Disulfram combined with copper induces leukemia stem cell apoptosis through TNF-α/ROS pathway

周勇; 钟文彬; 陈凯; 董慧娟; 闫道广; 周淑云; 徐兵

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Disulfram combined with copper induces leukemia stem cell apoptosis through TNF-α/ROS pathway

更新时间：2026-01-09

- Disulfram combined with copper induces leukemia stem cell apoptosis through TNF-α/ROS pathway
- Journal of Army Medical University Vol. 37, Issue 10, Pages: 984-989(2015)
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  CLC： R733.71;R966;R979.19
- Published：2015
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周勇,钟文彬,陈凯,董慧娟,闫道广,周淑云,徐兵.Disulfiram联合Cu通过上调TNF-α表达及蓄积ROS诱导白血病干细胞凋亡[J].陆军军医大学学报(原第三军医大学学报),2015,37(10):984-989.

　Zhou Yong,Zhong Wenbin,Chen Kai,Dong Huijuan,Yan Daoguang,Zhou Shuyun,Xu Bing.Disulfram combined with copper induces leukemia stem cell apoptosis through TNF-α/ROS pathway[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(10):984-989.
周勇,钟文彬,陈凯,董慧娟,闫道广,周淑云,徐兵.Disulfiram联合Cu通过上调TNF-α表达及蓄积ROS诱导白血病干细胞凋亡[J].陆军军医大学学报(原第三军医大学学报),2015,37(10):984-989. DOI：

　Zhou Yong,Zhong Wenbin,Chen Kai,Dong Huijuan,Yan Daoguang,Zhou Shuyun,Xu Bing.Disulfram combined with copper induces leukemia stem cell apoptosis through TNF-α/ROS pathway[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(10):984-989. DOI：

摘要

目的探讨Disulfiram联合Cu（DS/Cu）诱导急性髓系白血病（acute myeloblastic leukemia

AML)细胞凋亡及其相关分子机制。方法磁珠筛选CD34

CD38

的KG1α细胞作为AML干细胞，流式细胞术检测空白对照组、DS单药组（5 μmol/L）、DS/Cu组（5 μmol/L/0.5 μmol/L）、DS/Cu+NAC组（5 μmol/L/0.5 μmol/L/10 mmol/L）的AML干细胞24 h后凋亡比率及ROS水平；qRT-PCR检测上述各组TNF-α、CD40、TNFRSF11B、TNFRSF1B、HRK凋亡相关基因mRNA表达水平；Western blot检测各组处理后TNF-α的蛋白表达水平；予以中和抗体TNF-α mAb(2 μg/mL)和对照抗体IgG(2 μg/mL)预处理2 h后，流式细胞术检测空白对照组、DS/Cu组、TNF-α mAb组、TNF-α mAb+DS/Cu组、IgG组及IgG+DS/Cu组的AML干细胞24 h后的ROS水平。结果予以DS或DS/Cu处理CD34

CD38

KG1α细胞后，细胞凋亡率由空白对照组的(6.65±0.64)%分别上升至(11.87±1.30)%、(27.43±1.65)%，差异均具有显著性统计学意义（P=0.01和P0.01）；CD34

CD38

KG1α细胞ROS也分别上升了(1.39±0.12)倍和(2.81±0.11)倍，差异也具有统计学意义（P0.03和P0.01），并且DS/Cu组比DS组更明显（P=0.00）；应用NAC抑制DS/Cu诱导ROS蓄积后，细胞凋亡率由(27.43±1.65)%下降到(12.37±0.85)%（P0.01）。qRT-PCR结果显示DS及DS/Cu均上调TNF-α、CD40、TNFRSF1B、TNFRSF11B、HRK凋亡相关基因的mRNA水平（P0.05），且DS/Cu组较DS组上调的更明显（P0.05），但是予以NAC预处理2 h后，仅TNF-α基因水平依然高表达（P=0.73），而CD40、TNFRSF11B、TNFRSF1B、HRK基因水平均明显下调（P0.05）。为进一步证实TNF-α作用，分别予以TNF-α mAb(2 μg/mL)或IgG (2 μg/mL)预处理2 h后，TNF-α mAb+DS/Cu组相比DS/Cu组的ROS相对变化率由(2.78±0.25)倍下降到(1.28±0.17)倍（P=0.00），而IgG+DS

/Cu组相比DS/Cu组的ROS相对变化率无显著性差异（P=0.23），TNF-α mAb及IgG组相比空白对照组的ROS水平无显著性差异（P=0.09和P=0.50）。结论 DS/Cu可通过上调TNF-α表达及蓄积ROS诱导AML干细胞凋亡。

Abstract

Objective To investigate the molecular mechanism of disulfram/copper (DS/Cu)-induced reactive oxygen species (ROS) accumulation and apoptosis in leukemia stem cells (LSCs). Methods CD34

CD38

KG1α cells were separated from KG1α lines by magnetic cell sorting (MACS). Then

qRT-PCR was employed for determination of TNF-α

CD40

TNFRSF1B

TNFRSF11B and HRK mRNA levels

and Western blotting was used to detect TNF-α protein level. ROS level and cell apoptosis were assayed by flow cytometry. Results The apoptotic proportions of CD34

CD38

KG1α cells exposed to DS and DS/Cu were (11.87±1.30)% and (27.43±1.65)%

respectively (P=0.01

P0.01). DS and DS/Cu could increase ROS accumulation by 1.39±0.12 folds and 2.81±0.11 folds

respectively

(P=0.03

P0.01). However

cell apoptosis induced by DS/Cu was inhibited by pre-treatment of ROS inhibitor N-acetyl-L-cysteine (NAC) [(27.43±1.65)% vs (12.37±0.85)%

P0.01

]

. The mRNA levels of TNF-α

CD40

TNFRSF11B

TNFRSF1B and HRK were up-regulated after treatment with DS and DS/Cu (P0.05)

and the up-regulation induced by DS/Cu was more significantly (P0.05). Pre-treatment of NAC significantly down-regulated the CD40

TNFRSF11B

TNFRSF1 and HRK expression (P0.05)

but TNF-α expression was still at high level (P=0.726). To further confirm the role of TNF-α

ROS accumulation was inhibited by pre-treatment with neutralizing antibody TNF-α mAb (2.78±0.25 vs 1.28±0.17 folds

P=0.00)

while pre-treatment with IgG had no impact on ROS accumulation induced by DS/Cu (P=0.23). Conclusion DS/Cu induces leukemia stem cell apoptosis through TNF-α/ROS pathway.

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