BCR-ABL下游通路抑制剂对人慢性粒细胞白血病细胞的作用及BEZ235逆转伊马替尼耐药的研究

Effect of BCR-ABL downstream pathway inhibitors on human chronic myelogenous leukemia cells and BEZ235 reversing of imatinib resistance

目的探讨RAF抑制剂SB590885、JAK抑制剂AZD1480、PI3K-mTOR双靶点抑制剂BEZ235等BCR-ABL下游通路抑制剂体外对慢性粒细胞白血病(CML)细胞的作用及BEZ235对CML细胞增殖、凋亡及伊马替尼敏感性的影响。方法用不同浓度药物处理K562细胞,采用MTS法检测K562细胞的增殖抑制率,计算各药物48 h半数抑制浓度(IC50);采用流式细胞术Annexin V-FITC/PI双染色法检测细胞凋亡率,流式细胞术PI单染色法检测细胞周期。以不同浓度药物处理K562细胞、伊马替尼耐药的T315I突变的人CML KBM7R细胞、伊马替尼耐药的CML患者原代细胞,采用MTS法检测细胞增殖抑制率,分别计算各药物48 h的IC50。分别单用1.0μmol/L BEZ235、1.0μmol/L伊马替尼或两者联合处理KBM7R细胞或CML患者原代细胞,采用流式细胞术PI单染色法检测KBM7R细胞周期,流式细胞术Annexin V-FITC/PI双染色法检测CML患者原代细胞凋亡率,蛋白质印迹法检测KBM7R细胞p-AKT、cleaved Caspase-3、Cyclin D1蛋白的表达情况。结果SB590885、AZD1480、BEZ235均能抑制K562细胞的增殖,三者处理K562细胞48 h的IC50分别为(11.49±3.14)、(4.83±1.26)、(0.37±0.21)μmol/L。SB590885、AZD1480、BEZ235均能促进K562细胞的凋亡,与未经药物作用的对照组比较,细胞凋亡率均升高(均P<0.01)。SB590885和BEZ235诱导细胞G0/G1期阻滞(均P<0.05),AZD1480诱导细胞G2/M期阻滞(P<0.05)。BEZ235可抑制K562、KBM7R细胞以及患者原代细胞的增殖,其48 h的IC50分别为(0.37±0.21)、(0.43±0.27)、(0.49±0.24)μmol/L。与单用伊马替尼组相比,0.2μmol/L BEZ235与不同浓度伊马替尼联用可增加对K562、KBM7R细胞和患者原代细胞的增殖抑制,降低伊马替尼的IC50,其中,伊马替尼单用和联合BEZ235处理48 h后,K562细胞的伊马替尼IC50分别为(0.14±0.05)、(0.09±0.04)μmol/L(t=1.351,P=0.249),KBM7R细胞分别为(3.93±2.29)、(0.44±0.22)μmol/L(t=2.837,P=0.047),原代细胞分别为(3.12±1.53)、(0.39±0.23)μmol/L(t=3.925,P=0.042)。未经药物作用的对照组、单用1.0μmol/L BEZ235、单用1.0μmol/L伊马替尼及两药联合处理24 h后患者原代细胞凋亡率分别为(4.9±1.4)%、(13.1±3.2)%、(8.8±2.0)%、(40.6±6.0)%,差异有统计学意义(F=71.031,P<0.01)。与单用伊马替尼相比,BEZ235联合伊马替尼处理12 h的KBM7R细胞p-AKT、Cyclin D1蛋白的表达降低,cleaved Caspase-3蛋白的表达升高。结论BCR-ABL下游通路抑制剂可有效抑制K562细胞的增殖,促进细胞凋亡。BEZ235能够抑制K562细胞、伊马替尼耐药的T315I突变的KBM7R细胞以及伊马替尼耐药的CML患者原代细胞的增殖,促进细胞凋亡,与伊马替尼具有协同作用。

