安罗替尼通过核因子信号通路抑制非小细胞肺癌细胞增殖、迁移和侵袭并促进细胞凋亡  

Amlotinib inhibits cell proliferation,migration,invasion and promotes cell apoptosis in non-small cell lung cancer through nuclear factor-κB signaling pathway

作　　者：张全 曹志坤 丁成智 刘孟博 李基伟[1] 魏立[1] Zhang Quan;Cao Zhikun;Ding Chengzhi;Liu Mengbo;Li Jiwei;Wei Li(Department of Thoracic Surgery,Henan People’s Hospital,Zhengzhou 450000,China)

机构地区：[1]河南省人民医院胸外科,郑州450000

出　　处：《中华实验外科杂志》2024年第12期2778-2782,共5页Chinese Journal of Experimental Surgery

基　　金：河南省科技公关项目(232102310044)。

摘　　要：目的研究安罗替尼对非小细胞肺癌细胞增殖、迁移、侵袭、凋亡的影响, 并探究其可能的分子机制。方法体外培养人非小细胞肺癌细胞系A549, 将A549细胞随机分为药物空白组[0.1 %二甲基亚砜(DMSO)]、安罗替尼低、中、高剂量组(2.5、5和10 μmol/L)。各组加入相应药液, 继续培养24 h。通过噻唑蓝(MTT)法、细胞计数试剂盒(CCK-8)和5-乙炔基-2’-脱氧尿苷(EdU)染色实验检测细胞增殖情况, 细胞划痕实验、Transwell小室侵袭实验、原位缺口末端标记法(TUNEL)染色法分别检测各组细胞的迁移、侵袭能力和凋亡率, 通过蛋白质免疫印记(Western blot)实验检测各组细胞凋亡相关蛋白B淋巴细胞瘤相关蛋白X(bax)、B淋巴细胞瘤-2(bcl-2)、磷酸化κB抑制蛋白β(p-IκB)、IκB和磷酸化核因子κB p65亚基(p-NF-κB p65)蛋白的表达水平;利用NF-κB信号通路激动剂处理A549细胞, 并且分别通过EDU、细胞划痕实验、Transwell小室侵袭实验、TUNEL染色法检测各组细胞增殖、迁移、侵袭、凋亡的情况。结果安罗替尼高剂量组细胞的增殖能力、迁移率、侵袭能力均高于药物空白组(83.41±2.83比29.87±5.31、0.47±0.02比0.98±0.03、0.54±0.02比1.01±0.03、0.29±0.02比0.97±0.02、21.67±3.06比143.70±3.51, t=15.42、25.74、24.30、43.56、45.50, P均<0.05), p-IκB、p-NF-κB p65的表达水平以及p-IκB/IκB、p-NF-κB-p65/NF-κB-p65比值均显著降低(0.52±0.05比1.02±0.04、0.49±0.04比1.02±0.03、0.08±0.01比0.98±0.06、0.24±0.03比0.92±0.05, t=15.36、18.48、27.25、22.32, P均<0.05)。而细胞凋亡率、bax和bcl-2蛋白的表达水平均显著高于药物空白组(2.46±0.09比0.94±0.04、2.35±0.09比0.91±0.06, t=27.09、23.44, P<0.05), 并且具有明显的浓度依赖性。与安罗替尼高剂量+DMSO组比较, 安罗替尼高剂量+NF-κB激动剂组细胞增殖能力、迁移率、侵袭能力显著降低, 细胞凋亡率显著升高(0.96±0.03比0.49±0.03、0.57±ObjectiveTo investigate the effects of Anlotinib on the proliferation,migration,invasion,and apoptosis of non-small cell lung cancer(NSCLC)cells and explore its possible molecular mechanisms.MethodsHuman NSCLC cell line A549 was cultured in vitro and randomly divided into a drug blank group[0.1%dimethyl sulfoxide(DMSO)]and Anlotinib low-,medium-,and high-dose groups(2.5,5,and 10μmol/L,respectively).Each group was treated with the corresponding solution and cultured for 24 hours.Cell proliferation was detected using the methylthiazolyldiphenyl-tetrazolium bromide(MTT)assay,Cell Counting Kit-8(CCK-8),and 5-Ethynyl-2’-deoxyuridine(EdU)staining assay.Cell migration,invasion,and apoptosis were assessed using scratch assays,Transwell chamber invasion assays,and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining,respectively.Western blotting was performed to measure the expression of apoptosis-related proteins,including B lymphoma-associated X protein(bax),B lymphoma-2 protein(bcl-2),phosphorylated IκBβ(p-IκB),IκB,and phosphorylated nuclear factor-κB p65 subunit(p-NF-κB p65).A549 cells were treated with NF-κB pathway agonists,followed by the same proliferation,migration,invasion,and apoptosis assays.ResultsCompared with the drug blank group,the high-dose Anlotinib group exhibited significantly reduced proliferation,migration,and invasion abilities(83.41±2.83 vs.29.87±5.31,0.47±0.02 vs.0.98±0.03,0.54±0.02 vs.1.01±0.03,0.29±0.02 vs.0.97±0.02,21.67±3.06 vs.143.70±3.51,t=15.42,25.74,24.30,43.56,45.50,all P<0.05)and lower expression levels of p-IκB and p-NF-κB p65,as well as reduced p-IκB/IκB and p-NF-κB-p65/NF-κB-p65 ratios(0.52±0.05 vs.1.02±0.04,0.49±0.04 vs.1.02±0.03,0.08±0.01 vs.0.98±0.06,0.24±0.03 vs.0.92±0.05,t=15.36,18.48,27.25,22.32,all P<0.05).The apoptosis rate and expression levels of bax and bcl-2 were significantly higher(2.46±0.09 vs.0.94±0.04,2.35±0.09 vs.0.91±0.06,t=27.09,23.44,all P<0.05),demonstrating a clear dose-dependent effect.Compared w

关 键 词：安罗替尼 非小细胞肺癌 细胞增殖 细胞迁移 细胞侵袭 细胞凋亡 核因子ΚB信号通路 

分 类 号：R734.2[医药卫生—肿瘤]