Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou Avenue North 1838, Guangzhou 510515, China.
Biomed Pharmacother. 2010 Oct;64(8):550-8. doi: 10.1016/j.biopha.2010.03.003. Epub 2010 Jul 13.
This work aimed to explore the induction of cetuximab-resistant human nasopharyngeal carcinoma (hNPC) 5-8F/Erbitux cells and the mechanisms of drug resistance. The 5-8F cells with high EGFR expression and cetuximab sensitivity were screened and then induced by stepwise exposure to increasing doses of cetuximab. Western blot was conducted to detect protein levels. Our results are as follows: we successfully induced the cetuximab-resistant 5-8F/Erbitux hNPC cells. After treatment with cetuximab for 3 and 5 days, the RI was 1.2 and 1.1, respectively. The 5-8F/Erbitux cells showed cross-resistance to 5-FU (P<0.01) and some resistance to Taxol (P>0.05) as well as enhanced sensitivity to DDP (P>0.05). The cells had increased levels of P-gP, IGF-1R, P-IGF-1R, K-ras, H-ras, and PTEN protein expression (P<0.001), while survivin decreased (P<0.001). Through sequence alignments, gene mutations in the PTEN gene at exons 5, 7, and 8, as well as the H-ras and K-ras genes in codons 12, 13, 59, and 61, were not observed. After transfection with H-ras-shRNA plasmid, the 5-8F/Erbitux cells showed reduced levels of gene and protein expression of H-ras and elevated sensitivity to cetuximab. In conclusion, gene amplification and overexpression of H-ras was the major mechanism that caused resistance of 5-8F/Erbitux cells to cetuximab, while the overexpression of the H-ras gene was probably associated with the over-activity of the IGF-1R signaling pathway. Gene deletion or mutation of PTEN was not associated with resistance of 5-8F/Erbitux cells to cetuximab.
本研究旨在探索诱导西妥昔单抗耐药的人鼻咽癌细胞（hNPC）5-8F/Erbitux 细胞及其耐药机制。筛选具有高 EGFR 表达和西妥昔单抗敏感性的 5-8F 细胞，然后逐步暴露于递增剂量的西妥昔单抗诱导耐药。采用 Western blot 检测蛋白水平。结果如下：我们成功诱导了西妥昔单抗耐药的 5-8F/Erbitux hNPC 细胞。用西妥昔单抗处理 3 天和 5 天后，RI 分别为 1.2 和 1.1。5-8F/Erbitux 细胞对 5-FU（P<0.01）表现出交叉耐药，对紫杉醇（P>0.05）有一定耐药性，对顺铂（DDP）敏感性增强（P>0.05）。细胞 P-gP、IGF-1R、P-IGF-1R、K-ras、H-ras 和 PTEN 蛋白表达水平增加（P<0.001），而 survivin 表达降低（P<0.001）。通过序列比对，未观察到 PTEN 基因外显子 5、7 和 8、H-ras 和 K-ras 基因的密码子 12、13、59 和 61 发生基因突变。转染 H-ras-shRNA 质粒后，5-8F/Erbitux 细胞 H-ras 基因和蛋白表达降低，对西妥昔单抗敏感性增强。结论：H-ras 基因扩增和过表达是导致 5-8F/Erbitux 细胞对西妥昔单抗耐药的主要机制，而 H-ras 基因的过表达可能与 IGF-1R 信号通路的过度激活有关。PTEN 基因缺失或突变与 5-8F/Erbitux 细胞对西妥昔单抗耐药无关。
