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.