双靶点阻滞对肺腺癌细胞的协同抑制作用及其可能机制

摘要

Objective To determine the synergic effect of simultaneously blocking epidermal growth factor receptor ( EGFR) and cyclooxygenase-2 on the growth inhibition of human lung cancer A 549 cells.Methods The A549 cells were treated by gefitinib (5 μmol/L), celecoxib (25 μmol/L), or a combination of them at the same dosages respectively for 48 h, and the cells without treat-ment served as control .Cell growth was detected by trypan-blue exclusion assay , apoptosis rate and cell cycle were measured by flow cytometry and Hoechst33258 staining, expression of EGFR and COX-2 proteins was determined by Western blotting .Results With time and dose increase , both gefitinib and celecoxib showed stronger inhibitory effect on the growth of A 549 cells, and the combination of them had more significant inhibitory effect than the gefitinib and celecoxib groups (P<0.01).The combination group also had obvious higher apoptosis rate than the monotherapy groups (32.40%vs 7.12%and 8.43%, P<0.01).The proportion of the cells at S stage was (3.2 ±0.9)%in the combination group , significantly decreased when compared with those in the gefitinib and celecoxib groups [(37.4 ±1.6)%, (21.0 ±3.1)%, P<0.01].The combination group had obviously more cells arrested at G0/G1 stage than the monotherapy groups [(87.2 ±6.4)% vs (61.4 ±5.2)% and (51.8 ±4.7)%, P<0.01].The protein levels of EGFR and COX-2 were significantly lower in the combination group than the other 2 groups (P<0.05).Conclusion The combination treatment of gefitinib and celecoxib exerts inhibitory effect through blocking both EGFR-and COX-2-related pathways , and the regimen might provide a promising strategy for cancer therapy and chemoprevention in lung cancer .%目的：探讨联合阻断表皮生长因子受体（EGFR）和环氧合酶－2（COX－2）对肺腺癌A549细胞株的协同抑制作用及其可能机制。方法采用不同药物干预肺癌A549细胞株，分为4组：正常对照组、单药吉非替尼组、单药塞来昔布组、联合用药组。药物干预细胞48 h后台盼蓝（trypan blue）染色法检测药物对细胞生长的影响；以Hoechst33258染色法和流式细胞术观察用药前后细胞凋亡和细胞周期的变化。采用Western blot检测EGFR和COX－2蛋白的表达情况。结果随着时间和剂量增加，单药吉非替尼与塞来昔布对A549细胞的抑制作用增强，加药48 h，联合用药组抑制率明显高于单药组（P＜0．01）。联合用药组细胞凋亡率明显高于单独用药组（32．40％vs 7．12％和8．43％；P＜0．01）。联合用药组S期细胞比例为（3．2±0．9）％，较单药吉非替尼组[（37．4±1．6）％]和单药塞来昔布组[（21．0±3．1）％]明显减少（P＜0．01）；联合用药组G0／G1期细胞比例为（87．2±6．4）％，较单药吉非替尼组[（61．4±5．2）％]和单药塞来昔布组[（51．8±4．7）％]明显增加（P＜0．01）。与单独用药组比较，联合用药组EGFR和COX－2蛋白的表达明显减弱（P＜0．05）。结论联合用药通过EGFR和COX－2双靶点阻滞发挥作用，有望为肺癌的化学预防和治疗提供新的策略。