阿美替尼对非小细胞肺癌细胞增殖、侵袭和迁移的影响

Effect of almonertinib on the proliferation,invasion,and migration in non-small cell lung cancer cells

目的:肺癌是世界上最常见的恶性肿瘤之一,其病死率排在众多恶性肿瘤之首。非小细胞肺癌(nonsmall cell lung cancer,NSCLC)患者的病死率较高,总体5年存活率<15%。当NSCLC发生局部侵袭时,患者5年生存率仅为20%,而当发生远处转移时5年生存率更是低至4%。阿美替尼(almonertinib)是我国自主研发并拥有自主知识产权的创新药,其作为表皮生长因子受体(epidermal growth factor receptor,EGFR)酪氨酸激酶抑制剂主要用于EGFR T790M突变的局部晚期或转移性NSCLC患者。本研究旨在探讨阿美替尼对NSCLC细胞增殖、侵袭和迁移的影响。方法:体外培养NSCLC H1975、PC-9细胞,采用CCK-8法、细胞凋亡实验、Transwell实验检测阿美替尼对H1975、PC-9细胞增殖、凋亡、侵袭及迁移的影响;蛋白质印迹法检测侵袭迁移相关蛋白质的表达。结果:CCK-8实验结果显示阿美替尼呈时间和剂量依赖性抑制H1975、PC-9细胞增殖,PC-9细胞24、48 h的IC_(50)值分别为5.422、1.302μmol/L,H1975细胞24、48 h的IC_(50)值分别为4.803、2.094μmol/L。细胞凋亡实验结果显示1、2、4、8μmol/L阿美替尼分别处理PC-9和H1975细胞24 h时,细胞凋亡率分别为(8.82±3.22)%、(9.53±4.24)%、(13.62±3.69)%、(42.10±1.76)%和(9.81±0.90)%、(10.51±1.49)%、(15.34±3.50)%、(28.97±2.57)%。Transwell实验结果显示阿美替尼抑制H1975、PC-9细胞侵袭和迁移。蛋白质印迹法结果显示:与对照组相比,1、2、4μmol/L阿美替尼处理组PC-9和H1975细胞中MMP-9,MMP-2和Vimentin蛋白表达水平均明显降低,E-cadherin蛋白表达水平明显升高(均P<0.05)。裸鼠实验结果发现:与对照组和阳性对照奥希替尼(AZD9291)组相比,阿美替尼治疗组肿瘤生长被显著抑制,裸鼠体重减轻,肿瘤体积显著减小,肿瘤质量也明显减轻(均P<0.05)。结论:阿美替尼能够在体内外抑制H1975、PC-9细胞的增殖、侵袭、迁移,并促进H1975、PC-9细胞凋亡。其抑制侵袭的作用机制可能与抑制肿瘤细胞上皮间质转化和金属蛋白酶表达有关。

Objective:Lung cancer is one of the most common malignant tumors in the world,and its lethality ranks the first among many malignant tumors.For non-small cell lung cancer(NSCLC)patients,due to the high mortality rate,the overall 5-year survival rate is less than 15%.When NSCLC undergoes local invasion,the 5-year survival rate is only 20%,and it is even lower when distant metastasis occurs up to 4%.Almonertinib is an innovative drug independently researched and developed by China with independent intellectual property rights.As an epidermal growth factor receptor tyrosine kinase inhibitor,almonertinib is mainly used for locally advanced or metastatic NSCLC patients with epidermal growth factor receptor(EGFR)T790 M mutation.This study aims to investigate the effects of almonertinib on the proliferation,invasion and migration of NSCLC cells in vitro.Methods:NSCLC cells H1975 and PC-9 were cultured in vitro.The effects of almonertinib on the proliferation,apoptosis,invasion,and migration of H1975 and PC-9 cells were detected by CCK-8 assay,apoptotic assay and Transwell assay.The expression of invasion and migration related proteins was detected by Western blotting.Results:The CCK-8 experiment showed that almonertinib inhibited the proliferation of H1975 and PC-9 cells in a time-and dose-dependent manner.The IC_(50) values in PC-9 cells at 24 and 48 h were 5.422 and 1.302μmol/L,respectively.The IC_(50) values in H1975 cells at 24 and 48 h were 4.803 and 2.094μmol/L,respectively.Almonertinib(1,2,4,8μmol/L)-treated PC-9 and H1975 cells for 24 h resulted in apoptosis rate at(8.82±3.22)%,(9.53±4.24)%,(13.62±3.69)%,(42.10±1.76)%and(9.81±0.90)%,(10.51±1.49)%,(15.34±3.50)%,(28.97±2.57)%,respectively.The transwell experiment showed that almonertinib inhibited the invasion and migration of H1975 and PC-9 cells.Western blotting showed that compared with the control group,the expression levels of MMP-9,MMP-2 and vimentin protein in PC-9 and H1975 cells in 1,2 and 4μmol/L almonertinib treatment group were significantly lower,and the expression level of E-cadherin protein was significantly higher(all P<0.05).The experimental results of nude mice showed that compared with the control group and the positive control ositinib(AZD9291)group,the tumor growth was significantly inhibited,the weight of nude mice,the tumor volume and the tumor mass were significantly reduced in the almonertinib treatment group(all P<0.05).Conclusion:Almonertinib can inhibit the proliferation,invasion,and migration of NSCLCH1975 and PC-9 cells in vitro and vivo,and promote the apoptosis of H1975 and PC-9 cells.The underlying mechanism may be related to the inhibition of tumor cell epithelial mesenchymal transformation and metalloproteinase expression.