| ObjectiveIn this study,we investigated the effects of Gefitinib Combined with Celecoxib in cell morphology changes, apoptosis, and EGFR, COX-2 protein expression and EGFR mRNA expression, against human lung cancer A549 cell. Thus we try to provide new ideas for targeted therapy for lung cancer.MethodsThe cells were cultured in RPMI-1640 and divided into four groups:normal control group; 5μmol/L Gefitinib group; 25μmol/L Celecoxib group; 5μmol/L Gefitinib+25μmol/L Celecoxib group,and treated with drugs for 48 hours. Cellular morphological changes were observed under an inverted microscope,cell inhibition rate was detected by trypan-blue exclusion assay, the apoptosis rate and cell cycle were measured by a flow cytometer and Hoechst33258 staining method, the expression of EGFR,COX-2 protein and EGFR mRNA were determined by immunofluorescence and real-time RT-PCR. All the data above were expressed byχ±s,and were recorded and analyzed using t test,χ2 test, analysis of variance,with SPSS 11.5 software. The statistic significance were defined as P<0.05.Results1. Cellular morphological changes observed under an inverted microscope①Normal groups:Cell adherence was aequalis and in good condition, and various cell divisions were observed in the normal group.②Gefitinib monotherapy group:Cells slightly rounded, shape changed, cavitation and particle growed in number, the cell gap increased.③Clecoxib monotherapy group:Cells adherent slowed,and intracellular vacuoles and granules were observed.④Combination group:A great quantity of granules and vacuolus were observed in the combined treatment,with cell rounding and defluxion. And the cell debris and apoptotic bodies were observed.2.Cell vigor detected by methyl thiazolyl tetrazolium(MTT)Aftered treated by drugs for 48h, gefitinib and celecoxib inhibited the growth of A549 in does-and time-dependent manner, whose inhibition rates were respectively (24.8±2.1)%and (21.5±1.7)%.The growth inhibition rates in the combination group,were (58.2±4.6)%, much higher than the group treated by drug alone (P<0.01)3.The apoptosis rate treated by drugsAnnexin V/PI method showed that:early apoptosis rate in the control group was 0.66%, the apoptosis rate of the combined group was 33.9%,significantly higher than gefitinib or celecoxib alone, whose apoptotic rate was respectively 6.00% and 8.85%.Hoechst33258 staining showed that:The uniform nuclear staining, shape rules were observed in the normal cells. The apoptotic nuclei presented nuclear condensation,nuclear lobulation as rescent-shape or bean-shape, which even break intograins of rice. The apoptosis rate of the combined group was 32.4%, much higher than the single gefitinib group (7.12%) and the celecoxib group (8.43%)4. Cell cycle treated by drugsGefitinib and celecoxib both caused G0～G1 phase arrest. In contrast to each monotherapy group,S stage cell ratio decreased (P<0.01) and G0/G1 stage cell ratio increased (P<0.01),in each combined treament group.5. The expression of EGFR,COX-2 were determined by immunofluorescenceThe strong fluorescence intensity of EGFR and COX-2 can be seen in the normal control group.The fluorescence intensity was much weeker in the combined treatment, and was statistically different than the monotherapy group (P<0.05)6. EGFR mRNA determined by real-time RT-PCRAdding drugs for 48 h, the relative EGFR mRNA expression level of 25μmol/L celecoxib group and 5μmol/L gefitinib group were respectively (0.45+0.07) and (0.59+0.11), which were much higher than the normal control group (P<0.01). The relative EGFR mRNA expression level of the combined group was 0.28+0.05, which was much lower (P<0.05),compared with monotherapy groups.ConclusionsGefitinib combined with Celecoxib can significantly inhibit the growth of A549 cell, possibly by promoting apoptosis, G0-G1 arrest,and down-regulation of EGFR and COX-2 protein and mRNA expressions. |
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