摘要
背景与目的表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变是非小细胞肺癌(non-small cell lung cancer,NSCLC)最常见的驱动基因突变。为延长患者生存时间,NSCLC EGFR酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)耐药是目前急需解决的重大难题。本研究主要探究烟草烟雾(cigaree smoke,CS)诱导NSCLC发生吉非替尼耐药的机制。方法体外培养PC-9、A549细胞,分别经1μmol/L吉非替尼处理4 h、10%CS萃取物(CS extract,CSE)处理48 h。Western blot检测沉默蛋白3(Sirtuin 3,Sirt3)、超氧化物歧化酶2(superoxide dismutase 2,SOD2)蛋白表达,使用DCFH-DA探针检测细胞内活性氧(reactive oxygen species,ROS)水平,CCK-8试剂盒检测细胞活性,Ed U检测细胞增殖能力。Sirt3过表达质粒(OV-Sirt3)转染于PC-9和A549细胞中、N-乙酰半胱氨酸乙酯(N-acetylcysteine,NAC)作用于细胞后分别经1μmol/L吉非替尼处理4 h和10%CSE处理48 h。Western blot检测Sirt3、SOD2蛋白表达,DCFH-DA探针检测细胞中的ROS水平,CCK-8检测细胞活性。结果CSE均可促使PC-9、A549细胞对吉非替尼的半数抑制浓度(50%inhibitory concentration,IC_(50))提高(P<0.01),并且增强PC-9和A549细胞的增殖能力,提示CS可诱导NSCLC吉非替尼耐药;ROS参与CSE诱导的吉非替尼耐药(P<0.05);CSE诱导Sirt3、SOD2低表达(P<0.01),且Sirt3/SOD2与肺癌患者不良预后有关(P<0.05);OV-Sirt3的PC-9、A549细胞可逆转CSE诱导的吉非替尼耐药(P<0.05)且显著降低ROS生成;NAC可逆转CSE诱导的PC-9、A549细胞吉非替尼耐药(P<0.05)。结论ROS/Sirt3/SOD2通路参与了CS诱导的NSCLC吉非替尼耐药。
Background and objective Epidermal growth factor receptor(EGFR)gene mutations are the most common driver mutations in non-small cell lung cancer(NSCLC).To prolong the survival of the patients,EGFR tyrosine kinase inhibitors(TKIs)resistance in NSCLC is a major challenge that needs to be addressed urgently,and this study focuses on investigating the mechanism of cigarette smoke(CS)induced Gefitinib resistance in NSCLC.Methods PC-9 and A549 cells were cultured in vitro and treated with 1μmol/L Gefitinib for 4 h and 10%cigarette smoke extract(CSE)for 48 h.Western blot was used to detect Sirtuin 3(Sirt3)and superoxide dismutase 2(SOD2)protein expressions;DCFH-DA probe was used to detect intracellular reactive oxygen species(ROS);CCK-8 kit was used to detect cell activity,and EdU was used to detect cell proliferation ability.Sirt3 overexpression plasmid(OV-Sirt3)was transfected in PC-9 and A549 cells and treated with 1μmol/L Gefitinib for 4 h and 10%CSE for 48 h after N-acetylcysteine(NAC)action.The expressions of Sirt3 and SOD2 were detected by Western blot;the ROS level in the cells was detected by DCFH-DA probe,and the cell activity was detected by CCK-8.Results CSE induced an increase in the 50%inhibitory concentration(IC_(50))of both PC-9 and A549 cells to Gefitinib(P<0.01)and enhanced the proliferation of PC-9 and A549 cells,suggesting that CS induced Gefitinib resistance in NSCLC.ROS was involved in CSE-induced Gefitinib resistance(P<0.05).CSE induced low expressions of Sirt3 and SOD2(P<0.01),and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients(P<0.05).OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance(P<0.05)and significantly reduced ROS production.NAC reversed CSEinduced Gefitinib resistance in PC-9 and A549 cells(P<0.05).Conclusion The ROS/Sirt3/SOD2 pathway is involved in CSinduced Gefitinib resistance in NSCLC.
作者
訾亚婉
廖科
陈虹
Yawan ZI;Ke LIAO;Hong CHEN(Department of Pulmonary and Critical Care Medicine;Chongqing Key Laboratory of Ophthalmology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)
出处
《中国肺癌杂志》
CAS
CSCD
北大核心
2023年第4期245-256,共12页
Chinese Journal of Lung Cancer
基金
重庆市卫计委卫生适宜技术项目(No.2018jstg012)资助。
