多西他赛与吉非替尼不同时序应用对人肺腺癌细胞的作用

Sequence-dependent effect of docetaxel and gefitinib on the proliferation of lung adenocarcinoma cell lines A549 and PC-9

目的探讨多西他赛与吉非替尼不同时序应用对人肺腺癌细胞A549和PC-9的生长影响及其细胞学机制。方法 qPCR-HRM法检测人肺腺癌细胞EGFR和K-Ras基因突变,MTT法检测细胞增殖,Western blotting检测细胞信号蛋白及磷酸化表达,FCM法检测细胞周期变化。结果人肺腺癌A549细胞为EGFR基因野生型,PC-9细胞为EGFR第19外显子突变型。多西他赛和吉非替尼单药或联合用药均能抑制A549和PC-9细胞生长,呈浓度依赖性。多西他赛对A549和PC-9细胞生长的半数抑制浓度(IC50)分别为5.24×10-7和2.13×10-8mol/L,吉非替尼分别为1.28×10-5和4.58×10-8mol/L。在IC50浓度时,多西他赛序贯吉非替尼对A549和PC-9细胞的生长抑制率分别为60.00%和57.45%,均较单药组明显增高(P<0.05);而同时用药只对PC-9细胞有增效作用,抑制率为53.46%,较单药组明显增高(P<0.05)。多西他赛表现为增强A549、PC-9细胞EGFR和ERK磷酸化,吉非替尼表现为抑制,两药均抑制PC-9细胞IGF-1R磷酸化。多西他赛序贯应用吉非替尼显著抑制EGFR和ERK磷酸化,两药同时应用对抑制PC-9细胞的IGF-1R磷酸化具有增强作用。多西他赛将A549及PC-9细胞阻滞在G2期,吉非替尼将PC-9细胞明显阻滞在G1期。结论多西他赛序贯吉非替尼能够抑制EGFR野生型和突变型的A549及PC-9细胞生长,且呈增效作用,可能与影响细胞EGFR和ERK磷酸化有关;两药同时应用仅对PC-9细胞具有增效作用,可能与IGF-1R磷酸化抑制有关;不同时序应用的效果均可能与细胞周期相关。

Objective To assess the sequential administration of docetaxel and gefitinib on the cell proliferation of lung ade nocarcinoma A549 and PC9 cells and to probe its cellular mechanism. Methods M3T assay was used to measure the cell prolifera tion. The expression and phosphorylation of EGFR, ERK, AKT and IGF1R were determined by Western blotting. Results The EG FR gene of A549 and PC9 cells was widetype and the 19 exon mutation,respectively. Docetaxel dosedependently inhibited both A549 and PC9 cells proliferation with the ICs0 values of 5.24 x 107mol/L and 2. 13 x 108mol/L,while gefitinib inhibited A549 and PC9 cells dosedependently with the ICs0values of 1.28 x10Smol/L, and 4. 58 x 108mol/L. Sequential administration of gefitinib follow ing docetaxel remarkably enhanced the inhibition of docetaxel on the proliferation of both A549 （ inhibition rate 60. 00% , P 〈 0. 05 ） and PC9 cells（ inhibition rate 57.45% , P 〈0. 05 ）. Synergistic effects on the cell proliferation were observed only on PC9 cell （inhibition rate 53.46% , P 〈 0.05 ） , not on A549 cells, when cells were treated with docetaxel and gefitinib with the doses of IC50 concomitantly. The phosphorylation of EGFR and ERK induced by docetaxel on both A549 and PC9 cells was significantly suppressed by subsequent exposure to gefitinib. The phosphorylation of IGF1R on PC9 cells was significantly suppressed when ceils were treatded with docetaxel and gefitinib concomitantly. Cell cycle studies showed that docetaxel induced G2/M arrest for both the A549 and PC9 cells,which enhanced 49. 58% （P 〈 O. 05 ） and 30. 13% （ P 〈 O. 05 ） respectively. Gefitinib significantly induced G0/G~ arrest for PC-9 cells （ increase 29. 96% , P 〈 0. 05）, not for A549 cells. Conclusion Regardless of EGFR status, the synergistic effects on the proliferation of both EGFR wild-type and mutant NSCLC cells were observed when gefitinib was sequentially administrated following docetaxel, and the phosphorylation of both EGFR and ERK may contribute to this additive effects. The concomitant treatment of gefitinib with docetaxel exerted significant additive effects of cell proliferation only on PC-9 cells, and the suppression of IGF-1R phosphorylation may contribute to this synergistic effects. The sequential treatment of gefitinib followed by docetaxel 4exert no significant additive effects on the cell proliferation and resulted in the accumulation of cells in G0/Gl phase, which may decrease the effectiveness of docetaxel in subsequent cycles.