吉非替尼与培美曲塞联合时序对EGFR不同类型人肺腺癌细胞作用观察

Effect of different sequence administratation of gefitinib and pemetrexed on human lung adenocacinoma cell lines with different EGFR gene types

目的:通过体外研究吉非替尼不同时序联合培美曲塞对EGFR不同类型的人肺腺癌细胞A549和PC-9的生长抑制作用及其对细胞凋亡的影响,探讨吉非替尼与培美曲塞的最佳用药方式。方法:选取EGFR野生型A549和EGFR突变型PC-9人肺腺癌细胞为研究对象,分为对照组、培美曲塞单药72h组、吉非替尼单药72h组、培美曲塞24h序贯吉非替尼48h组、吉非替尼48h序贯培美曲塞24h组和吉非替尼同步联合培美曲塞72h组。MTT法检测不同给药方式对细胞增殖抑制作用,AnnexinⅤFITC/PI双标记染色法和TUNEL法检测不同给药方式对细胞凋亡的影响。结果:吉非替尼联合培美曲塞、培美曲塞序贯吉非替尼均可以增强吉非替尼和培美曲塞两药对2种细胞的增殖抑制效应,而吉非替尼序贯培美曲塞的生长抑制率较2个单药降低。对PC-9细胞,各浓度下吉非替尼联合培美曲塞组的生长抑制率分别为40.61%、50.15%、62.96%、76.09%和86.09,高于培美曲塞序贯吉非替尼组,P<0.001;对A549细胞,各浓度下培美曲塞序贯吉非替尼组的生长抑制率分别为43.89%、50.83%、65.71%、76.22%和85.91%,高于吉非替尼联合培美曲塞组,P<0.001。AnnexinⅤFITC/PI双标记染色实验中,吉非替尼联合培美曲塞组对PC-9细胞的总凋亡率最高,为70.2%,较其他各组差异有统计学意义,P<0.001。培美曲塞序贯吉非替尼组对A549细胞的总凋亡率最高,为97.4%,较其他各组差异有统计学意义,P<0.001。TUNEL实验中,吉非替尼联合培美曲塞组对PC-9细胞的总凋亡率最高,为82.99%,P<0.001。培美曲塞序贯吉非替尼组对A549细胞的总凋亡率最高,为82.69%,P<0.001。结论:吉非替尼联合培美曲塞对EGFR突变型的PC-9细胞增殖抑制和诱导凋亡作用最强,在EGFR野生型的A549细胞中,培美曲塞序贯吉非替尼的增殖抑制和诱导凋亡作用最优,表明吉非替尼和培美曲塞两药的最佳联用方式可能与EGFR基因突变状态有关。

OBJECTIVE： To study the antiproliferative and apoptotic effect of different sequences of combined ge fitinib and pemetrexed on human lung adenocacinoma cell lines A549 and PC-9 with different EGFR gene types in vitro,to explore the optimal combination schedule of gefitinib and pemetrexed respectively. METHODS： Human lung adenocacino- ma cell lines with wide-type EGFR gene（A549） and mutant-type EGFR gene（PC-9） were used in vitro in different experi- ment groups including control group,pemetrexed for 72 h group,gefitinib for 72h group,pemetrexed for 24h sequence by gefitinib for 48h group,gefitinib for 48h group sequence by pemetrexed for 24 h,gefitnib concomitant with pemetrexed for 72 h. MTT assay was used to measure the cell proliferation. The characteristics of cell apoptosis were analyzed by Annexin V FITC/PI double marking staining assay and TUNEL assay. RESULTS： In the both cell lines, the antiproliferative effects of the pemetrexed-gefitinib sequence and concomitant administration of the two drugs （pemetrexed gefitinib） were increased,whereas the effect of the gefitinib-pemetrexed sequence was reduced. In PC-9 cell, the growth inhibitory rates of pemetrexed＋gefitinib were 40.61%,50. 15%,62. 96%,76.09%,86.09%,which were higher than that of thepemetrexed-gefitinib sequence （P〈0. 001）. In A549 cell, the growth inhibition rates of pemetrexed-gefitinih sequence were 43.89%,50.83% ,65.71% ,76.22% ,85.91%,which were higher high than that in the others （P〈0. 001）. By Annexin V FITC/PI double making staining assay,the apoptotic rate of pemetrexed-l-gefitinib was 70.2% on the PC-9 cell,which was higher than that of others （P〈0. 001）. Whereas on A549 cell,the apoptosis rate of pemetrexed-gefitinib sequence was higher than that of other groups, 97.4 % （P〈 0. 001）. By TUNEL assay, gefitinib-pemetrexed had the highest apoptosis rate on PC-9 cell,82.99% （P〈0. 001）. In contrast,the apoptosis rate of gefitinib-pemetrexed sequence was the highest on A549 cell,82.69% （p〈 0. 001 ）. CONCLUSIONS： Pemetrexed ＋ gefitinib shows the most significant antiproliferative and apoptotic effects in mutant-type EGFR gene PC-9 cell,whereas pemetrexed-gefitinib sequence is the best in wild-type EGFR gene A549 cell. The study indicates that the optimal combination schedule of gefitinib and pemetrexed may be relat- ed to EGFR gene mutation.