桑皮苷A逆转K562/阿霉素耐药细胞化疗多药耐药的研究

Reversal effect of mulberroside A on the multidrug resistance in K562/adriamycin-resistant cells

目的研究桑皮苷A对人白血病耐药细胞K562/阿霉素耐药细胞(K562/ADM)化疗耐药的影响。方法实验分为溶媒(DMSO)对照组、阳性对照组(10μmol·L^(-1)维拉帕米处理48 h,再用5μg·mL^(-1)阿霉素孵育1 h或2μg·mL^(-1)罗丹明123孵育1.5 h)、阿霉素单用组(5μg·mL^(-1)阿霉素孵育1 h)、阿霉素合用桑皮苷A组(用5,10,20μmol·L^(-1)桑皮苷A处理48 h,再用5μg·mL^(-1)阿霉素孵育1 h)、罗丹明123合用桑皮苷A组(用5,10,20μmol·L^(-1)桑皮苷A处理48 h,再用2μg·mL^(-1)罗丹明123孵育1.5 h)。用噻唑蓝(MTT)实验初步确定桑皮苷A的逆转耐药作用;再以细胞内阿霉素蓄积实验,罗丹明123(Rho123)蓄积及外排实验进一步研究桑皮苷A的逆转耐药作用;分别用聚合酶链反应(PCR)和蛋白质印迹(Western blot)法检测桑皮苷A对K562/ADM细胞中P-糖蛋白(P-gp)mRNA、蛋白表达及磷脂酰肌醇3-激酶-蛋白激酶B-雷帕霉素靶蛋白(PI3K-Akt-mTOR)通路相关蛋白的影响。结果桑皮苷A能增加K562/ADM细胞对阿霉素的敏感性,桑皮苷A(5~20μmol·L^(-1))与阿霉素(5~160μg·mL^(-1))联合处理可明显降低耐药细胞对阿霉素的半数抑制浓度(IC50),逆转倍数分别为1.59,2.20和5.48倍。阿霉素胞内蓄积实验结果显示桑皮苷A能显著升高K562/ADM细胞中阿霉素浓度,其中10,20μmol·L^(-1)桑皮苷A可使胞内阿霉素平均荧光值升高2.3倍和4.3倍;同时,Rho123胞内蓄积和外排实验发现桑皮苷A不仅能增加细胞内Rho123的蓄积,还能减少Rho123的外排,其中10和20μmol·L^(-1)桑皮苷A可分别使胞内Rho123的平均荧光值增加至2.1倍和2.6倍。与对照组相比,桑皮苷A处理可浓度依赖性地降低细胞内P-gp的mRNA和蛋白表达水平,其中10与20μmol·L^(-1)桑皮苷A可分别使P-gp mRNA水平显著下降36.90%和65.40%。桑皮苷A能显著抑制K562/ADM细胞中p-PI3K、p-Akt、p-mTOR蛋白水平。结论桑皮苷A可通过抑制K562/ADM细胞中PI3K-Akt-mTOR通路,进而下调细胞内P-gp的表达水平,以达到提高肿瘤细胞对阿霉素的化疗敏感性和逆转细胞多药耐药的作用。

Objective To investigate the effect of Mulberroside A(Mul A)on drug resistance to chemotherapy in K562/adriamycin-resistant(K562/ADM)leukemia cells.Methods The experiment was divided into vehicle(DMSO)control group,positive control group(treated with 10μmol·L^(-1) verapamil for 48 h and then incubated for 1 h with 5μg·mL^(-1) adriamycin or incubated for 1.5 h with 2μg·mL^(-1) Rho123),adriamycin alone group(incubated for 1 h with 5μg·m L^(-1)adriamycin),adriamycin+Mul A group(treated with 5,10,20μmol·L^(-1)Mul A for 48 h and then incubated for 1 h with 5μg·m L^(-1)adriamycin)and Rho123+Mul A group(treated with 5,10,20μmol·L^(-1)Mul A for 48 h and then incubated for 1.5 h with 2μg·m L^(-1)Rho123).The methyl thiazolyl tetrazolium(MTT)assay was used to initially identify the reversal effect of Mul A on the MDR in adriamycin-resistant K562/ADM cells.In order to further evaluate the reversal effect of Mul A,the intracellular accumulation of adriamycin and Rhodamine 123(Rho123)were measured using multilabel counter.In addition,the efflux of Rho123 was also measured.Polymerase chain reaction(PCR)and Western Blot were used to study the expression of P-glycoprotein(P-gp)and the phosphatidylinositol 3 kinase-protein kinase B-mammalian target of rapamycin(PI3K-Akt-m TOR)pathway related proteins,respectively.Results Compared with the vehicle-treated group,Mul A can significantly increase the sensitivity of K562/ADM cells to adriamycin.Combination of Mul A(5-20μmol·L^(-1))and adriamycin(10-160μg·m L^(-1))can significantly reduce the IC50(50%inhibitory concentration)of drug-resistant cells to adriamycin.The reversal fold were 1.59,2.20 and 5.48 after treated with Mul A at 5,10 and 20μmol·L^(-1),respectively.Moreover,intracellular adriamycin-associated mean fluorescence intensity was increased 2.3 and 4.3 folds after exposure to Mul A at 10μmol·L^(-1)and 20μmol·L^(-1)compared with control group(P<0.05).A dose-dependent increase of intracellular Rho123 and decrease of Rho123 efflux were also observed in K562/ADM cells incubation with Mul A.Intracellular Rho123-associated mean fluorescence intensity was increased 2.1 and 2.6 folds after exposure to Mul A at 10μmol·L^(-1)and 20μmol·L^(-1)compared with the control group(P<0.05).In addition,Mul A could significantly inhibit the mRNA and protein expression of P-gp as well as the protein level of p-PI3K,p-Akt and p-m TOR.Compared with the control group,10μmol·L^(-1)and 20μmol·L^(-1)Mul A could significantly reduce the level of P-gp mRNA by 36.90%and 65.40%,respectively.The levels of p-PI3K,p-Akt and p-MTOR proteins in K562/ADM cells were significantly inhibited by Mul A.Conclusion Mul A can enhance the sensitivity of K562/ADM cells to chemotherapy and reverse its MDR,by means of inhibiting the PI3K-Akt-m TOR signaling pathway and the expression of P-gp.