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Study on resistance of lung cancer cells to inhibitor of EGF receptor tyrosine kinase

肺癌细胞对EGF受体酪氨酸激酶抑制剂耐药的研究

基本信息

批准号：
20590077
负责人：
ITO Fumiaki
金额：
$ 3.08万
依托单位：
Setsunan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

Despite the dramatic efficacy of gefitinib and erlotinib in non-small cell lung cancer (NSCLC) patients with EGFR mutations, all patients ultimately develop resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). A secondary mutation in EGFR (T790M) and MET amplification have been identified as major mechanisms of acquired resistance to EGFR-TKIs. However, it is still important to identify additional mechanisms of resistance and to overcome acquired resistance to EGFR-TKIs in NSCLC patients. We previously reported that EGFR-TKI AG1478 induces apoptosis in PC-9 cells, a gefitinib-sensitive human NSCLC cell line with a mutation (delE746-A750) in tyrosine kinase domain of their EGFR. In this study, we repeatedly treated PC-9 cells with a high concentration of AG1478 (500 nM) and isolated AG1478-resistant cell lines. Expression level of ErbB3 in these resistant cells was extremely low as compared with that of PC-9 cells. Furthermore, PC-9 cells became resistant to AG1478, when their Erb … More B3 expression was down-regulated by siRNA. Therefore, the apoptosis-inducing action of AG1478 was correlated with the expression level of ErbB3. These results indicate that ErbB3 served to couple EGFR to the cell survival pathway in PC-9 cells and that the resistant cells could find a way to effectively activate survival signaling independent of.We next treated PC-9 cells with a lower concentration of AG1478 (50 nM) and isolated another series of AG1478-resistant cell lines. In PC-9 cells, AG1478 decreased the expression of the MAPK phosphatase-1(MKP-1) and intensively stimulated phosphorylation of JNK. Further, AG1478 induced the accumulation of proapoptotic Bcl-2 family protein Bim in mitochondria. However, in the resistant cell lines, expression level of MKP-1 was still high after AG1478 treatment, and neither JNK phosphorylation nor accumulation of Bim was increased. These results indicate that translocation of Bim to mitochondria through the MKP-1/JNK pathway is critical for EGFR-TKI-induced apoptosis in PC-9 cells and that continuous high-level expression of MKP-1 leads to resistance to EGFR-TKIs. Less

尽管吉非替尼和厄洛替尼对 EGFR 突变的非小细胞肺癌 (NSCLC) 患者具有显着疗效，但所有患者最终都会对 EGFR 酪氨酸激酶抑制剂 (EGFR-TKI) 产生耐药性。 EGFR (T790M) 二次突变和 MET 扩增已被确定为 EGFR-TKI 获得性耐药的主要机制。然而，确定其他耐药机制并克服 NSCLC 患者对 EGFR-TKI 的获得性耐药仍然很重要。我们之前报道过 EGFR-TKI AG1478 诱导 PC-9 细胞凋亡，PC-9 细胞是一种吉非替尼敏感的人 NSCLC 细胞系，其 EGFR 酪氨酸激酶结构域存在突变 (delE746-A750)。在本研究中，我们用高浓度AG1478（500 nM）反复处理PC-9细胞并分离出AG1478耐药细胞系。与PC-9细胞相比，这些耐药细胞中ErbB3的表达水平极低。此外，当 siRNA 下调 Erb … 更多 B3 表达时，PC-9 细胞对 AG1478 产生耐药性。因此，AG1478的凋亡诱导作用与ErbB3的表达水平相关。这些结果表明，ErbB3 将 EGFR 与 PC-9 细胞中的细胞生存途径偶联，并且耐药细胞可以找到一种独立于有效激活生存信号传导的方法。接下来，我们用较低浓度的 AG1478 (50 nM) 处理 PC-9 细胞，并分离出另一系列 AG1478 耐药细胞系。在 PC-9 细胞中，AG1478 降低 MAPK 磷酸酶-1 (MKP-1) 的表达并强烈刺激 JNK 的磷酸化。此外，AG1478 诱导促凋亡 Bcl-2 家族蛋白 Bim 在线粒体中积累。然而，在耐药细胞系中，AG1478处理后MKP-1的表达水平仍然较高，JNK磷酸化和Bim积累均未增加。这些结果表明，Bim 通过 MKP-1/JNK 途径易位至线粒体对于 EGFR-TKI 诱导的 PC-9 细胞凋亡至关重要，并且 MKP-1 的持续高水平表达导致对 EGFR-TKI 的耐药。较少的