抗EGFR单抗联合EGFR酪氨酸激酶抑制剂对人肺腺癌细胞凋亡的影响及其机制

Effects of anti-EGFR monoclonal antibody cetuximab in combination with the EGFR tyrosine kinase inhibitor gefitinib on apoptosis of human pulmonary adenocarcinoma cells and their action mechanism

目的:目前抗表皮生长因子受体(epidermal growth factor receptor,EGFR)的小分子酪氨酸激酶抑制剂吉非替尼(gefitinib)和抗EGFR单克隆抗体西妥昔单抗(cetuximab)在肺癌的临床应用中颇为广泛。鉴于这两种药物均针对EGFR分子靶点,因此本研究旨在就上述两种药物联合用药对人肺腺癌细胞凋亡的影响及其分子机制进行探讨。方法:吉非替尼和西妥昔单抗单独或联合用药作用于人肺腺癌细胞株A549和SPC-A-1后,用碘化丙啶标记,采用流式细胞术观察细胞凋亡情况,活细胞计数试剂盒测定各组细胞增殖抑制情况,Western印迹法检测两种药物对EGFR下游信号通路蛋白[磷酸化蛋白激酶B(phosphorylated Akt,p-Akt)、磷酸化EGFR(phosphorylated EGFR,p-EGFR)和磷酸化丝裂原激活蛋白激酶(phosphorylated mitogen-activated protein kinase,p-MAPK)]在蛋白水平表达的影响。结果:吉非替尼或西妥昔单抗单独作用后,A549和SPC-A-1细胞均明显凋亡,同时细胞增殖受到不同程度的抑制。Western印迹法检测p-Akt、p-EGFR和p-MAPK蛋白表达量均较不用药对照组下降。吉非替尼和西妥昔单抗联合作用后,肺腺癌细胞的凋亡、增殖以及在EGFR分子层次表现出较单一用药更为显著的作用。结论:吉非替尼和西妥昔单抗两种药物之间具有良好的协同作用,联合用药可能在临床治疗非小细胞肺癌中具有较大的应用潜力。

Objective：The small molecule epidermal growth factor receptor （EGFR）-tyrosine kinase inhibitor （gefitinib） and anti-EGFR monoclonal antibody （cetuximab） were widely used in the treatment of lung cancer. Because EGFR was the target of both gefitinib and cetuximab, this study decided to explore the effects of the combined administration of gefitinib and cetuximab on apoptosis of human pulmonary adenocarcinoma cells and elucidate their molecular mechanism.Methods：Human pulmonary adenocarnoma cells A549 and SPC-A1 were treated with gefitinib and cetuximab alone or in combination. After being labeled with propidium iodide （PI）, apoptosis of cells was observed by using flow cytometry.Cell counting Kit-8（CCK8）was utilized to measure the inhibition of cell proliferation in each group. Western blotting was performed to detect the protein expressions of phosphorylated Akt （p-Akt）, phosphorylated EGFR （p-EGFR） and phosphorylated mitogen-activated protein kinase （p-MAPK）.Results：The A549 and SPC-A-1 cells had apparent apoptotic features after treatment with gefitinib and cetuximab alone and the proliferations of the two cell lines were inhibited to different degrees. The expressions of p-Akt, p-EGFR, and p-MAPK proteins were decreased compared with control group. The combined treatment with gefitinib and cetuximab resulted in a synergistic effect in inducing apoptosis, inhibiting cell proliferation and decreasing the expressions of p-Akt, p-EGFR and p-MAPK proteins.Conclusion：Gefitinib and cetuximab have better synergistic effects. Combined treatment has a great potential in the treatment of non-small cell lung cancer in clinic.