姜黄素衍生物C15对白血病K562/A02细胞多药耐药的逆转作用

Reversal effects of curcumin derivative C15 on multidrug resistance of leukemia K562/A02 cells

目的:探讨姜黄素衍生物C15对人白血病K562/A02细胞多药耐药(multidrug resistance,MOR)的逆转作用及其作用机制。方法:四甲基偶氮唑蓝(MTT)法检测细胞增殖;流式细胞术检测P-糖蛋白(P-gp)外排泵功能和细胞周期;免疫印迹法检测蛋白表达;P-gp-GloTM Assay System试剂盒检测P-gp ATP水解酶(ATPase)活性。结果:C15对K562/A02细胞半数抑制浓度(IC50)大于50μmol·L-1。对K562/A02细胞无明显细胞毒的浓度为2.5,5.0,10.0μmol·L-1的C15逆转对K562/A02细胞对阿霉素(ADR)耐药的倍数分别为2.60,5.39,11.39,对长春新碱(vincristine,VCR)耐药的倍数分别为4.50,18.07,124.35,但是对非P-gp底物的化疗药物顺铂(cisplatin,CIS)和敏感细胞K562基本无逆转效果。2.5,5.0,10.0μmol·L-1 C15可以增加耐药细胞K562/A02胞内罗丹明123(Rh-123)的蓄积量分别为1.93,2.30,2.47倍。C15增加阿霉素(adriamycin,ADR)在K562/A02细胞中的蓄积水平,降低P-gp介导的Rh-123外排速率。2.5,10.0μmol·L-1 C15与300 nmol·L-1 VCR联合作用后,可使K562/A02细胞的G2/M期比例从9.36%增加到67.57%和69.38%。C15对P-gp蛋白和ATPase的活性没有抑制作用。结论:C15可能是P-gp的非衣物型抑制剂,且具有逆转K562/A02细胞MDR的作用,该作用与其抑制细胞P-gp的外排泵功能有关。

OBJECTIVE To investigate reversal effects of curcumin derivative C15 on multidrug resistance（MDR）in K562/A02 cells,and explore underlying mechanisms.METHODS Cell proliferation was detected by MTT method.P-gp function and cell cycle were measured by flow cytometry.Expression of proteins was detected by Western blot.Activity of P-gp ATPase was measured by P-gp-GloTM Assay System kit.RESULTS IC50 values of C15 on K562/A02 cells were greater than 50mol·L^-1.Compared with control,reversal fold of multidrug resistance to ADR or VCR were 2.60,5.39 and 11.39 folds or4.50,18.07 and 124.35 folds by 2.5,5.0and 10.0mol·L^-1 C15 in K562/A02 cells,respectively.However,it almost had no effect on non-P-gp substrate cisplatin（CIS）or its sensitive K562 cells.The accumulation of Rh-123 by 2.5,5.0and 10.0mol·L^-1 C15 were 1.93,2.30 and 2.47 folds when compared with control.In addition,C15 could increase accumulation of ADR and decreased P-gp-mediated efflux rate of Rh-123.2.5 and 10 mol·L^-1 C15 arrested G2/M phase of K562/A02 from9.36%to 67.57% and 69.38% when jointly treated with 300 nmol·L^-1 VCR.C15 had no inhibitive effects on expression of P-gp at protein level and on hydrolysis activity of ATPase of P-gp.CONCLUSION C15 may be a run-substrate inhibitor of P-gp,and hare the reversal effect on MDR can reverse multidrug resistance in K562/A02 cells,which is related with inhibition of efflux function of P-gp.