In Vitro Study On The Antiproliferative And Apoptotic Effects Of Different Sequences Of Combined Gefitinib And Pemetrexed Or Elemene On Human Lung Adenocarcinoma Cell Lines

Objective:(1)The study investigated the antiproliferative and apoptotic effects of different sequences of combined gefitinib and pemetrexed on human lung adenocacinoma cell lines A549and PC-9with different EGFR gene types in vitro,to expound the optimal combination schedule of gefitinib and pemetrexed respectively.(2)The other study investigated the antiproliferative and apoptotic effects of different sequences of combined gefitinib and elemene on human lung adenocacinoma cell lines A549and PC-9with different EGFR gene types in vitro,to research the synergy and sensitivity-increasing effects of elemene at determination of gefitinib, and the optimal combination schedule of both drugs.Methods:Human lung adenocacinoma cell lines with wide-type EGFR gene(A549) and mutant-type EGFR gene(PC-9) were used as in vitro models.This study used some methods to define the different effects of various schedules of gefitinib with pemetrexed or elemene treatment on cell growth and apoptosis, including:(1)The antiproliferative activities of gefitinib,pemetrexed and elemene single-agents in various concentrations and times were determinated via MTT assay.Based on this,concentration-growth inhibition curves were drawed and half-inhibitory concentrations(IC50) were calculated.(2)Cell viability of various schedules of gefitinib with pemetrexed or elemene were determinated via MTT assay.Then,the coefficient of drug in interaction(CDI) or combination index(CI) was used as a quantitative measure of the degree of interaction between different drugs.(3)The characteristics of cell apoptosis were analyzed by Annexin-V FITC/PI double marking staining method.(4)The dyed nucleuses of apoptotic cells were observed by TUNEL assay.Results:In part I,(1)In our evaluation of the antiproliferative effects of gefitinib and pemetrexed treatment on A549and PC-9cells,we observed dose-response and time-response growth inhibition effects.The IC5o values of gefitinib treatment on A549and PC-9cells for72h were8.642±0.261and0.044±0.002μmol/L,with nearly200times difference.The IC50values of pemetrexed treatment on A549and PC-9cells for72h were0.346±0.104and0.982±0.111μg/ml.(2)In the both cell lines,the antiproliferative effects of the pemetrexed-gefitinib sequence and concomitant administration of the two drugs(gefitinib+pemetrexed) increased with statistical differences when compared with pemetrexed and gefitinib single-agent(P<0.05), whereas the effects of the gefitinib-pemetrexed sequence reduced.In mutant-type EGFR gene PC-9cell,the growth inhibitory effects of the gefitinib+pemetrexed was more potent than the pemetrexed-gefitinib sequence(P<0.05), and the gefitinib+pemetrexed had a synergistic interaction.In contrast,the pemetrexed-gefitinib sequence had a better antiproliferative effect in wide-type EGFR gene A549cell and presented a synergistic interaction.(3)The apoptotic rate of gefitinib+pemetrexed was the highest in PC-9cell,and pemetrexed-gefitinib sequence was the highest in A549cell, but the inductions of apoptosis of gefitinib-pemetrexed sequence in both the cells were worst. In part Ⅱ,(1)In our evaluation of the antiproliferative effects of gefitinib and elemene treatment on A549and PC-9cells,we observed dose-response and time-response growth inhibition effects.The IC50values of elemene treatment on A549and PC-9cells for72h were26.461±2.309and27.830±0.614μg/ml.(2)The growth inhibition rates of various schedules of gefitinib with elemene were higher than two single-agents, having a characteristic of the gefitinib combined with elemene(gefitinib+elemene)>elemene-gefitinib sequence> gefitinib-elemene sequence.The antiproliferative effects of the gefitinib+elemene with the Cl value less than1,which meaned a synergistic interaction,were more potent than the other groups and the difference had a statistical significance. The IC50of gefitinib on PC-9and A549cells reduced68.33%and69.16%when gefitinib combined with elemene concurrently.(3)All the various schedules of gefitinib with elemene enhanced the inductions of apoptosis,with the most prominent effect in the gefitinib+elemene group(P<0.05).Conclusion:In part Ⅰ, the antiproliferative and apoptotic effects of the pemetrexed-gefitinib sequence and concomitant administration of the two drugs(gefitinib+pemetrexed) increased on A549and PC-9cells.wheras the effects of the gefitinib-pemetrexed sequence reduced.The simultaneous administration of gefitinib and pemetrexed showed the most significant antiproliferative and apoptotic effects in mutant-type EGFR gene PC-9cell,but in wide-type EGFR gene A549cell these effects of the sequence of pemetrexed followed by gefitinib were the most prominent. In part Ⅱ, the growth inhibitory and apoptotic effects of three different sequences of combined gefitinib and elemene treatment on A549and PC-9cells enhanced.The concomitant administration of gefitinib and elemene had the highest growth inhibitory and apoptotic rates on different EGFR gene types,resulted in a synergistic effect.Elemene at determination of gefitinib had a synergy and sensitivity-increasing effect when gefitinib and elemene combined.