奥希替尼联合抗血管生成靶向药对人肺腺癌细胞抑制作用的实验研究

目的:通过体外细胞学实验初步探究奥希替尼联合两种不同机制的抗血管靶向治疗药物（贝伐珠单抗或阿帕替尼）治疗表皮生长因子受体（EGFR）敏感突变和T790M耐药突变肺腺癌细胞的抗肿瘤活性及其作用机制。方法:培养人肺腺癌细胞PC-9（E19 del）和H1975（E21 L858R/E20 T790M），CCK-8法检测奥希替尼及抗血管靶向治疗药物（贝伐珠单抗或阿帕替尼）单药或联合处理肺腺癌细胞48 h后的抑瘤率；蛋白质印迹法检测EGFR及其下游AKT和ERK信号通路蛋白表达情况。结果:PC-9和H1975肺腺癌细胞对奥希替尼敏感且呈剂量依赖性。奥希替尼联合抗血管生成靶向药物（贝伐珠单抗，阿帕替尼）较同等浓度的单药奥希替尼可增加对PC-9和H1975细胞株的抑瘤率（P<0.05）。低浓度奥希替尼联合高浓度阿帕替尼（1 000 nmol/L）的抑制作用与高浓度奥希替尼相当（P>0.05）。随着联合的阿帕替尼浓度的升高，对PC-9和H1975细胞抑瘤率也有一定程度的提高（P<0.05）。不同处理因素对PC-9细胞的抑制率均高于对H1975细胞（P<0.01）。随着奥希替尼浓度的上升，p-EGFR、p-AKT、p-ERK磷酸化蛋白表达逐渐降低。结论:奥希替尼联合贝伐珠单抗或阿帕替尼会进一步增强对EGFR敏感突变或T790M突变肺腺癌细胞的杀伤作用。奥希替尼与阿帕替尼联合使用具有很强的抑瘤活性，具有很好的应用前景。奥希替尼单药或与抗血管形成药联合作用可能是进一步下调EGFR及其下游AKT和ERK信号通路的活化。

Study of the inhibition of osimertinib in combination with anti-angiogenesis target drug on human lung adenocarcinoma cell lines

Objective:To investigate the anti-tumor activity and mechanism of osimertinib combined with anti-angiogenesis target drugs (bevacizumab or apatinib) in the treatment of PC-9 (E19 del) and H1975 (E21 L858R/E20 T790M) the two lung adenocarcinoma cell lines through in vitro experiment.Methods:The human lung adenocarcinoma cells lines of PC-9 and H1975 were detected by CCK-8 method after culturing and exposing 48 hours.Western blotting was used to detect protein expression of EGFR and its downstream signaling pathway (AKT and ERK).Results:PC-9 and H1975 lung adenocarcinoma cell lines were sensitive to osimertinib and in a dose-dependent manner.Osimertinib combined with anti-angiogenesis target drugs (bevacizumab or apapatinib) increased tumor inhibition rate，the result had statistical significance comparing to single osimertinib at the same concentration，P<0.05.Low concentration of osimertinib combined with high concentration of apatinib (1 000 nmol/L) could increase tumor inhibition rate（P<0.05) or almost equally (P>0.05) comparing to high concentration of osimertinib monotherapy.Osimertinib was constant，with the increase of the concentration of apatinib，the tumor inhibition rate also increased to some extent (P<0.05)，but bevacizumab did not show a good trend.The inhibitory rate of PC-9 cell line was higher than that of H1975 cell line with any different drug concentrations treatment，the result had statistical significance，P<0.01.Western blotting suggested that the protein expression of p-EGFR，p-AKT and p-ERK were decreased with the increasing of osimertinib concentration and the result had statistical significance(P<0.05).Conclusion:Osimertinib combined with anti-vascular targeting drugs (bevacizumab or apatinib) could enhance anti-tumor activity of EGFR-sensitive mutant or T790M mutant lung adenocarcinoma cells.Osimertinib combined with apatinib has a strong anti-tumor activity and a good application prospects.The mechanism of osimertinib monotherapy or combined with bevacizumab or apatinib may be due to further down regulation of EGFR and its downstream AKT and ERK signaling pathway activation.