多西他赛与吉非替尼序贯应用对人肺腺癌细胞H1975存活及凋亡通路的研究

Sequence-dependent Effect of Docetaxel with Gefitinib on the Proliferation and Apoptosis of Lung Adenocarcinoma Cell H1975

背景与目的表皮生长因子受体(epidermal growth factor receptor,EGFR)酪氨酸激酶抑制剂(tyrosinekinase inhibitors,TKIs)被用于治疗进展性晚期非小细胞肺癌(non-small cell lung cancer,NSCLC),然而最初接受EGFR-TKIs治疗有反应的患者,大部分会在10个月左右出现获得性耐药。绝大多数报告称T790M的突变是产生获得性耐药的主要原因,约占获得性耐药的50%。本研究旨在探索多西他赛和吉非替尼序贯应用对肺腺癌细胞H1975增殖和凋亡通路的作用。方法 M法检测细胞的增殖。等效线图法和联合指数(combination index,CI)法评估多西他赛和吉非替尼序贯作用的效价。流式细胞术检测细胞凋亡和周期分布,Hoechest 33258染色法检测凋亡形态。化学比色发光法检测Caspases的活性。结果等效线图法和联合指数法均显示多西他赛序贯吉非替尼组较其它序贯作用组明显抑制了细胞增殖,增加了细胞的凋亡。细胞周期分布实验结果显示与吉非替尼序贯多西他赛组主要把细胞抑制在G0/G1期相比较,多西他赛序贯吉非替尼组主要把细胞抑制在G2/M期。在肺腺癌H1975中,所有序贯模型组都主要通过活化Caspase-8/Caspase-3来诱导激活细胞凋亡通路。结论先用多西他赛再用吉非替尼治疗模式可能是TKIs耐药后T790M突变肺癌的一个新选择。

Background and objective Epidermal growth factor receptor（EGFR）tyrosine kinase inhibitors（TKIs）such as gefitinib and erlotinib show promising therapeutic effects in patients with advanced non-small cell lung cancer（NSCLC）.However,despite an initial response to EGFR-TKIs treatment among responsive patients,most inevitably acquire resistance after a progression-free period of about 10 months.The percentage of T790M in TKI acquired-resistant patients in most studies is around 50%.The aim of this study is to assess the effects of the sequential administration of docetaxel and ge-fitinib on cell proliferation and apoptosis of lung adenocarcinoma cell H1975. Methods An MTT assay was used to measure cell proliferation.The potency of the sequential administration of docetaxel and gefitinib were determined by isobolograms and combination index（CI）.Cell apoptosis and cycle distribution were determined by flow cytometry.The Hoechst 33258 method was used to observe the apoptotic morphology.Chemical colorimetric luminescence was used to measure the caspase activity.Results The isobolograms and CI showed that the sequential administration of docetaxel following gefitinib remark-ably inhibits cell proliferation and cell apoptosis compared with other sequential administration models.The cycle distribution results indicate that sequential docetaxel administration following gefitinib blocked the cells in the G2/M phase but not in the G0/G1.The activation of the Caspase-8/Caspase-3 cascade is mainly involved in the apoptotic pathway of lung adenocarcinoma cell H1975 in all sequential administration models.Conclusion The docetaxel administration following gefitinib might be a new stratagy for lung cancer with T790M mutation after having EGFR-TKIs resistance.