Sequence-dependent Effects Of Pemetrexed And Gefitinib On Lung Adenocarcinoma Cells

Objective To obtain the optimal combination schedule of gefitinib and pemetrexedthrough investigating sequence-dependent influence of growth and apoptosis on PC-9and A549cells respectively and to explore the possible mechanisms according todetection of the cell cycle and the protein expression of phosphorylation of EGFR,AKT, ERK, and TS.Methods1、MTT assay was used to measure the proliferation inhibition of differentdose of gefitinib and pemetrexed alone for24h,48h,72h. Dose-effect curve weredrawn according to the growth inhibition rates and drug concentration andhalf-inhibitory concentrations (IC50) were calculated.2、MTT assay was used tomeasure the sequence-dependent proliferation inhibition effect of pemetrexed andgefitinib on PC-9and A549cells with various concentration.3、Flow cytometryusing annexin V-FITC/PI staining was employed to measure the sequence-dependenteffect of pemetrexed and gefitinib on cell apoptosis and cell cycle.4、Western blotwas used to measure the sequence-dependent effect of protein expressions ofphosphorylation of EGFR, AKT, ERK and TS.Results1、The anti-proliferation effect of pemetrexed and gefitinib were observeddose-dependent and time-dependent on PC-9and A549cells. The IC50values ofgefitinib on PC-9and A549cells for72h were0.049±0.004μmol/L,8.937±0.356μmol/L respectively，nearly200times differences. The IC50values ofpemetrexed on PC-9and A549cells for72h were0.483±0.040μmol/L,2.742±0.505μmol/L respectively.2、The anti-proliferation and inducing apoptosiseffect of sequential pemetrexed followed by gefitinib and cocurrent pemetrexed and gefitinib was increased compared with pemetrexed and gefitinib alone(P<0.05) andthe effect of sequential pemetrexed followed by gefitinib is more significant(P<0.05).The anti-proliferation and inducing apoptosis effect of sequential gefitinib followedby pemetrexed had no significant difference compared with pemetrexed and gefitinibalone.2、Pemetrexed induced the phosphorylation of EGFR, AKT and ERK, Whilegefitinib significantly suppressed the phosphorylation of EGFR，AKT，ERK and TSexpression. Sequential pemetrexed followed by gefitinib and concurrent pemetrexedand gefitinib has a more significant effect in decreasing the expressions of p-EGFR，p-AKT and p-ERK.3、The gefitinib mainly blocked the cells in G0/G1phase andpemetrexed blocked the cells in S phase(p<0.05). The percentage of cells in G2/Mphase increased in sequential pememtrexed followed by gefitinib and cocurrentpemetexed and gefitinib groups(p<0.05).Conclusion Both sequential pemetrexed followed by gefitinib and concurrentpemetrexed and gefitinib has a synergistic effects in anti-proliferation，inducingapotosis and decreasing the expression of p-EGFR，p-AKT and p-ERK on PC-9andA549cells and sequential pemetrexed followed by gefitinib has a more siganificanteffect.