Erlotinib Combined With Celecoxib Inhibits The Growth Of Lung Cancer A549 Cells By Simultaneously Blocking EGFR And COX-2

ObjectiveA lot of reasearches have reported that epidermal growth factor receptor (EGFR) and Cyclooxy-genase-2(COX-2) is expressed frequently in non-small cell lung carcinoma (NSCLC), most likely to contribute to the biological characteristics of NSCLC, including development,invasion and metastasis. This study was to determine the interaction of Erlotinib and Celecoxib against human lung cancer A549 cell and its possible mechanism. Thus we can provide new ideas for targeted therapy of non-small cell lung cancer.MethodsA549 cells were observed under an inverted microscope,MTT was used to measure the growth inhibitory effects of A549 cells by Erlotinib and Celecoxib treated with drugs for 48 hours.. The cell apoptosis was studied byTUNEL and Hoechst33258 staining method. The cell cycle was detected by flow cytometer,and the expression of EGFR,COX-2 were determined by immunofluorescence. All the data above were expressed by x±s,and were analyzed using t 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 without drugs:Cell adherence was in good condition, the cell gap is cloesd and and various cell divisions without the cell debris and apoptotic bodies were observed.②50μmol Erlotinib monotherapy group:Cells adherent slowed, shape changed, the cell debris were observed with cell rounding and defluxion..cavitation and particle growed in number, the cell gap increased. ③25μmol Clecoxib monotherapy group:Cells slightly smalled,Cells adherent slowed, the cell gap increased.,and shape changed, intracellular vacuoles and granules were observed,and apoptotic bodies were observed④50μmol Erlotinib and 25μmol Clecoxib Combinated group:, shape changed,Cells rounded, the cell gap increased,Cells adherent slowed,a lot of granules and vacuolus were observed,. And the cell debris and apoptotic bodies were observed.anywhere.2.Cell vigor detected by methyl thiazolyl tetrazolium(MTT)Erlotinib and celecoxib inhibited the growth of A549 was asssayed in dose-and time-dependent manner,50μmol Erlotinib monotherapy group inhibition rates was (34.35±2. 77)% and 25μmol Clecoxib monotherapy group was (26.66±2.12)% 50μmol Erlotinib and 25μmol Clecoxib Combinated group inhibition rates was (50.33±3.27)%, The growth inhibition rates in the combination group were much higher than the group treated by drug alone (P<0.05)3.The apoptosis rate treated by drugsHoechst33258 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 (63.37±2.12)%.much higher than the single Erlotinib group (32.56±1.67%) and the celecoxib group (25.67±1.98)%.TUNEL method showed that:the signs of the apoptosis cells means the uniform nuclear staining without the cytoplasm staining. The kill rates of A549 cells treaded with the control group,50μmol Erlotinib and 25μmol celecoxib alone were (0.95±0.24)%, (32.56±1.67)% and (25.67±1.98)% respectivelyand. the combined group was(42.13±1.4)%,significantly higher than any other group (P<0.05).Annexin V/PI method showed that:early apoptosis rate in the control group was (0.86±1.3)%, the apoptosis rate of the combined group was (52.47±2.3)%,significantly higher than Erlotinib or celecoxib alone, whose apoptotic rate was respectively (30.24±1.7)% and (22.13±1.2)%. 4, Cell cycle treated by drugs50μmol Erlotinib and 25μmol celecoxib both caused G0/G1 stage cell ratio increased and S stage cell ratio decreased.The combined treament group in contrast to each monotherapy group, GO-G1 phase arrest significantly (P＜0.05) (P＜0.05)5. The expression of EGFR,COX-2 were determined by immunofluorescenceEGFR and COX-2 highly expreses in the nuclear and cytoplasm. The 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)ConclusionsThe results suggest that combination treatment with Erlotinib and Celecoxib showed significant synergistic inducing apoptosis and G1 phase arrest,owing to down-regulating expressing both EGFR and COX-2.