山奈酚逆转慢性粒细胞白血病K562/ADM细胞耐药性及相关机制

Kaempferol in reversing drug resistance of chronic myelogenous leukemia K562/ADM cells and its related mechanism

目的探讨山奈酚逆转耐多柔比星(ADM)的慢性粒细胞白血病K562/ADM细胞耐药性及相关机制。方法采用四甲基偶氮唑盐(MTT)法检测ADM作用24 h对K562和K562/ADM细胞的毒性,计算24 h ADM半数抑制浓度(IC50)及耐药倍数。MTT法检测山奈酚作用24 h对K562/ADM细胞的毒性,计算24 h山奈酚5%抑制浓度(IC5)、10%抑制浓度(IC10),以二者确定后续实验山奈酚两组浓度,未经山奈酚处理的细胞为空白对照组。采用MTT法检测空白对照组及山奈酚干预组ADM作用24 h的细胞抑制情况,计算各组24 h的细胞抑制情况,计算各组24 h ADM IC50,空白对照组与山奈酚组IC50的比值为山奈酚逆转耐药倍数。应用流式细胞术检测山奈酚作用后K562/ADM细胞内ADM荧光强度。采用蛋白质印迹法检测分别经山奈酚、p38-MAPK信号通路特异性抑制剂SB202190、山奈酚和SB202190联用后的K562/ADM细胞耐药蛋白[P糖蛋白(P-gp)、多药耐药相关蛋白1(MRP1)、磷酸化p38(p-p38)、总p38(t-p38)]表达情况。结果ADM处理24 h后,K562、K562/ADM细胞IC50值分别为(0.9±0.6)、(28.1±3.5)μg/ml;相对于K562细胞,K562/ADM细胞对ADM作用24 h的耐药倍数为31.16倍。MTT法检测结果显示,山奈酚呈剂量依赖性抑制K562/ADM细胞增殖,依据IC5和IC10,确定以0.5、1.0μmol/L山奈酚进行后续实验。山奈酚和ADM共同作用24 h后,空白对照组、0.5μmol/L山奈酚组、1.0μmol/L山奈酚组K562/ADM细胞24 h ADM IC50分别为(33.7±5.7)、(21.4±0.6)、(15.9±1.8)μg/ml(F=30.85,P<0.05;组内两两比较,均P<0.05),0.5μmol/L山奈酚组、1.0μmol/L山奈酚组K562/ADM细胞24 h逆转耐药倍数分别为1.58、2.12倍。流式细胞术检测示,空白对照组、0.5μmol/L山奈酚组、1.0μmol/L山奈酚组K562/ADM细胞内ADM平均荧光强度(MFI)分别为138.4±8.9、154.3±2.2、165.7±4.8,差异有统计学意义(F=161.48,P<0.05)。与空白对照组比较,0.5、1.0μmol/L山奈酚处理24 h后,K562/ADM细胞的P-gp、MRP1、p-p38蛋白相对表达量均降低(均P<0.05),而t-p38蛋白表达量差异无统计学意义(P>0.05);SB202190可降低P-gp、MRP1、p-p38蛋白的相对表达量(均P<0.05);SB202190联合不同浓度山奈酚作用后,K562/ADM细胞P-gp、MRP1、p-p38蛋白的相对表达量未下降(P>0.05)。结论山奈酚可能通过抑制p38-MAPK通路降低K562/ADM细胞P-gp、MRP1的相对表达量,实现增加细胞内ADM的蓄积,从而逆转K562/ADM细胞的耐药性。

Objective To investigate the drug resistance of kaempferol reversed adriamycin(ADM)-resistant K562/ADM cells in chronic myelogenous leukemia(CML)and its related mechanism.Methods Methyl thiazolyl tetrazolium(MTT)method was used to detect the toxicity of ADM on K562 and K562/ADM cells for 24 h.The half inhibitory concentration(IC50)of ADM and the drug resistance multiple for 24 h were calculated.MTT method was used to detect the toxicity of kaempferol on K562/ADM cells for 24 h.The 5%inhibitory concentration(IC5)and 10%inhibitory concentration(IC10)of kaempferol for 24 h were calculated to determine the concentration of kaempferol in the subsequent experiments.And the cells untreated by the kaempferol were selected as the control group.The cell inhibition after the treatment of ADM for 24 h of the blank control group and kaempferol intervention group was detected by using MTT method.And then the cell inhibition for 24 h and ADM IC50 for 24 h in the above groups were calculated.The ratio of IC50 in the blank control group and kaempferol group was the reversal drug resistance multiple of kaempferol.The fluorescence intensity of ADM in K562/ADM cells treated by kaempferol was detected by using flow cytometry.Western blotting was used to detect the expressions of P-glycoprotein(P-gp),multidrug resistance-associated protein 1(MRP1),phosphorylated p38(p-p38),and total p38(t-p38)protein in K562/ADM cells after the treatment of kaempferol,the specific inhibitor of p38-MAPK signaling pathway SB202190,and the combination of kaempferol and SB202190.Results After the treatment of ADM for 24 h,the IC50 value of K562 and K562/ADM cells was(0.9±0.6),(28.1±3.5)μg/ml,respectively.The drug resistance multiple of K562/ADM cells on the treatment of ADM for 24 h was 31.16 compared with the K562 cells.MTT method showed that kaempferol inhibited the proliferation of K562/ADM cells in a dose-dependent manner.According to the IC5 and IC10,0.5μmol/L and 1.0μmol/L kaempferol were determined to do the subsequent experiments.After the combined interaction of kaempferol and ADM for 24 h,the ADM IC50 of K562/ADM cells in the blank control group,0.5μmol/L kaempferol group and 1.0μmol/L kaempferol group was(33.7±5.7),(21.4±0.6),(15.9±1.8)μg/ml,respectively(F=30.85,P<0.05),and there was a statistical difference of pairwise comparison(both P<0.05).The reversal drug resistance multiple of K562/ADM cells for 24 h in 0.5μmol/L kaempferol group and 1.0μmol/L kaempferol group was 1.58 and 2.12,respectively.Flow cytometry results showed that the mean fluorescence intensity(MFI)of ADM in the blank control group,0.5μmol/L kaempferol group and 1.0μmol/L kaempferol group was 138.4±8.9,154.3±2.2,165.7±4.8,respectively,and the difference was statistically significant(F=161.48,P<0.05).Compared with the blank control group,after treatment of K562/ADM cells with 0.5μmol/L and 1.0μmol/L kaempferol for 24 h,the relative expressions of P-gp,MRP1 and p-p38 protein were decreased in K562/ADM cells(all P<0.05),but there was no statistical difference in the expression of t-p38 protein(P>0.05);SB202190 could reduce the relative expressions of P-gp,MRP1 and p-p38 protein(all P<0.05);after the treatment of SB202190 combined with different concentration of kaempferol,the relative expressions of P-gp,MRP1 and p-p38 protein in K562/ADM cells did not decrease(P>0.05).Conclusions Kaempferol can decrease the relative expressions of P-gp and MRP1 in K562/ADM cells by inhibiting p38-MAPK pathway,so as to increase the concentrations of ADM and to reverse the drug resistance of K562/ADM cells.