姜黄素衍生物C15对白血病K562/A02细胞多药耐药的逆转作用

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-Glo^TM 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50 μmol·L^-1。对K562/A02细胞无明显细胞毒的浓度为2.5,5.0,10.0 μmol·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。2.5,5.0,10.0 μmol·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。2.5,10.0 μmol·L^-1 C15与300 nmol·L^-1VCR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。

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

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-Glo^TM Assay System kit.RESULTS IC₅₀ values of C15 on K562/A02 cells were greater than 50 mol·L^-1. Compared with control, reversal fold of multidrug resistance to ADR or VCR were 2.60, 5.39 and 11.39 folds or 4.50, 18.07 and 124.35 folds by 2.5, 5.0 and 10.0 mol·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, 5.0 and 10.0 mol·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 from 9.36% 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.