Effect Of Different Combination Of Pemetrexed And Erlotinib On The Lung Adenocarcinoma A549Cells

Objective:To investigate the effect of combination and sequensial application of pemetrexed and erlotinib on the proliferation and apoptosis of human lung adenocarcinoma A549cells and to explore its possible mechanisms.Methods:After A549cells were treated with pemetrexed or erlotinib alone, combination and sequensial application for72hours, cell proliferation rate was measured by MTT assay and combination index was calculated. Cell cycle distribution and apoptosis rate were detected by flow cytometry. The expressions of phosphor-extracellular regulated protein kinase1/2(p-ERKl/2), phosphor-AKT (p-AKT) and phosphor-epidermal growth factor receptor (p-EGFR) were detected by Western blotting.Results:1. The growth inhibitory rate of A549cells treated with pemetrexed and erlotinibThe half inhibitory concentration of A549cells after treatment with erlotinib and pemetrexed were (11.89±0.73) u mol/L and (508.62±17.36) nmol/L respectively. The combination index of erlotinib combined with pemetrexed group was1.14±0.07, The combination index of erlotinib following pemetrexed group was0.36±0.02, The combination index of pemetrexed following erlotinib group was1.95±0.18. The application of erlotinib following pemetrexed on A549cells proliferation had synergistic effect while pemetrexed following or combined with erlotinib had antagonism effect.2. The morphological changes of A549cells after treatment with erlotinib and pemetrexedThe A549cells of control group were spindle shaped, monolayer and vigorous growth, its cell gap is clear; The decrease in size of A549cells and a small amount of suspended dead cells could be seen in erlotinib group, erlotinib combined with pemetrexed group and erlotinib followed by pemetrexed group. The shape of A549cells of pemetrexed group and erlotinib following pemetrexed group turned to be more larger,irregular and there were more dead cells too. The A549cells of erlotinib following pemetrexed group showed a large amount of cell debris and dead cells in suspension.3. The apoptosis morphological changes of A549cells after treatment with erlotinib and pemetrexedThe nucleus of A549cells of control group were integrity and uniformly dispersed; The A549cells of each dosing group all have some morphological changes such as nuclear staining,brightness enhancement and chromatin condensation. The most obvious apoptosis morphological changes of A549cells could be seen in erlotinib following pemetrexed group.4. Effects of different combination of erlotinib and pemetrexed on the cell cycle distribution of A549cellsCompared with control group,The S phase of A549cells after treatment with pemetrexed and erlotinib following pemetrexed were increased(P<0.05); The G1phase of A549cells after treatment with erlotinib and pemetrexed following or combined with erlotinib were increased(P<0.05)5. Effects of different combination of erlotinib and pemetrexed on the apoptosis rate of A549cellsBoth pemetrexed and erlotinib could induce the apoptosis of A549cells,and the apoptosis rate of A549cells after treatment with erlotinib following pemetrexed were significantly increased.6. Effects of different combination of erlotinib and pemetrexed on the expressions of p-ERK1/2(phosphorylation extracellular regulated protein kinase1/2), p-AKT and p-EGFR (phosphorylation epidermal growth factor receptor) in A549cells. Combined with erlotinib group,the apoptosis rate of A549cells after treatment with erlotinib following pemetrexed were increased(P<0.05),The expression levels of p-EGFR and p-AKT in A549cells after treatment with erlotinib following pemetrexed were down-regulated(P<0.05), The expression levels of P-ERK1/2in A549cells after treatment with erlotinib following pemetrexed had no significant differences (P>0.05)Conclusion:The application of erlotinib following pemetrexed on A549cells proliferation has synergistic effect. This effect may be related to the expression of phosphatidylinositol3-kinase(PI3K)-AKT signal pathway.