新藤黄酸与细胞外信号调节激酶抑制剂PD98059联合应用对肺腺癌A549细胞生长的影响

目的研究新藤黄酸(Gambogenic acid,GNA)与细胞外信号调节激酶(extracellular signal regulatedkinase,ERK)特异性抑制剂PD98059联合应用对肺腺癌A549细胞生长的影响。方法新藤黄酸与ERK特异性抑制剂PD98059处理肺腺癌A549细胞,倒置显微镜观察细胞形态变化;甲基噻唑基四唑(methyl th-iazolyl tetrazolium,MTT)法检测细胞存活率;流式细胞仪检测细胞内活性氧的变化;蛋白印迹法(Westernblot)检测ERK、p-ERK蛋白的表达。结果 MTT法结果表明GNA在2.5μmol/L对肺腺癌A549细胞的增殖有抑制作用;与PD98059合用24h,GNA的抑制作用更加明显。倒置显微镜观察显示GNA作用肺腺癌A549细胞24h后,细胞固缩变圆,体积变小,部分细胞呈半贴壁状态;GNA和抑制剂PD98059合用后,多数细胞固缩变圆,脱落,并悬浮于培养液中;少数细胞体积增大、肿胀、破裂呈坏死状。流式细胞仪检测结果表明,20μmol/L PD98059+2.5μmol/L GNA共同作用于A549细胞24h后,与GNA 2.5μmol/L组比较细胞内活性氧生成增加。Western blot法检测表明,PD98059和GNA联合作用A549细胞24h后,p-ERK蛋白表达与GNA组比较显著降低。结论 GNA能诱导肺腺癌A549细胞凋亡,联合应用ERK抑制剂PD98059后可减少ERK蛋白的活化,增加肺腺癌A549细胞的凋亡;结果初步表明GNA诱导肺腺癌A549细胞的凋亡可能与调节ERK信号通路有关。

Effects of Gambogenic Acid Combined with Extracellular Signal Regulated Kinase Specific Inhibitor PD98059 on Growth of Lung Adenocarcinoma A549 Cells

Objective To investigate the effects of Gambogenic acid(GNA) combined with extracellular signal regulated kinase(ERK) specific inhibitor PD98059 on the growth of pulmonary adenocarcinoma A549 cells.Methods A549 cells were treated with GNA and PD98059.Morphologic changes of A549 cells were detected by inverted microscope.Cell viability was measured by methyl thiazolyl tetrazolium(MTT) staining.The changes of intracellular reactive oxygen species(ROS) were determined by flow cytometry,and the expressions of ERK and P-ERK proteins were detected by western blotting method.Results MTT staining results showed that 2.5 μmol/L GNA significantly inhibited the proliferation of A549 cells,its inhibitive effects were obviously enhanced in combination with PD98059 for 24 h.After treated with GNA for 24 h,inverted microscope showed that A549 cells became pyknotic,round,and smaller,some A549 cells were in a semi-adherence state.After treated with GNA combined with PD98059 for 24 h,most A549 cells became pyknotic and round,then dropped out and suspended in the medium;a few A549 cells became larger,swelled,ruptured,and became into necrosis.Flow cytometry showed that the production of ROS in A549 cells treated with 2.5 μmol/L GNA was significantly more than that in A549 cells treated with 20 μmol/L PD98059 plus 2.5 μmol/L GNA for 24 h.Western blotting detection showed that the expression levels of p-ERK protein were significantly decreased in A549 cells treated with PD98059 plus GNA in comparison with those in A549 cells treated with GNA for 24 h.Conclusion GNA can induce apoptosis of pulmonary adenocarcinoma A549 cells.The combined use of ERK specific inhibitor PD98059 and GNA can reduce the activation of ERK protein,and increase the apoptosis of A549 cells.The preliminary results indicated that the GNA-induced apotosis of A549 cells may be related to regulating ERK pathway.