胰岛素样生长因子1受体信号通路与西妥昔单抗在鼻咽癌耐药机制中的关系

Relationship between the insulin-like growth factor 1 receptor signaling pathway and the resistance of nasopharyngeal carcinoma to cetuximab

目的 建立西妥昔单抗耐药的人鼻咽癌细胞系5-8F/Erbitux,初步探讨胰岛素样生长因子1受体（IGF-1R）信号通路与西妥昔单抗在鼻咽癌耐药机制中的关系.方法 以表皮生长因子受体（EGFR）高表达且西妥昔单抗抑制率较高的人鼻咽癌5-8F细胞株为研究对象,采用逐步增加剂量法诱导建立西妥昔单抗耐药细胞5-8F/Erbitux.采用四甲基偶氮唑蓝（MTr）法检测西妥昔单抗对5-8F/Erbitux细胞的半数抑制浓度（IC50）,计算耐药指数（RI）.计数法描绘5-8F/Erbitux和5-8F细胞的生长曲线,计算倍增时间（TD）.以流式细胞仪检测5-8F/Erbitux和5-8F细胞的细胞周期.采用MTT法检测5-8F/Erbitux细胞对5-氟尿嘧啶（5-Fu）、顺铂（DDP）及紫杉醇（TAX）的交叉耐药谱.采用Western blot法检测5-8F/Erbitux和5-8F细胞中P糖蛋白（P-gp）、IGF-1R及磷酸化IGF-1R（p-IGF1R）的表达.采用实时荧光定量PCR法检测5-8F/Erbitux和5-8F细胞中多药耐药基因（MDR1）的表达量.结果 成功建立西妥昔单抗耐药的人鼻咽癌细胞系5-8F/Erbitux,西妥昔单抗作用3 d和5 d时的RI分别为1.2和1.1.5-8F和5-8F/Erbitux细胞的TD分别为26.63 h和142.30 h.与5-8F细胞比较,5-8F/Erbitux细胞中G0/G1期比例显著增高（P＜0.001）,S期比例显著下降（P＜0.001）.5-8F/Erbitux对5-Fu存在交叉耐药（RI=3.95,P＜0.01）,对TAX和DDP不存在交叉耐药（RI分别为1.14和0.68,均P＞0.05）.与5-8F细胞相比,5-8F/Erbitux细胞中P-gp、IGF-1R及p-IGF-1R蛋白的表达水平均显著升高（均P＜0.001）.MDR1基因在5-8F细胞中未检测到,而在5-8F/Erbitux细胞中仅有很微量的表达.结论 西妥昔单抗耐药细胞5-8F/Erbitux不存在与传统化疗药物相似的多药耐药性;过度活化的IGF-1R信号通路可能是5-8F/Erbitux细胞耐药的机制之一.

Objective To establish a cetuximab-resistant human nasopharyngeal carcinoma 5-8F/Erbitux cell line and preliminarily study the relationship between the insulin-like growth factor 1 receptor （IGF-1R） signaling pathway and the resistance of nasopharyngeal carcinoma to cetuximab. Methods A nasopharyngeal cancer cell line, 5-8F, with high epidermal growth factor receptor （EGFR） expression and cetuximab sensitivity, was selected as study object. The cetuximab-resistant 5-8F/Erbitux cell line was induced by stepwise selection after exposure to increasing doses of cetuximab. The IC5o was determined by 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay and the resistance index （RI）was calculated. The growth curves of 5-8F and 5-8F/Erbitux cells were plotted and the doubling times were calculated by cell counting assay. The cell cycle was detected by flow cytometry. Cross-resistance profiles of 5-8F/Erbitux cells to 5-Fu, Taxol and DDP were tested by MTT assay. Expression levels of P-gP, IGF-1R and P-IGF-1R of 5-8F and 5-8F/Erbitux cells were determined by Western blot analysis and MDR1 gene by real-time fluorescent quantitative PCR. Results A cetuximab-resistant human nasopharyngeal carcinoma cell line 5-8F/Erbitux was successfully established and their resistance index （RI） were 1.2 and 1.1,respectively, at 3 d and 5 d of the cetuximab treatment. The doubling times of 5-8F and 5-8F/Erbitux cells were 26.63 h and 142.30 h, respectively. Flow cytometry demonstrated that 5-8F/Erbitux cells showed an increased population at G0/G1 phase （ P 〈 0. 001 ） and reduced population at S phase （ P 〈 0. 001 ）,compared with 5-8F cells. The 5-8F/Erbitux cells showed cross-resistance to 5-Fu （ RI = 3.95, P 〈0. 01 ）and some resistance to Taxol as well as enhanced sensitivity to DDP （P 〉0.05 for all）. The 5-8F/Erbitux cells also had increased levels of P-gP, IGF-1R and P-IGF-1 R compared with 5-8F cells （ P 〈 0. 001 for all）. Expression of MDR1 gene was not detected in 5-8F cells and only very weak expression in 5-8F/Erbitux cells. Conclusion Cetuximab-resistant 5-8F/Erbitux cells have no common multidrug resistance like that induced by traditional chemotherapeutic drugs. The excessive activation of the IGF-1R signaling pathway is probably one of the mechanisms that caused resistance of 5-8F/Erbitux cells to cetuximab.