雷公藤甲素联合吉非替尼对EGFR突变NSCLC细胞的协同效应分析-中国药房

雷公藤甲素联合吉非替尼对EGFR突变NSCLC细胞的协同效应分析

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篇名：	雷公藤甲素联合吉非替尼对EGFR突变NSCLC细胞的协同效应分析
TITLE：	Analysis of the synergistic effect of triptolide combined with gefitinib on EGFR-mutant NSCLC cells
摘要：	目的探讨雷公藤甲素（TPL）联合表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）吉非替尼对EGFR突变非小细胞肺癌（NSCLC）细胞的协同效应及潜在机制。方法体外培养人NSCLC细胞系H1975（EGFRT790M/L858R突变的耐药细胞系）和H1299（EGFR野生型的非耐药细胞系），采用MTT法检测细胞存活率并通过联合用药指数（CI）评价TPL和吉非替尼的联用效应。将H1975细胞分为空白组，低、高浓度TPL组（5、15nmol/L），吉非替尼组（2μmol/L），低浓度TPL+吉非替尼组、高浓度TPL+吉非替尼组（5nmol/LTPL+2μmol/L吉非替尼、15nmol/LTPL+2μmol/L吉非替尼），采用流式细胞术检测其凋亡及周期分布情况；利用分子对接技术预测TPL与EGFR的结合能力，并采用流式细胞术检测细胞中磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）通路及自噬相关蛋白[微管相关蛋白1轻链3α（MAP1LC3A）、MAP1LC3B]的表达情况。结果TPL对H1975和H1299细胞的增殖均有一定的抑制作用，且有浓度、时间依赖趋势；5或15nmol/LTPL联合吉非替尼作用48h对H1975细胞的增殖均有协同抑制效应（CI＜1），而对H1299细胞则无协同作用（CI＞1）。与空白组比较，各药物组的细胞凋亡率和G0/G1期细胞的比例均显著升高，S期、G2/M期（个别TPL组除外）细胞的比例均显著降低，且联用组效果更优（P＜0.05）。分子对接结果显示，TPL的羟基可与EGFRT790M/L858R突变编码产物的Thr854残基形成氢键。与空白组比较，各药物组细胞中通路相关蛋白的表达均显著下调，自噬相关蛋白的表达均显著上调，且联用组效果更优（P＜0.05）。结论TPL联合吉非替尼可协同抑制EGFR突变NSCLC细胞的增殖活性，其作用机制可能与下调PI3K/Akt/mTOR通路和诱导细胞自噬有关。
ABSTRACT：	OBJECTIVE To investigate the synergistic effect of triptolide （TPL） combined with epidermal growth factor receptor tyrosine kinase inhibitor （EGFR-TKI） gefitinib on EGFR-mutated non-small cell lung cancer （NSCLC） cells and its potential mechanism. METHODS Human NSCLC cell lines H1975 （EGFR T790M/L858R mutated drug-resistant cell lines） and H1299 （EGFR wild-type non-drug-resistant cell lines） were cultured in vitro. MTT method was used to detect cell activity， and the effect of combined medication was evaluated by the combination index （CI）. The H1975 cells were divided into blank group， low- concentration and high-concentration groups of TPL （5 nmol/L or 15 nmol/L）， gefitinib group （2 μmol/L）， low-concentration and high-concentration groups of TPL+gefitinib （5 nmol/L TPL+2 μmol/L gefitinib， 15 nmol/L TPL+2 μmol/L gefitinib）. Flow cytometry was used to detect the apoptosis of H1975 cells and the distribution of the cell cycle. Molecular docking studies were used to predict the binding ability of TPL to EGFR. The expressions of phosphatidylinositol 3 kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） pathway and autophagy-related proteins [microtubule-associated protein 1 light chain 3α （MAP1LC3A）， MAP1LC3B] in H1975 cells were detected by flow cytometry. RESULTS TPL had a strong inhibitory effect on the proliferation of H1975 and H1299 cells in a time-dependent and dose-dependent manner. Forty-eight hours treatment of 5 or 15 nmol/L TPL combined with gefitinib had a synergistic inhibitory effect on the proliferation of H1975 cells （CI＜1）， while there was no synergistic inhibitory effect on H1299 cells （CI＞1）. Compared with the blank group， the apoptosis rate and the proportion of H1975 cells at G0/G1 phase were increased significantly in administration groups， while the proportions of cells at S phase and G2/M phase （except for several TPL groups） were decreased significantly， and the combination group had better effects （P＜0.05）. Molecular docking studies showed that the hydroxyl radical of TPL could form hydrogen bonds with the Thr854 residue of the product encoded by EGFR T790M/L858R mutation. Compared with the blank group， the expressions of pathway-related proteins were down-regulated significantly in administration groups， while those of autophagy-related proteins were up-regulated significantly， and the combination group had better effects （P＜0.05）. CONCLUSIONS TPL combined with gefitinib can synergically inhibit the proliferation activity of EGFR-mutated NSCLC cells， the mechanism of which may be related to the down-regulation of PI3K/Akt/ mTOR pathway and induction of autophagy.
期刊：	2023年第34卷第18期
作者：	张艺；郭芬
AUTHORS：	ZHANG Yi，GUO Fen
关键字：	雷公藤甲素；吉非替尼；表皮生长因子受体；酪氨酸激酶抑制剂；非小细胞肺癌；磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物
KEYWORDS：	triptolide； gefitinib； epidermal growth factor receptor； tyrosine kinase inhibitor； non-small cell lung cancer；
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