EGFR/IGFR-1β异二聚体形成对吉非替尼抑制结肠癌细胞增殖的影响

Effect of EGFR/IGFR-1β heterodimerization on anti-proliferative activity of gefitinib against colon carcinoma cells

目的:观察EGFR(epidermal growth factor receptor)和IGFR-1β(insulin-like growth factor receptor-1β)的相互作用,探讨IGFR-1β对吉非替尼(gefitinib)抑制结肠癌细胞增殖的影响。方法:以免疫共沉淀法和Western blotting检测EGFR和IGFR-1β之间以及受体与下游信号通路蛋白AKT、MAPK的结合。以IGFR-1酪氨酸激酶抑制剂AG1024和吉非替尼单独或联合作用于3种结肠癌细胞(LoVo,HT29,HCT116细胞),MTT法检测细胞增殖率。结果:LoVo细胞(吉非替尼敏感型)在有或无吉非替尼作用下均不出现与EGFR结合的IGFR-1β条带,HT29细胞(吉非替尼中度敏感型)在吉非替尼作用下可见该条带,而HCT116细胞(吉非替尼耐受型)在有或无吉非替尼作用下均可见条带。LoVo细胞的吉非替尼作用组、HT29细胞的联合用药组以及HCT116细胞的AG1024作用组EGFR结合的AKT、MAPK显著减少(P<0.05)。LoVo细胞在吉非替尼组的细胞增殖率较对照组明显下降(P<0.05),AG1024单独组的增殖率无明显降低,联合用药组与吉非替尼单独组相比较并未进一步降低;HT29细胞在单独应用吉非替尼或AG1024时增殖率均无明显降低,联合用药组的增殖率显著下降(P<0.05);HCT116细胞在吉非替尼作用下增殖率无明显降低,但在AG1024作用下增殖率显著降低(P<0.05),联合用药组与AG1024单独作用组相比较并未进一步降低。结论:结肠癌细胞耐受吉非替尼作用可能或者部分可能与EGFR/IGFR-1β异二聚体形成激活IGFR-1β信号通路有关,抑制IGFR-1β活性在一定程度上可以提高结肠癌细胞对吉非替尼的敏感性。

Objective：To observe the interaction of EGFR with IGFR-1β and to investigate the effect of EGFR inhibitor IGFR-1β on the anti-proliferative activity of gefitinib against colon carcinoma cells. Methods：Interactions between EGFR and IGFR-1β and their association with their downstream signal proteins AKT and MAPK were examined by immunoprecipitation and Western blotting analysis. MTT assay was used to measure cell proliferation of colon carcinoma cells after treatment with IGFR-1 tyrosine kinase inhibitor AG1024,gefitinib or both. Results：Irrespective of gefitinib treatment,EGFR immunoprecipitates from LoVo cells （the gefitinib-sensitive cell line） failed to display IGFR-1β band,whereas HCT116 cells （the gefitinib-resistant cell line） displayed the band and the band became more obvious after treatment with gefitinib. And IGFR-1β band was seen in gefitinib-treated HT29 cells （the gefitinib-moderate-sensitive cell line） but not in non-treated ones. Decreases in Akt and MAPK binding were found in LoVo cells treated with gefitinib,in HT29 cells treated with gefitinib plus AG1024,and in HCT116 cells treated with AG1024 （P〈0.05）. Gefitinib alone significantly decreased the proliferation rate of LoVo cells compared with control group （P〈0.05）,and AG1024 alone did not decrease the proliferation rate of LoVo cells; furthermore,gefitinib combined with AG1024 failed to further decrease the proliferation rate of LoVo cells compared with gefitinib group; AG1024 or gefitinib treatment alone did not decrease the proliferation rate of HT29 cells,but combined treatment led to a significant decrease （P〈0.05）; and AG1024 but not gefitinib alone significantly reduced the proliferation rate of HCT116 cells （P〈0.05）,whereas combined treatment did not result in a synergistic effect compared with AG1024 group. Conclusion：Resistance of colon carcinoma cells to gefitinib might partly arise from activation of IGFR-1β signaling pathway through the formation of EGFR/IGFR-1β heterodimerization,and administration of AG1024 blocking IGFR-1β activation might increase the sensitivity of colon carcinoma cells to gefitinib.