吉非替尼联合槲皮素对非小细胞肺癌PC9/GR细胞增殖的协同抑制作用

Synergistic inhibitory effect of gefitinib in combination with quercetin on proliferation of non-small cell lung cancer PC9/GR cells

目的:探究吉非替尼(getinib,Gef)联合槲皮素(quercetin,Que)对非小细胞肺癌(non-small cell lung cancer,NSCLC)PC9/Gef耐药(getinibresisitant,GR)细胞的增殖抑制作用及其可能的作用机制。方法:应用MTT法检测1.562 5、3.125、6.25、12.5、25、50和100μmol/L Gef或7.812 5、15.625、31.25、62.5、125、250和500μmol/L Que单药以及2药联合对PC9/GR细胞增殖的影响,计算Gef和Que对PC9/GR细胞的半数抑制浓度(half inhibitory concentration,IC50)值以及2药联用时的联合指数(combination index,CI)。40μmol/L Gef、90μmol/L Que和40μmol/L Gef+90μmol/L Que处理PC9/GR细胞后,在荧光显微镜下观察各组细胞的形态学变化,FCM法检测各组细胞的凋亡率,蛋白质印迹法检测各组细胞中磷酸化蛋白激酶B(phosphorylated-protein kinase B,p-PKB,又称p-Akt)及剪切型caspase-3蛋白的表达水平。结果:不同浓度的Gef和Que均可抑制PC9/GR细胞的增殖(P值均<0.01),IC50值分别为(41.26±2.38)μmol/L和(88.82±2.06)μmol/L;Gef联合Que时,Gef的IC50值为(17.49±0.77)μmol/L,Que的IC50值为(75.56±2.15)μmol/L,不同浓度的Gef与Que联合时的CI值均<1,表现为协同效应。荧光显微镜下观察可见,Gef联合Que组PC9/GR细胞凋亡的形态学改变更明显;Gef联合Que组PC9/GR细胞凋亡率高于Gef和Que单药组(P值均<0.01);Gef联合Que组PC9/GR细胞中p-Akt蛋白的表达水平低于Gef和Que单药组(P值均<0.01),而剪切型caspase-3蛋白的表达水平则高于Gef和Que单药组(P值均<0.01)。结论:Gef联合Que可显著抑制PC9/GR细胞增殖,2药具有协同作用,其机制可能与促进细胞凋亡和下调p-Akt蛋白的表达水平有关。

Objective： To investigate the inbitory effect of gefitinib （Gef） in combination with quercetin （Que） on the proliferation of non-small cell lung cancer （NSCLC） PC9/Gef-resistant （GR） cells, and to explore the possible mechanism. Methods： The effects of different concentrations of Gef （1.562 5, 3.125, 6.25, 12.5, 25, 50 and 100 μmol/L） and Que （7.812 5, 15.625, 31.25, 62.5, 125, 250 and 500 μmol/L） and their combination on the proliferation of PC9/GR cells were detected by MTT assay. The half inhibitory concentration （IC50） and combination index （Cl） values of Gef in combination with Que were calculated. After PCg/GR cells were treated with 40 μmol/L Gel, 90 μmol/L Que or 40 μmol/L Gef plus 90μmol/L Que, the morphological changes of PC9/GR cells were observed under a fluorescent microscope, the apoptotic rate of PCg/GR cells was detected by FCM, and the expression levels of phosphorylated protein kinase B （p-PKB, p-Akt） and cleaved-caspase-3 protein were detected by Western blotting. Results： Different concentrations of Gef and Que could inhibit the proliferation of Pcg/ GR cells （all P 〈 0.01） with ICso value of 41.26_＋2.38 ~mol/L and 88.82_＋2.06 ~mol/L, respectively. When treatment with Gef in combination with Que, the ICs0values of Gef and Que were 17.49_＋0.77 ~mol/L and 75.56_＋2.15 ~mol/L, respectively, and their combination treatment exhibited synergistic effect with the CI values which were smaller than those of Gef and Que alone treatment. The PC9/GR cells in the combination treatment group showed more obviously apoptotic changes under a fluorescence microscope. The apoptotic rate in combination treatment group was significantly higher than those in Gef and Que alone treatment groups （all P 〈 0.01）. The expression level of p-Akt in PCg/GR cells in combination treatment group was lower than those in Gel and Que alone treatment groups （both P 〈 0.01）, whereas the expression level of cleaved-caspase-3 protein in PC9/GR cells in combination treatment group was higher than those in Gef and Que alone treatment groups （both P 〈 0.01）. Conclusion： Gef in combination with Que can significantly inhibit the proliferation of PC9/GR cells and exert a synergistic effect. The potential mechanism may be related to apoptosis induction and down-regulation of the expression level of p-Akt protein.