Background: 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 1 µmol/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 1 µmol/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 (IC50) 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 CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05).
Conclusions: The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.
【中文题目：烟草烟雾通过ROS/Sirt3/SOD2通路 诱导NSCLC细胞吉非替尼耐药】 【中文摘要：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（cigarette 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1 µmol/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，EdU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1 µmol/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, IC50）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。 】 【中文关键词：肺肿瘤；烟草烟雾；ROS；Sirt3/SOD2；吉非替尼耐药】.
Keywords: Cigarette smoke; Gefitinib resistance; Lung neoplasms; ROS; Sirt3/SOD2.