Objective To explore the effects of BCR-ABL downstream pathway inhibitors,such as RAF inhibitor SB590885,JAK inhibitor AZD1480,PI3K-mTOR double target inhibitor BEZ235 on chronic myelogenous leukemia(CML)cells,and the effect of BEZ235 on the proliferation,apoptosis of CML cells and the sensitivity of imatinib in vitro.Methods K562 cells were treated with different concentrations of the drugs.MTS method was applied to detect the proliferation inhibition rate of K562 cells,and 50%inhibitory concentration(IC50)of all drugs for 48 h was calculated.The cell apoptosis rate was tested by using flow cytometry with Annexin V-FITC/PI double staining.The cell cycle was tested by using flow cytometry with PI staining.K562 cells,imatinib-resistant and T315I-mutant human CML KBM7R cells and imatinib-resistant CML primary cells of patients were treated with different concentrations of the drugs.MTS method was used to test the proliferation inhibition of cells,and IC50 of all drugs for 48 h was evaluated.KBM7R cells or primary cells of CML patients were treated with 1.0μmol/L BEZ235,1.0μmol/L imatinib or the combination of both,respectively.Flow cytometry with PI staining was used to detect the cell cycle of KBM7R cells.Flow cytometry with Annexin V-FITC/PI double staining was used to detect the cell apoptosis rate in CML primary cells.The expressions of p-AKT,cleaved Caspase-3 and Cyclin D1 proteins were detected by using Western blot.Results SB590885,AZD1480 and BEZ235 could inhibit the proliferation of K562 cells,and the IC50 after the treatment of K562 cells for 48 h was(11.49±3.14),(4.83±1.26)and(0.37±0.21)μmol/L,respectively.SB590885,AZD1480 and BEZ235 could promote the apoptosis of K562 cells.The cell apoptosis rates were increased compared with the control group without drug treatment(all P<0.01).SB590885 and BEZ235 induced G0/G1 block(both P<0.05).AZD1480 induced G2/M block(P<0.05).BEZ235 could inhibit the proliferation of K562 cells,KBM7R cells and CML primary cells,and their IC50 for 48 h was(0.37±0.21),(0.43±0.27)and(0.49±0.24)μmol/L,respectively.Compared with imatinib alone,the different concentrations of imatinib combined with 0.2μmol/L BEZ235 could increase the proliferation inhibition of K562 cells,KBM7R cells and CML primary cells,and could reduce the IC50 of imatinib.After the treatment of imatinib alone and combination with BEZ235 for 48 h,the imatinib IC50 of K562 cells was(0.14±0.05)and(0.09±0.04)μmol/L(t=1.531,P=0.249),the imatinib IC50 of KBM7R cells was(3.93±2.29)and(0.44±0.22)μmol/L(t=2.837,P=0.047),the imatinib IC50 of the primary cells was(3.12±1.53)and(0.39±0.23)μmol/L(t=3.925,P=0.042).The cell apoptotic rate of the primary cells was(4.9±1.4)%,(13.1±3.2)%,(8.8±2.0)%and(40.6±6.0)%,respectively in the control group without drug treatment,1.0μmol/L BEZ235,1.0μmol/L imatinib and the combination of 1.0μmol/L BEZ235 and 1.0μmol/L imatinib after the treatment of 24 h(F=71.031,P<0.01).Compared with imatinib alone,the expressions of p-AKT and Cyclin D1 proteins were decreased,and the expression of cleaved Caspase-3 protein was increased after the treatment of KBM7R cells for 12 h in the combination group of both BEZ235 and imatinib.Conclusions BCR-ABL downstream pathway inhibitors can effectively inhibit the proliferation and promote the apoptosis of K562 cells.BEZ235 can inhibit the proliferation and promote the apoptosis of K562 cells,imatinib-resistant and T315I-mutant human KBM7R cells and imatinib-resistant CML primary cells of patients,which has a synergistic effect to imatinib.