低氧诱导因子-1α抑制剂逆转K562/A02细胞多药耐药机制研究

Effect of hypoxia inducible factor1-α inhibitor on reversal of multidrug resistance of K562/A02 cell line

目的 探讨低氧诱导因子抑制剂3-（5＇-Hydroxymethyl-2＇-furyl）-1-benzylindazoh（YC-1）对人白血病耐阿霉素细胞K562/A02的耐药逆转作用,并探讨逆转机制.方法 采用MTT法检测不同浓度YC-1和阿霉素（ADM）单独或联合使用48 h对K562/A02细胞和K562细胞增值抑制效应及耐药逆转效应;用流式细胞术检测0、5、10和20 μmol/L YC-1单独或联合1 mg/L阿霉素作用K562/A02细胞48 h后细胞凋亡率及联合应用后细胞内阿霉素浓度;半定量RT-PCR检测各组细胞低氧诱导因子-1α（HIF-1α）、mdr1基因mRNA表达变化;Western blot方法检测各组细胞HIF-1α、P-糖蛋白（P-gp）表达变化.结果 K562与K562/A02细胞对阿霉素的IC50值分别为（1.56±0.07）mg/L和（42.98±3.15）mg/L,耐药倍数为27.55倍.予5、10和20μmol/L YC-1作用后,K562/A02细胞对阿霉素的耐药倍数分别为24.63、16.38和10.71倍;0、5、10和20μmol/L YC-1单独或联合1 mg/L阿霉素处理K562/A02细胞48 h后,凋亡率分别为（1.9±0.9）%、（4.9±0.9）%、（5.8±1.1）%和（9.3±1.4）%与（2.3±0.7）%、（8.2±1.2）%、（19.0±1.7）%和（34.5 ±2.4）%.0、5、10和20 μmol/L YC1联合1 mg/L阿霉素处理K562/A02细胞48 h后细胞内阿霉素荧光强度分别为232±33、1300±219、1961±240和3342±269;随着YC-1浓度增加,HIF-1α mRNA表达没有明显差异,mdr1 mRNA逐渐下调,HIF-1±和P-gp表达均下调.结论 YC-1可以通过抑制HIF-1α蛋白表达,下调mdr1 mRNA水平和P-gp水平,增加细胞内阿霉素药物浓度,部分逆转K562/A02细胞耐药.

Objective To study the reversal effect of the hypoxia inducible factor（ HIF）-1α inhibitor,YC-1 ,on muitidrug resistance of K562/A02 cells and its mechanism. Methods Pre- and post- incubation with adriamycin （ADM） alone or in combination with YC-1 for 48 h, the proliferation capacity of K562/A02 and K562 cells were evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treated with 0,5,10 and 20 μmol/L YC-1 alone or in combination with 1 mg/L ADM and intracellular ADM concentration were analyzed by flow cytometry（FCM）. The mRNA levels of HIF-1α and mdr1 genes were determined by semi-quantitative RT-PCR. The protein levels of HIF-1α and P-glycoprotein （P-gp） were detected by Western blot. Results The IC50 of ADM for K562 and K562/A02 cells were （ 1.56 ± 0.07 ） mg/L and （42.98 ±3.15） mg/L respectively. The resistance of K562/A02 cells to ADM was 27.55- fold higher of that of K562cells. After treatment with YC-1 （5μmol/L, 10μmol/L, 20 μmol/L） for 48h, the resistances of K562/A02cells to ADM were 24.63-, 16.38- and 10.71- fold increase respectively. After treatment of K562/A02 cell with YC-1（0 μmol/L, 5 μmol/L, 10 μmol/L, 20 μmoL/L） alone or in combination with 1 mg/L ADM for 48 h, the apoptotic rates were （ 1.9 ± 0. 9） %, （4.9 ± 0. 9 ） %, （ 5.8 ± 1.1 ） %, and （ 9.3 ± 1.4 ） % and（2.3 ± 0.7 ） %, （8.2 ± 1.2） %, （ 19.0 ± 1.7 ） %, and （ 34.5 ± 2.4 ） % respectively. The intracellular flucorescence intensity of ADM were 232 ±33, 1300 ±219, 1961 ±240 and 3342 ±269 in the combined treatment group. With the increase in YC-1 concentration, the levels of mdr1 mRNA reduced, while that ofHIF-1α mRNA had no obvious change.Furthermore.the expressions of HIF-1α and P-gp were also decreased in K562/A02 cells.Conclusion YC-1,as a HIF-1 inhibitor,cau reverse multidrug resistance of K562/A02cells through down-regulating HIF-1α and p-gp.