Jiang Hui-Nan, Liu Zhuo-Gang, Hu Rong
Department of Hematology, Affiliated Shengjing Hospital, China Medical University, Shenyang 110020,Liaoning Province,China.
Department of Hematology, Affiliated Shengjing Hospital, China Medical University, Shenyang 110020,Liaoning Province,China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2017 Oct;25(5):1342-1349. doi: 10.7534/j.issn.1009-2137.2017.05.011.
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.
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.
The IC 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 were 2.70%±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 for 24 h were 31.92%±3.29%, 49.57%±6.87%, 55.16%±0.78% and 71.94%±3.89%. The IC 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 combination of DNR with Embelin, expression of XIAP and BCL-2 mRNA was significantly down-regulated(P<0.05), expression of BAX mRNA was significantly up-regulated(P<0.05), while there was no obvious change of MDR1 mRNA expression(P>0.05).
The down-regulation of XIAP contributes to enhance the effect of DNR on K562/D cells, the mechanism of Embelin-reversing the drug-resistence of K562/D cells to DNR does not relate with P-gp and MDR1 mRNA.
探讨紫铆因逆转K562/D细胞对柔红霉素耐药的机制及其与P-糖蛋白（P-gp）和多药耐药基因1（MDR1）mRNA的关系。
采用MTT法检测并比较单独使用柔红霉素（DNR）及DNR联合紫铆因处理后的细胞增殖率。采用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率，蛋白质免疫印迹法（Western blot）检测单独使用DNR及联合紫铆因处理后K562/D细胞中X连锁凋亡抑制蛋白（XIAP）、半胱天冬酶-3（Caspase-3）、B细胞淋巴瘤/白血病-2（BCL-2）、Bcl-2相关X蛋白（BAX）和P-gp的表达。采用实时定量聚合酶链反应（qRT-PCR）检测XIAP、BCL-2、BAX和MDR1 mRNA。
DNR处理24 h后，K562和K562/D细胞的半数抑制浓度（IC）分别为2.177 μg/ml和69.43 μg/ml，耐药指数为31.89；3、10、30、100和300 μg/ml紫铆因处理K562/D细胞24 h的增殖抑制率分别为2.70%±1.08%、10.92%±4.89%、28.13%±2.09%、36.56%±3.24%和43.59%±1.16%；0.1、1、10和100 μg/ml DNR联合10 μg/ml紫铆因处理K562/D细胞24 h的增殖抑制率分别为31.92%±3.29%、49.57%±6.87%、55.16%±0.78%和71.94%±3.89%，IC分别为2.11 μg/ml，逆转指数为32.91。0.1 μg/ml DNR单独处理及联合10 μg/ml和30 μg/ml紫铆因处理K562/D细胞24 h的凋亡率分别为12.06%±0.95%、27.54%±0.59%和39.59%±1.57%。Western blot结果显示，DNR与紫铆因联合作用后，Caspase-3表达明显下调（P<0.05），且Z-VAD-FMK可拮抗药物联合对细胞的增殖抑制作用，同时XIAP和BCL-2蛋白表达明显下调（P<0.05），BAX蛋白表达明显上调（P<0.05），而P-gp表达无明显变化（P>0.05）。RT-PCR结果显示，DNR与紫铆因联合作用后，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），而MDR1 mRNA表达无明显变化（P>0.05）。
XIAP表达下调有助于增强DNR对K562/D细胞的作用，紫铆因逆转K562/D细胞对DNR耐药的机制与P-gp和MDR1 mRNA无关。
