伊曲康唑联合阿霉素对急性髓系白血病细胞增殖和凋亡的影响

[Effects of itraconazole plus doxorubicin on proliferation and apoptosis in acute myeloid leukemia cells].

作者信息

Wang Jing, Xu Xiaojun, Zhou Ruiqing, Guo Kunyun

机构信息

Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.

Department of Hematology, Zhongshan Hospital, Sun Yat-sen University, Zhongshan 528403, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2015 Jan 27;95(4):299-305.

PMID:25877249

Abstract

OBJECTIVE

To explore the effects of itraconazole (ITC) plus adriamycin (ADM) on proliferation and apoptosis of acute myeloid leukemia cells in vitro.

METHODS

The growth inhibition effects of different concentrations of ITC, ADM or ITC (2, 6, 15 µmol/L) plus ADM (0.30, 0.75 µmol/L) were detected by CCK8 assay in KG1α and primary adult acute leukemia cells. Different concentrations of ITC for 7, 14 and 21 days were applied for observing the effect on colony formation. After 48 h treatments with 6 µmol/L ITC, 0.75 µmol/L ADM or ITC (6 µmol/L) plus ADM (0.75 µmol/L), the morphological changes of cells were observed by Wright staining. Flow cytometry was used to detect cell apoptotic rate, mitochondrial membrane potential and cell cycle arrest. And the expression levels of Sonic Hedgehog (Shh) signal pathway-related proteins Shh and glima-associated oncogene homdog1 (Gli1) were determined by Western blot.

RESULTS

ITC and ADM inhibited the proliferation of KG1α and primary adult acute leukemia cells and ITC reduced the colony-formation ability of KG1α cells both in dose-dependent manners. Compared with control or single drug group, the changes of cell morphology were more apparent in combined group. Weeb coefficient test revealed a synergistic effect (D ≤ 70%C) of 0.75 µmol/L ADM plus 6 µmol/L ITC. When KG1α cells were treated with 6 µmol/L ITC plus 0.75 µmol/L ADM, the apoptotic rate was 31.72% ± 1.58%. And it was significantly higher than that in control, ITC and ADM groups (4.17% ± 0.74%, 4.33% ± 1.12%, 9.53% ± 1.15%, P < 0.01). Detection of mitochondrial membrane potential showed that low red-fluorescent cells (P3) of combined group were obviously higher than that in control, ADM and ITC single drug groups (18.80% ± 0.96% vs 5.00% ± 0.38%, 9.70% ± 0.43%, 7.10% ± 0.77%, P < 0.01) . KG1α cells were obviously arrested in G2 phase in combined group and it showed no statistical significance compared with control or single drug group. Western blot showed that the expression levels of Shh and Gli1 decreased with rising concentration of ITC.

CONCLUSIONS

ITC plus ADM can obviously inhibit cell proliferation and increase KG1α cell apoptotic rate, mitochondrial damage and G2 phase retardation. And ITC inhibits the Shh signal pathways.

摘要

目的

探讨伊曲康唑（ITC）联合阿霉素（ADM）对急性髓系白血病细胞体外增殖及凋亡的影响。

方法

采用CCK8法检测不同浓度的ITC、ADM或ITC（2、6、15μmol/L）联合ADM（0.30、0.75μmol/L）对KG1α细胞和原代成人急性白血病细胞的生长抑制作用。应用不同浓度的ITC处理7、14和21天，观察其对集落形成的影响。用6μmol/L ITC、0.75μmol/L ADM或ITC（6μmol/L）联合ADM（0.75μmol/L）处理细胞48小时后，通过瑞氏染色观察细胞形态变化。采用流式细胞术检测细胞凋亡率、线粒体膜电位及细胞周期阻滞情况。通过蛋白质免疫印迹法检测音猬因子（Shh）信号通路相关蛋白Shh和胶质瘤相关癌基因同源物1（Gli1）的表达水平。

结果

ITC和ADM均呈剂量依赖性抑制KG1α细胞和原代成人急性白血病细胞的增殖，ITC还降低了KG1α细胞的集落形成能力。与对照组或单药组相比，联合组细胞形态变化更明显。Weeb系数检验显示0.75μmol/L ADM加6μmol/L ITC具有协同作用（D≤70%C）。当KG1α细胞用6μmol/L ITC加0.75μmol/L ADM处理时，凋亡率为31.72%±1.58%，显著高于对照组、ITC组和ADM组（4.17%±0.74%、4.33%±1.12%、9.53%±1.15%，P<0.01）。线粒体膜电位检测显示，联合组低红色荧光细胞（P3）明显高于对照组、ADM单药组和ITC单药组（18.80%±0.96%对5.00%±0.38%、9.70%±0.43%、7.10%±0.77%，P<0.01）。联合组KG1α细胞明显阻滞于G2期，但与对照组或单药组相比无统计学意义。蛋白质免疫印迹法显示，Shh和Gli1的表达水平随ITC浓度升高而降低。