贝伐珠单抗联合奥希替尼协同抑制非小细胞肺癌细胞增殖及机制

Bevacizumab Combined with Osimertinib in Inhibiting the Proliferation of Non-small Cell Lung Cancer Cells and Its Mechanism

目的研究贝伐珠单抗(Bev)联合奥西替尼(Osi)对人非小细胞肺癌细胞株(A549)的增殖抑制作用、诱导凋亡作用,并探讨其分子机制。方法体外培养非小细胞肺癌A549细胞,并分成4组:空白对照组、Bev组、Osi组及联合处理组(Bev+Osi)。利用CCK-8实验检测各组处理后的细胞增殖情况;细胞凋亡情况、活性氧水平变化通过流式细胞术实验检测;细胞凋亡相关蛋白和信号通路相关蛋白的表达通过蛋白免疫印迹法进行检测。结果CCK-8结果显示,联合处理组对A549细胞的增殖抑制作用最强,对人胚肺成纤维MRC-5细胞几乎无毒副作用,与单独用药处理组相比,差异有统计学意义(P<0.05)。流式细胞术显示,联合处理组对A549细胞的诱导凋亡作用更强,较2个单药处理组分别高出45.7%和42.6%;联合处理组能够上调A549细胞中的活性氧水平,加入N-乙酰半胱氨酸后,诱导凋亡能力恢复到2个单药处理组相近的水平。蛋白免疫印迹法结果显示,联合处理组能够抑制ERK的磷酸化水平,上调p38的磷酸化水平。结论Bev与Osi联合使用能够上调细胞中ROS水平,调控p38/ERK信号通路,降低线粒体膜电位,引发A549细胞凋亡,联合使用具有明显的优势,有望成为肺癌治疗领域的重要策略。

Objective To study the proliferation inhibitory effect of bevacizumab(Bev)combined with osimertinib(Osi)on human non-small cell lung cancer cell line(A549),and to explore its molecular mechanism.Methods Cultured non-small cell lung cancer A549 cells in vitro and divided them into four groups:blank control group,Bev group,Osi group and combination group(Bev+Osi).The cell proliferation was detected by CCK-8 assay.Cell apoptosis and changes in reactive oxygen levels were detected by flow cytometry experiments.The expression of apoptosis-related proteins and signaling pathway-related proteins was detected by Western Blotting.Results CCK-8 results showed that the combined treatment group had the strongest inhibitory effect on the proliferation of A549 cells and the least toxic side effects on Human embryonic lung fibrosis MRC-5 cells.Compared with the single drug group,the difference was statistically significant(P<0.05).Flow cytometry showed that the combined treatment group had a stronger apoptosis-inducing effect on A549 cells,which was 45.7%and 42.6%higher than the two single-drug treatment groups,respectively.The combined treatment group was able to up-regulate the level of reactive oxygen species in A549 cells.After adding N-acetylcysteine,the ability to induce apoptosis was restored to a level similar to that of the two single-drug treatment groups.Western Blotting results showed that the combined treatment group can inhibit the phosphorylation level of ERK and up-regulate the phosphorylation level of p38.Conclusion The combination of Bev and Osi can upregulate ROS levels in cells,regulate the p38/ERK signaling pathway,reduce mitochondrial membrane potential,and induce apoptosis in A549 cells.The combination has obvious advantages and is expected to become an important strategy in the field of lung cancer treatment.