更高,Eth在低剂量下能够显著抑制SGC7901/DDP细胞的增殖及克隆形成,并改变SGC7901/DDP细胞的形态、圆度、面积。Eth处理组的细胞核明显皱缩,细胞凋亡率显著增加;Eth能下调caspase-9、caspase-3蛋白前体的表达,促进PARP切割,表明Eth诱导SGC7901/DDP细胞发生凋亡;Eth处理组细胞的GFP-LC3呈斑点化聚集;Eth能促进自噬相关蛋白LC3-Ⅱ的表达上调,表明Eth活化SGC7901/DDP细胞自噬;EThis study aims to investigate the effect of ethoxysanguinarine(Eth) on cisplatin(DDP)-resistant human gastric cancer cells and decipher the underlying mechanism. The human gastric cancer cell line SGC7901 and the DDP-resistant cell line SGC7901/DDP were used as the cell models. Western blot was employed to determine the expression levels of multidrug resistance-related proteins, and methyl thiazolyl tetrazolium(MTT) assay to detect the proliferation of SGC7901 and SGC7901/DDP cells exposed to DDP. After treatment with different concentrations of Eth, the proliferation of SGC7901 and SGC7901/DDP cells was detected by MTT assay, trypan blue exclusion assay, colony formation assay, and high-content imaging and analysis system. The apoptosis of SGC7901/DDP cells was detected by flow cytometry with Annexin V-FITC/PI staining. GFP-LC3 transfection was carried out to detect the effect of Eth on the autophagy of SGC7901/DDP cells. The expression levels of the multidrug resistance-related protein P-glycoprotein(P-gp), the apoptosis-related proteins [caspase-9, caspase-3, and poly(ADP-ribose) polymerase(PARP)], the autophagy-related protein light chain 3-Ⅱ(LC3-Ⅱ), the key effectors [mammalian target of rapamycin(mTOR), 70 kDa ribosomal protein S6 kinase(P70 S6 K), and 4 E binding protein 1(4 E-BP1)] of the mammalian target of rapamycin complex 1(mTORC1) signaling pathway, cancerous inhibitor of protein phosphatase 2A(CIP2A), and protein kinase B(Akt) were measured by Western blot. The mRNA level of CIP2A in the SGC7901/DDP cells exposed to Eth for 24 h was analyzed by RT-qPCR. After SGC7901/DDP cells were transfected with CIP2A expression vector pcDNA3.1-HA-CIP2A and treated with different concentrations of Eth, MTT assay was used to determine the prolife-ration of SGC7901/DDP cells and Western blot to detect the expression levels of related proteins. The interaction sites of Eth and CIP2A were predicted by molecular docking. The affinity between Eth and CIP2A was determined by drug affinity responsive target stabilit

关 键 词：乙氧基血根碱 胃癌 CIP2A 自噬 mTORC1 凋亡 

分 类 号：R285[医药卫生—中药学]