阻断表皮生长因子受体和环氧合酶-2信号途径对肺腺癌细胞的抗增殖作用

Inhibitory effect of blocking both epidermal growth factor receptor and cyclooxygenase-2 pathway on proliferation of lung cancer A549 cells

目的:探讨表皮生长因子受体抑制剂吉非替尼(gefitinib)联合新型环氧合酶-2(cyclooxygenase-2,COX-2)抑制剂塞来昔布(celecoxib)对肺腺癌A549细胞株的抑制作用及其可能机制。方法:肺腺癌A549细胞培养于RPMI 1640培养液中,实验分为正常对照组、吉非替尼5μmol/L组、塞来昔布25μmol/L组、吉非替尼5μmol/L加塞来昔布25μmol/L组。药物干预细胞48 h后,倒置相差显微镜观察细胞形态学变化,锥虫蓝拒染法检测药物对细胞生长的影响,Annexin V/PI法和Ho-echst33258染色法检测细胞凋亡,流式细胞术检测药物作用周期,免疫荧光和Real-time PCR法检测EGFR、COX-2蛋白的表达及EGFR mRNA的表达情况。结果:吉非替尼联合塞来昔布组相比单药组,A549细胞明显出现大量颗粒和空泡,细胞变圆并开始脱落。吉非替尼与塞来昔布单药对A549细胞抑制作用具有时间和剂量依赖性,加药48 h时吉非替尼(5μmol/L)联合塞来昔布(25μmol/L)组抑制率为(58.2±4.6)%,明显高于单药组(P<0.01)。联合用药组A549细胞凋亡率明显高于单独用药组(33.9%vs6.0%,8.8%),其S期细胞明显减少、G0/G1期明显增加(P<0.01);EGFR、COX-2蛋白的表达明显减弱(P<0.05),EGFR mRNA相对表达量(0.28±0.05)明显高于单独用药组(P<0.05)。结论:吉非替尼和塞来昔布联合对肺腺癌A549细胞增殖的抑制具有明显协同作用,其可能机制是诱导凋亡、增强G0/G1期阻滞和进-步下调活化的EGFR与COX-2的表达。

Objective：To explore the inhibitory effects of gefitinib （epidermal growth factor receptor inhibitor） combined with celecoxib （cyclooxygenase-2 inhibitor） against human lung cancer A549 cells and the possible mechanism. Methods： A549 ceils were cultured in RPMI 1640 medium and were divided into 4 groups： normal control group, 5 μmol/L gefitinib group, 25 μmol/L celecoxib group, and 5 μmol/L gefitinib ＋ 25μmol/L celecoxib group. The morphological changes of A549 cells were observed under inverted microscope 48 h after treatment ; the effects of drugs on growth of A549 Cells were detected by MTT assay; the apoptosis and cell cycles of A549 cells were measured by Annexin V/PI and Hoechst 33258 staining, respectively; and the expression of EGFR protein, COX-2 protein, and EGFR mRNA were determined by immunofluorescence and real-time PCR. Results： Compared with gefitinib and celecoxib groups, many granules and vacuoles were observed in the gefitinib and celecoxib combination group, and cells became round and there was defluxion. Both gefitinib and celecoxib inhibited the growth of A549 cells in a time- and dose-dependent manner. After treatment for 48 h, the inhibitory rate was （58.2 ±4.6）% in the combination group, which was significantly higher than those of the other two groups. Apoptosis rate in the combination group was also significantly higher than those in the other two groups （33.9% vs 6.0%, 8.8% ）, and the cell proportion in S phase significantly decreased and in G0/G1 phases significantly increased（P 〈0.01 ）. EGFR protein, COX-2 protein, and EGFR mRNA expression in A549 cells was significantly decreased in the combination treatment group compared with those in the other two groups （ P 〈 0.05 ）.Conclusion ： Gefitinib and celecoxib can synergistically inhibit the growth of A549 cells, possibly through promoting apoptosis, G0/G1 arrest, and down-regulating activated EGFR and COX-2 expression.