麦角甾醇与吉非替尼联合用药协同抑制非小细胞肺癌A549和PC-9细胞增殖

目的研究麦角甾醇(ERG)与吉非替尼(GEF)联合用药协同对非小细胞肺癌A549(GEF耐药细胞)和PC-9(GEF敏感细胞)细胞的抑制作用。方法将人肺癌细胞A549和PC-9细胞分别分为对照组、ERG 5~160μmol·L-1单药组、GEF 5~160μmol·L-1(A549)或0.625~20μmol·L-1(PC-9)单药组及不同浓度ERG+GEF联合用药组,MTT法测定细胞增殖;Hoechst 33258观察细胞核形态变化;流式细胞术测定细胞凋亡率及细胞周期;Western印迹法检测蛋白质丝氨酸苏氨酸激酶(Akt),磷酸化AKT(p-Akt),表皮生长因子受体(EGFR),磷酸化EGFR(p-EGFR)蛋白的表达。结果在不同作用时间点,与ERG和GEF单药组相比,相应浓度两药联用组对A549和PC-9细胞的增殖抑制率提高(P<0.01),表现出明显的协同效应(q>1.15);Hoechst 33258凋亡染色结果显示,与单药组相比,联用组A549细胞和PC-9细胞各给药组细胞出现明显核分叶、碎裂、凋亡样小体和核大小不一等典型凋亡样特征。与相应浓度GEF和ERG单药组相比,ERG+GEF联用(10+10)μmol·L-1和(40+40)μmol·L-1组A549细胞凋亡率升高(P<0.01),ERG+GEF(40+5)μmol·L-1和(80+10)μmol·L-1组PC-9细胞凋亡率显著升高(P<0.01);两药联用组(20+20)μmol·L-1A549细胞的p-EGFR/EGFR蛋白比值显著降低(P<0.01),两药联用组(40+5)μmol·L-1PC-9细胞的p-Akt/Akt和p-EGFR/EGFR蛋白比值显著下调(P<0.05,P<0.01);细胞周期结果显示,对于A549细胞,两药联用改变了G0/G1,G2/M和S期细胞比例,主要将细胞阻滞在G0/G1期。对于PC-9细胞,两药联用未改变G0/G1,G2/M和S期细胞比例。结论 ERG与GEF联用具有协同抑制A549和PC-9细胞增殖的作用,诱导细胞凋亡,其作用机制可能是抑制EGFR信号通路相关蛋白。

Co-administration of ergosterol and gefitinib inhibits proliferation of non-small cell lung cancer A549 and PC-9 cells

OBJECTIVE To study the inhibitory effect of ergosterol(ERG)combined with gefitinib(GEF)on non-small cell lung cancer A549(GEF-resistant cells)and PC-9(GEF-sensitive cells)cells.METHODS Human lung cancer cells A549 and PC-9 cells were divided into control group,ERG 5-160μmol·L-1 single drug group,GEF 5-160μmol·L-1 or 0.625-20μmol·L-1 single drug group and different concentrations of ERG+GEF combined drug groups.MTT method was used for in vitro proliferation inhibition test.Hoechst 33258 was used to observe the changes in nuclear morphology.Flow cytometry was used to determine the apoptosis rate and cell cycle,while Western blotting was used to detect the expressions of Akt,p-Akt,epiodermal grouth factor receptor(EGFR)and p-EGFR proteins.RESULTS At different time points,compared with the ERG and GEF single drug groups,the corresponding concentra?tion of the two-drug combination groups increased the proliferation inhibitory rate of A549 and PC-9 cells(P<0.01),showing a significant synergistic effect(q>1.15).The results of Hoechst 33258 apoptosis staining showed that A549 cells and PC-9 cells in the combination groups showed typical apoptotic characteristics,such as obvious nuclear lobes,fragmentation,apoptotic bodies,and different nuclear sizes.The apoptosis rate of A549 cells in ERG+GEF(10+10)μmol·L-1 and(40+40)μmol·L-1 groups was higher than that in the corresponding concentration of GEF and ERG single drug groups(P<0.01).And the apoptosis rate of PC-9 cells in ERG+GEF(40+5)μmol·L-1 and(80+10)μmol·L-1 groups was significantly higher(P<0.01).The p-EGFR/EGFR protein expression ratio of A549 cells in the two-drug combination groups(20+20)μmol·L-1 was significantly reduced(P<0.01).The p-Akt/Akt and p-EGFR/EGFR protein expression ratios of PC-9 cells(40+5)μmol·L-1 in the two-drug combination groups were significantly reduced(P<0.05,P<0.01).Cell cycle results showed that for A549 cells,the combination of the two drugs changed the ratio of G0/G1,G2/M,and S phase cells,mainly blocking the cells in G0/G1 phase.For PC-9 cells,the combination of the two drugs did not change the ratio of G0/G1,G2/M,or S phase cells.CONCLUSION The combination of ERG or GEF synergistically inhibits the proliferation of A549 and PC-9 cells and induces apoptosis,its mechanism of action may be related to the inhibition of EGFR signaling pathway related proteins.