多西他赛与吉非替尼序贯应用对人肺腺癌细胞SPC-A1生长及信号蛋白的影响

Sequence-dependent Effect of Docetaxel with Gefitinib on the Proliferation and Signal Protein Expression of Human Lung Adenocarcinoma Cell SPC-A1

背景与目的已经证明:化疗联合表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor recep- tor-tyrosine kinase inhibitors,EGFR-TKIs)与单独化疗比较治疗晚期非小细胞肺癌并不能增加疗效,但机制尚未完全明了。本研究通过观察多西他赛与吉非替尼不同时序应用对人肺腺癌SPC-A1细胞生长及对EGFR及其信号蛋白ERK、 AKT和胰岛素样生长因子(insulin-like growth factor,IGF)1型受体(IGF-1R)表达及磷酸化的影响,探索二者序贯用药增效的可能性及机制。方法 qPCR-HRM法检测细胞EGFR和K-ras基因突变;MTT检测细胞增殖;Western blot技术检测细胞EGFR、ERK、AKT、IGF-1R表达及磷酸化。结果 SPC-A1细胞EGFR和K-ras基因均无突变;与单药多西他赛或吉非替尼比较,多西他赛与吉非替尼同时应用及先吉非替尼后序贯多西他赛对细胞生长抑制作用均无明显差异, 但先多西他赛后应用吉非替尼对其生长抑制作用明显增强。多西他赛和吉非替尼分别增强和抑制EGFR和ERK磷酸化。多西他赛诱导的EGFR和ERK磷酸化明显被序贯应用的吉非替尼抑制,但不能被同时应用的吉非替尼抑制。吉非替尼抑制EGFR和ERK磷酸化作用不能被同时或序贯应用的多西他赛逆转。多西他赛与吉非替尼不同时序应用对 AKT及其磷酸化均无明显影响。多西他赛下调IGF-1R表达,吉非替尼对IGF-1R表达无明显影响。与多西他赛比较, 多西他赛序贯吉非替尼对IGF-1R表达无明显影响。结论多西他赛序贯吉非替尼对无EGFR突变的肺腺癌细胞SPC-A1 生长抑制作用明显增强,其机制可能与影响细胞EGFR和ERK磷酸化有关,与AKT磷酸化及IGF-1R表达无关。

Background and objective It has been proven that chemotherapy combined with epidermal growth factor receptor-tyrosine kinase inhibitors（EGFR-TKIs） could not increase response for advanced non-small cell lung cancer（NSCLC） ,but its cellular mechanism was not well known. The aim of this study is to assess the effects of sequential administration of docetaxel and gefitinib on the cell proliferation and signal pathway of lung adenocarcinoma cell SPC-A1 and its cellular mechanism. Methods The mutation of EGFR and K-ras gene were examined by qPCR-HRM. MTT assay was used to measure the cell proliferation. The expression and phosphorylation of EGFR,ERK,AKT and IGF-1R were determined by Western blot. Results No EGFR or K-ras gene mutation was found in SPC-A1 cells. Compared with docetaxel or gefitinib alone,no synergistic effects on the cell proliferation were observed in cells treated with docetaxel and gefitinib concomitantly or gefitinib followed by docetaxel. However,sequential administration of gefitinib following docetaxel could remarkably increase the inhibition of docetaxel on cell proliferation. Docetaxel increased,and gefitinib decreased,the phosphorylation of EGFR and ERK respectively. The suppression of pEGFR and pERK induced by gefitinib could not be activated by docetaxel,whether simultaneously or subsequently. No significant effects on the expression of AKT and p-AKT were found when docetaxel and gefitinib were administered simultaneously or sequentially. Docetaxel decreased the expression of IGF-1R. Conclusion The phosphorylation of both EGFR and ERK,not the phosphorylation of AKT or the expression of IGFR,may contribute to the synergistic effects of EFGR-TKI following chemotherapy on the cell proliferation of NSCLC.