Embelin逆转K562/D细胞对柔红霉素耐药与P-gp及MDR1 mRNA无关  

Embelin Reverses the Multi-drug Resistance of K562/D to Daunorubicin Independently of P-gp and MDR1 mRNA

作　　者：蒋卉男[1] 刘卓刚[1] 胡荣[1] 

机构地区：[1]中国医科大学附属盛京医院血液科,辽宁沈阳110020

出　　处：《中国实验血液学杂志》2017年第5期1342-1349,共8页Journal of Experimental Hematology

摘　　要：目的:探讨XIAP抑制剂Embelin逆转K562/D细胞对柔红霉素(daunorubicin,DNR)耐药的机制以及与P-gp及MDR1 mRNA的关系。方法:应用MTT法检测不同浓度DNR及联合Embelin对K562及K562/D细胞增殖抑制情况的影响;应用Annexin V-FITC/PI复染流式细胞术检测细胞凋亡的改变;用Western blot方法检测XIAP、Caspase-3、P-gp、BCL-2及BAX的蛋白表达情况;用RT-PCR检测XIAP、MDR1、BCL-2及BAX的mRNA表达情况。结果:DNR作用于K562与K562/D细胞系24 h的IC_(50)值分别为2.177μg/ml和69.43μg/ml,耐药指数为31.89;分别用浓度为3、10、30、100及300μg/ml的Embelin作用于K562/D细胞系24 h,其增殖抑制率分别为2.70%±1.08%、10.92%±4.89%、28.13%±2.09%、36.56%±3.24%及43.59%±1.16%;用0.1、1、10及100μg/ml DNR联合10μg/ml Embelin作用于K562/D细胞系24 h,细胞增殖抑制率分别为31.92%±3.29%、49.57%±6.87%、55.16%±0.78%和71.94%±3.89%,其IC_(50)值为2.11μg/ml,逆转指数为32.91。用Annexin V-FITC/PI双染流式细胞术检测凋亡率。单独应用0.1μg/ml DNR作用于K562/D细胞系24 h的细胞凋亡率为12.06%±0.95%,而联合10和30μg/ml Embelin作用于K562/D细胞系24 h的细胞凋亡率分别为27.54%±0.59%和39.59%±1.57%;Western Blot结果显示,DNR联合Embelin后,Caspase-3的表达明显下降(P<0.05),且用抑制剂Z-VADFMK可以抵消药物联合应用对细胞的增殖抑制作用。同时,XIAP和BCL-2蛋白表达明显下降(P<0.05),BAX蛋白表达明显升高(P<0.05),而P-gp的表达无改变;RT-PCR结果显示,DNR联合Embelin后,XIAP和BCL-2 mRNA表达明显下调(P<0.05),BAX mRNA表达明显上调(P<0.05),MDR1 mRNA表达无明显变化(P>0.05)。结论:降低XIAP表达有助于增强DNR对K562/D细胞的作用;Embelin逆转K562/D细胞对DNR耐药机制与P-gp及MDR1 mRNA无关。Objective： To investigate the mechanism of reversing drug resistance of K562/D cells to daunorubicin by Embelin and its relationship with P-gp and MDR1 mRNA. Methods： MTT assay was used to detect and compare the cell proliferation rate of treating with DNR alone and DNR combined with Embelin. Flow cytometry with Annexin V-FITC/PI double staining was used to detect cell apoptosis rate,Western blot was used to detect the expression of XIAP,Caspase-3,BCL-2,BAX and P-gp of K562/D cells after using DNR alone and combining with Embelin. Quantitative real-time PCR was used to detect XIAP,BCL-2,BAX and MDR1 mRNA. Results： The IC50 of K562 and K562/D cells treated with DNR for 24 h were 2. 177 μg/ml and 69. 43 μg/ml,respectively. The drug-resistance index was 31. 89;The proliferation inhibition rates of K562/D cells treated with Embelin of 3,10,30,100 and 300 μg/ml for 24 h were2. 70% ± 1. 08%,10. 92% ± 4. 89%,28. 13% ± 2. 09%,36. 56% ± 3. 24% and 43. 59% ± 1. 16%; The proliferation inhibition rates of K562/D cells treated with DNR of 0. 1,1,10 and 100 μg/ml combined with 10 μg/ml Embelin for24 h were 31. 92% ± 3. 29%,49. 57% ± 6. 87%,55. 16% ± 0. 78% and 71. 94% ± 3. 89%. The IC_（50） was 2. 11 μg/ml respectively. The reverse index was 32. 91. The apoptosis rates of K562/D cells treated with 0. 1 μg/ml DNR alone or combined with Embelin of 10 μg/ml and 30 μg/ml for 24 h were 12. 06% ± 0. 95%,27. 54% ± 0. 59% and 39. 59% ±1. 57%,respectively. The results of Western blot showed that after combination of DNR with Embelin,the expression of Caspase-3 was significantly down-regulated（ P〈0. 05）,moreover,the prolifiration inhibition effect of drug combination on cells could be countreacted by Z-VAD-FMK,at the same time the expression of XIAP and BCL-2 protein was significantly down-regulated（P〈0. 05）,the expression of BAX protein was significantly up-regulated（P〈0. 05）,while there was no change of P-gp expression later（ P〈0. 05）. The results of RT-PCR showed that after combin

关 键 词：EMBELIN K562/D细胞 柔红霉素 P-GP MDR1 MRNA 

分 类 号：R733.72[医药卫生—肿瘤] R979.1[医药卫生—临床医学]