联合应用尼洛替尼和汉防己甲素逆转K562/A02细胞耐药与诱导凋亡的研究

Inducing Apoptosis and Reversal Effect of Nilotinib in Combination with Tetrandrine on Multidrug Resistance of K562/A02 Cell Line

本研究旨在探讨联合应用尼洛替尼(nilotinib)和汉防己甲素(tetrandrine,Tet)逆转K562/A02细胞耐药与诱导凋亡的相关性及其机制。应用MTT法检测细胞敏感性,计算尼洛替尼和Tet干预后的柔红霉素(DNR)的半数抑制浓度(IC50),用流式细胞术(FCM)检测细胞凋亡率,RT-PCR检测bax、survivin mRNA的表达及Western blot分析BAX、SURVIVIN蛋白的表达情况。结果表明:5 nmol/L尼洛替尼或1.0μml/L Tet单独作用48小时后,DNR对K562/A02细胞的IC50分别为(5.71±0.72)mg/L和(6.52±0.43)mg/L,两药联合应用时DNR对K562/A02细胞的IC50降为(3.12±0.13)mg/L。单用尼洛替尼或Tet均能增加经DNR作用的K562/A02细胞的凋亡率,两药联合作用时凋亡率增高更明显。单用5 nmol/L尼洛替尼或1.0μml/L Tet 48小时后可上调bax mRNA及其蛋白的表达,且两药联合作用时上调幅度明显。单用5 nmol/L尼洛替尼或1.0μml/LTet48小时后可下调survivin mRNA及其蛋白表达水平,且两药联合作用时下调幅度明显。结论:K562/A02细胞对DNR耐药,单独应用尼洛替尼和Tet均可部分逆转K562/A02细胞对DNR的耐药,并可增加K562/A02细胞的凋亡率,两药联用具有明显的协同作用,其机制可能与上调bax mRNA及其蛋白的表达和下调survivin mRNA及其蛋白的表达有关。

This study was aimed to investigate the relevance of nilotinib in combination with tetrandrine（Tet） on reversing multidrug resistance and inducing apoptosis of K562/A02 cell line and its mechanism.Methyl-thiazol tetrazolium（MTT） assay was employed to examine the pharmacological effect of nilotinib or Tet alone on K562/A02 cell line,the IC50 of daunorubicin（DNR） on K562/A02 cell line treated with nilotinib and Tet was calculated;the flow cytometry（FCM） was employed to detect the apoptosis rate of K562/A02.The expression of bax/survivin mRNA was determined by RT-PCR,and the expression of bax/survivin protein was assayed by Western blot.The results showed that after being treated by 5 nmol/L nilotinib or 1.0 μml/L Tet for 48 hours,IC50 of DNR to K562/A02 was 5.71±0.72 mg/L or 6.52±0.43 mg/L,respectively,while in their combined treatment,IC50 decreased to 3.12±0.13 mg/L.Nilotinib or Tet alone could increase DNR-inducing apoptosis rate of K562/A02 cell,while the apoptosis rate of K562/A02 increased remarkably in combination treatment of nilotinib with Tet.After being treated with 5 nmol/L nilotinib or 1.0 μml/L Tet alone for 48 hours,the expressions of bax mRNA and BAX protein was up-regulated,while both effects were more obvious in combination treatment of nilotinib with Tet.Treatment with 5 nmol/L nilotinib or 1.0 μmol/L Tet alone for 48 hours down-regulated the expression of survivin mRNA and its protein,while treatment of nilotinib in combination with Tet had more significant effect on down-regulation of their expression.It is concluded that the K562/A02 cells are resistant to DNR,nilotinib or Tet alone both can partially reverse resistance of K562/A02 cells to DNR,increase the apoptosis rate of K562/A02 cells.Combination of nilotinib with Tet shows obvious synergistic action,mechanism of which may associate with up-regulation of bax mRNA and BAX protein expressions and down-regulation of survivin mRNA and its protein expressions.