伊马替尼联合依维莫司对小鼠垂体瘤细胞AtT-20的作用研究

Effects of glivec combined with everolimus on mouse pituitary AtT-20 cells

目的垂体瘤药物治疗目前主要针对泌乳素腺瘤和生长激素腺瘤,对于其他类型垂体瘤疗效差,因此新的治疗药物亟待发现。文中探讨伊马替尼联合依维莫司对小鼠垂体瘤细胞AtT-20的作用效果及分子作用机制。方法单独使用伊马替尼、依维莫司以及联合作用于AtT-20细胞。采用细胞增殖-毒性检测试剂盒(CCK-8)检测细胞的增殖抑制,实时荧光定量PCR检测细胞丝氨酸苏氨酸蛋白激酶(AKT)和细胞外调节蛋白激酶(ERK)的mRNA表达水平,Western blot检测AKT和ERK蛋白的磷酸化情况。结果伊马替尼与依维莫司均呈浓度和时间依赖性抑制At T-20细胞增殖,但两者联合作用效果为相互拮抗[联合指数CI分别为(1.13±0.06)、(1.12±0.03)和(1.07±0.03)];单独和联合用药对AKT、ERK的mRNA及蛋白表达水平无明显抑制作用(P>0.05);伊马替尼和依维莫司均可上调AKT、ERK的磷酸化水平,联合用药对p-AKT、p-ERK水平的促进作用明显强于单药(P<0.05)。结论伊马替尼和依维莫司对AtT-20细胞均有抑制作用,但联合用药表现出了拮抗效应。推测其机制为伊马替尼与依维莫司通过阻断信号通路抑制细胞增殖时均负反馈活化AKT、ERK,从而在联合用药时相互拮抗。

Objective Medication for pituitary adenomas is mainly targeted on the prolactin-secreting and growth-hormone types and shows poor therapeutic effects on other adenomas.Therefore,new drugs urgently need to be developed for this purpose.This study was to investigate the effects of glivec and everolimus on mouse pituitary AtT-20 cells and their molecular mechanisms in vitro.Methods Mouse pituitary AtT-20 cells were incubated with glivec or everolimus or combination of both and their inhibitory effect on the proliferation of the cells was measured by CCK-8 assay.The mRNA levels of AKT and ERK were determined by q-PCR and the expressions of the phosphorylated AKT（p-AKT） and ERK（p-ERK） were detected by Western blot.Results Used alone,both glivec and everolimus inhibited the proliferation of the AtT-20 cells in a time-and dose-dependent manner,but their combination produced a mutually antagonistic effect,with combination index values of 1.13 ± 0.06,1.12 ± 0.03,and 1.07 ± 0.03 respectively.The two agents,either used alone or in combination,induced no significantly inhibitory effects on the mRNA and protein expressions of AKT and ERK（P〈0.05）.Both glivec and everolimus up-regulated the expressions of p-AKT and p-ERK,and their combination manifested an even stronger effect（P〈0.05）.Conclusion Both glivec and everolimus inhibit the proliferation of AtT-20 cells when administered alone,but their combination produces an antagonistic effect.Their action mechanism might be that when targeting some signaling pathways to inhibit cell proliferation,glivec,as well as everolimus,induces a feedback activation of AKT and ERK.