EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells
EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells
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DOI：
10.1016/j.bbrc.2015.03.162
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发表时间：
2015-05-22
影响因子：
3.1
通讯作者：
Miyazawa, Keisuke
中科院分区：
文献类型：
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作者：
Sugita, Shohei;Ito, Kentaro;Miyazawa, Keisuke
吉非替尼（Gefitinib， GEF）是一种EGFR酪氨酸激酶抑制剂，在非小细胞肺癌（NSCLC）细胞系中，如通过EGFR基因突变表达组成型激活的EGFR激酶的PC-9细胞，以及具有野生型EGFR的A549和H226细胞，可有效诱导自噬。出乎意料的是，gef诱导的自噬也在非nsclc细胞中被观察到，如小鼠胚胎成纤维细胞（MEF）和不表达EGFR的白血病细胞系K562和HL-60。用CRISPR/Cas9系统敲除A549细胞中的EGFR基因后，GEF处理后仍能诱导细胞自噬，说明GEF诱导细胞自噬不是通过抑制EGFR激酶活性来介导的。GEF和克拉霉素（CAM）（一种具有抑制自噬通量作用的大环内酯类抗生素）联合治疗可增强NSCLC细胞系的细胞毒作用，尽管单独使用CAM治疗无细胞毒性。GEF处理诱导内质网（ER）应激相关基因如CHOPI、GADD153和GRP78上调。在PC-9细胞中敲除CHOP和敲除CHOP的MEF对GEF的敏感性均低于对照组。在培养基中添加CAM后，gef诱导的内质网应力加载进一步明显，而单独添加CAM则没有效果。这些数据表明，GEF诱导的自噬具有细胞保护功能，并表明使用CAM进行GEF治疗的潜在治疗可能性。此外，研究表明，响应GEF启动自噬的细胞内信号可以与EGFR完全分离，但可能存在未知的GEF诱导自噬的靶分子。(C) 2015年作者。Elsevier Inc.出版。
Gefitinib (GEF), an inhibitor for EGFR tyrosine kinase, potently induces autophagy in non-small cell lung cancer (NSCLC) cell lines such as PC-9 cells expressing constitutively activated EGFR kinase by EGFR gene mutation as well as A549 and H226 cells with wild-type EGFR. Unexpectedly, GEF-induced autophagy was also observed in non-NSCLC cells such as murine embryonic fibroblasts (MEF) and leukemia cell lines K562 and HL-60 without EGFR expression. Knockout of EGFR gene in A549 cells by CRISPR/Cas9 system still exhibited autophagy induction after treatment with GEF, indicating that the autophagy induction by GEF is not mediated through inhibiting EGFR kinase activity. Combined treatment with GEF and clarithromycin (CAM), a macrolide antibiotic having the effect of inhibiting autophagy flux, enhances the cytotoxic effect in NSCLC cell lines, although treatment with CAM alone exhibits no cytotoxicity. GEF treatment induced up-regulation of endoplasmic reticulum (ER)-stress related genes such as CHOPI GADD153 and GRP78. Knockdown of CHOP in PC-9 cells and Chop-knockout MEF both exhibited less sensitivity to GEF than controls. Addition of CAM in culture medium resulted in further pronounced GEF-induced ER stress loading, while CAM alone exhibited no effect. These data suggest that GEF-induced autophagy functions as cytoprotective and indicates the potential therapeutic possibility of using CAM for GEF therapy. Furthermore, it is suggested that the intracellular signaling for autophagy initiation in response to GEF can be completely dissociated from EGFR, but unknown target molecule(s) of GEF for autophagy induction might exist. (C) 2015 The Authors. Published by Elsevier Inc.