米哚妥林对P糖蛋白介导的多药耐药的逆转作用

目的评价新型激酶抑制剂类抗癌药米哚妥林逆转P糖蛋白(P-gp)介导肿瘤细胞多药耐药的作用,并探讨其可能机制。方法米哚妥林0.5,1和5μmol·L-1分别加入P-gp高表达的K562/A02和K562细胞中培养72 h,MTS法测定细胞存活率以检测细胞毒性。米哚妥林0.125,0.25和0.5μmol·L-1在K562/A02和K562细胞中与无毒剂量的P-gp底物多柔比星、紫杉醇或长春新碱共培养72 h,MTS法测定细胞存活率以检测逆转耐药作用。米哚妥林0.5和10μmol·L-1与P-gp荧光底物罗丹明123在耐药和敏感细胞中共同孵育30 min后用流式细胞术分析底物积累的变化。米哚妥林0.5μmol·L-1与耐药和敏感细胞共同孵育72 h,Western印迹法检测耐药蛋白和信号分子的表达,定量PCR检测MDR1基因表达的变化。将米哚妥林与P-gp膜蛋白共孵育,化学发光法测定剩余ATP的量,检测米哚妥林对P-gp ATP酶活性的影响。结果米哚妥林对P-gp高表达的K562/A02和亲本敏感细胞K562的细胞毒性无明显的差异,其中米哚妥林0.5μmol·L-1时2种细胞的存活率均达80%以上。米哚妥林0.5μmol·L-1在细胞水平即可有效逆转K562/A02对多种底物的耐药,而对敏感细胞无显著作用;米哚妥林能显著抑制P-gp的外排作用,增加底物Rh-123在K562/A02细胞中的积累,且效果好于阴性对照维拉帕米0.5和10μmol·L-1;米哚妥林对P-gp基因和蛋白表达以及AKT和ERK的表达与磷酸化水平均无影响;米哚妥林对P-gp的ATP酶活性具有明显的抑制作用。结论米哚妥林可抑制P-gp介导的药物外排并逆转P-gp介导的肿瘤多药耐药。

Reversal effect of midostaurin on P-glycoprotein-mediated multidrug resistance

OBJECTIVE To determine the potency of midostaurin to sensitize P-glycoprotein(P-gp)-mediated multidrug resistance. METHODS K562/A02 or K562 cells were treated with midostaurin 0.5, 1 and 5 μmol·L^-1 for 72 h, and determined by MTS assay to evaluate the cytotoxicity effect of midostaurin in sensitive and resistant cells. The effect of inhibitors on drug resistance was accessed by exposing cells to a low concentration (≤IC₁₀) of anticancer drugs such as doxorubicin, vincristine and paclitaxel in the absence or presence of midostaurin 0.25 and 0.5 μmol·L^-1 followed by MTS assay. Rh-123 accumulation was detected by FACS in K562/A02 or K562 cells following 30 min incubation in the absence or presence of midostaurin or verapamil 0.5 and 10 μmol·L^-1. The effect of midostaurin on the protein and mRNA expression level was determined separately by Western blotting and qPCR. Midostaurin was firstly incubated with P-gp rich crude membranes and MgATP at 37℃ for 40 min before luminescence was initiated by ATP detection buffer, and then the remaining ATP was detected after incubation at room temperature for 40 min. RESULTS Midostaurin showed equal cytotoxicity in both K562/A02 and K562 cells, and the cell survival rate reached 80% after midostaurin 0.5 μmol·L^-1 for 72 h. Midostaurin 0.5 μmol·L^-1 significantly potentiated the cytotoxicity of doxorubicin, paclitaxel and vincristine to K562/A02 rather than to P-gp negative parental cell K562. Midostaurin 0.5 and 10 μmol·L^-1 remarkably increased the accumulation of Rh-123 in K562/A02 cells and had better effect than verapamil under the same condition, but had no effect on sensitive cells. However, the ATPase activity assay showed that P-gp-mediated ATP hydrolysis could be inhibited by midostaurin. Midostaurin 0.5 μmol·L^-1 for 72 h did not influence the expression of P-gp at the protein and mRNA level, and had little impact on the total and phosphorylated forms of AKT and ERK1/2. CONCLUSION Midostaurin reverses multidrug resistance by inhibiting the efflux function of P-gp, unlikely to be a substrate of P-gp.