人肺腺癌厄洛替尼耐药细胞系PC-9/ER的建立及其特性

Establishment and characterization of a erlotinib—drug resistant variant of human lung adenocarcinoma cell line PC-9/ER

目的:构建厄洛替尼耐药人肺腺癌细胞模型PC-9/ER,观察单用人表皮生长因子受体(epithelial growth factor receptor,EGFR)酪氨酸激酶抑制剂厄洛替尼或联合胰岛素样生长因子受体1(insulinlike growth factor receptor 1,IGFIR)酪氨酸激酶抑制剂苦鬼臼毒(picropodophyllotoxin,PPP)作用于该细胞后,该细胞对厄洛替尼耐药性的影响,并探讨耐药相关机制。方法:选择人肺腺癌细胞株PC-9,采用逐步递增厄洛替尼浓度的方法体外诱导构建耐药株PC-9/ER,CCK-8法检测耐药指数;细胞计数法绘制PC-9和PC-9/ER的生长曲线,并计算出两细胞系的倍增时间;流式细胞术检测两细胞系的细胞周期;Western Blot法检测p-EGFR及p-IGFIR的表达水平,并进一步检测厄洛替尼及PPP单独或联合作用于PC-9/ER后,各组Akt,ERK,p-Akt及p-ERK的表达水平。结果:PC-9/ER细胞株的耐药指数是72.3。细胞生长曲线显示,PC-9/ER细胞生长较亲代细胞慢,PC-9与PC-9/ER细胞的倍增时间分别为32.9及36.9 h(P=0.003)。与PC-9相比,PC-9/ER细胞的G1期细胞增多(P=0.001),而S期细胞则明显下降(P:0.015)。Western Blot结果表明,PC-9/ER细胞中p-IGF1R表达比亲代细胞明显增多(P=0.016),而p-EGFR无明显变化(P=0.152)。在亲代及耐药细胞系,厄洛替尼联合PPP组均较其他组更能显著的抑制细胞增殖(P<0.05);且Western Blot法表明联合用药组EGFR下游磷酸化的Akt、ERK的表达水平明显减少。结论:成功构建了人肺腺癌厄洛替尼耐药细胞株PC-9/ER,IGF1R通路可能与肺腺癌EGFR-TKI获得性耐药有关。

Objective： To establish a human lung adenocarcinoma cell line PC-9/ER that is resistant to erlotinib; to observe the effect of erlotinib alone or in combination with PPP （a tyrosine kinase inhibitor of insulin-like growth factor receptor 1） on PC-9/ER and to discuss its possible mechanism. Methods： A erlotinib resistant human lung adenocarcinoma cell line PC-9/ER was induced by continuously exposing human lung adenocarcinoma cell line PC-9 to gradually increased doses of erlotinib. The drug resistant ability to erlotinib was measured by CCK-8 assay. The growth curve and cell cycle of PC-9 and PC-9/ER were compared. The expression of Akt, ERK, p-EGFR, p-IGF1R, p-Akt and p-ERK was measured by Western Blot in different groups. Results： The drug resistant index of PC-9/ER to erlotinib was 72.3. Double time of PC-9 and PC-9/ER were 32.9 and 36.9 h （P=0.003）, respectively, as evaluated by the growth curve. Figures in G1 phase was decreased in PC-9 than PC-9/ER （P=0.001）. The expression ofp-IGFIR significantly increased in PC-9/ER （P=0.016） but not p-EGFR （P=0.152）. More significant inhibition of cell proliferation than other groups was observed in combination group that erlotinib combination with PPP （P〈0.05）. The expression ofp-Akt and p-ERK was decreased in combination group. Conclusion： The resistant cell model PC-9/ER is established and IGF1R may associate with drug resistance to EGFR-TKI.