Disulfiram Combined with Copper Induces Acute Myeloid Leukemic Stem-like KG1a Cell Apoptosis through TNF-a/ROS Pathway

Acute myeloid leukemia (AML) is a heterogeneity disease initiating from a rare population of cells known as leukemia stem cells (LSCs), which have been a major hurdle for the success of acute myeloid leukemia chemotherapy. Therefore, new drugs targeting LSCs is urgently needed.

Disulfiram (DS) has been used clinically as a safe anti-alcoholism drug for over 6 decades. Recent studies demonstrated that disulfiram combined with cooper (DS/Cu) have the antitumor activity in a wide range of cancer cell lines. CD34⁺ CD38^- KG1a cells were previous found to have the characteristics of stem cells. Here, we explored whether DS/Cu could induce acute myeloid leukemic stem-like KG1a cells apoptosis and further investigated the molecular mechanism.

CD34⁺ CD38^- KG1a cells were sorted from KG1a cell lines by magnetic activated cell sorting (MACS). Flow cytometry (FCM) analysis confirmed the percentage of CD34⁺ CD38^- KG1a cells was 95.37±1.84%. To determine the effect of DS/Cu induced apoptosis in LSC-like KG1a cells, CD34⁺ CD38^- KG1a cells were exposed to DS (5nM) with or without Cu (0.5nM) for 24h. FCM analysis showed the apoptotic proportion of CD34⁺ CD38^- KG1α cells exposed to DS was 11.87±1.30%, while 27.43±1.65% to DS/Cu (P=0.00). To explore the role of ROS in DS/Cu induced apoptosis, ROS levels were detected using DCFH-DA after KG1a cells being treated with DS and DS/Cu for 24h. DS and DS/Cu could induce ROS acumulation by 1.39±0.115 fold and 2.81±0.109 fold, respectively (P<0.01). However, cell apoptosis induced by DS/Cu was inhibited by pre-treatment of ROS inhibitor N-Acety-L-Cysteine (NAC,10mM) [(27.43±1.65)% vs. (12.37±0.85)%, P<0.01].This result showed that DS and DS/Cu could induce leukemia stem-like KG1a cells apoptosis by induced ROS acumulation.

To explore the upstream pathway of ROS accumulation induced by DS and DS/Cu in CD34+CD38-KG1α cells, We examined the expression of apoptosis-related moleculars include TNF-a,CD40, TNFRSF11B, TNFRSF1B, Hrk in LSC-like KG1a cells exposed to DS, DS/ Cu and DS/Cu+NAC for 24h. DS and DS/Cu up-regulated TNF-a, CD40, TNFRSF11B, TNFRSF1B, Hrk expression at the mRNA level (P<0.05), and the up-regulation by DS/Cu was more drastically (P<0.05). However, Pre-treatment of NAC significantly suppress the CD40, TNFRSF11B, TNFRSF1B, Hrk expression induced by DS/Cu (P<0.05), TNF-a expression was still at high level (P=0.73). The expression of TNF-a at protein levels was also confirmed by western bolt. These results thus demonstrate that CD40, TNFRSF11B, TNFRSF1B, Hrk was downstream target genes of ROS, and TNF-a may be able to regulate ROS accumulation induced by DS/Cu in leukemia stem-like KG1a cells.

To further investigate the role of TNF-a in DS/Cu induced accumulation of ROS, CD34⁺ CD38^- KG1α cells were pretreated with neutralizing antibody TNF-a mAb (2μg/mL) , control antibody IgG (2μg/mL) for 2h, then cells were exposed to DS/Cu for 24h. ROS accumulation induced by DS/Cu was inhibited by pretreatment with TNF-a mAb (2.78±0.25 vs. 1.28±0.17 folds, P=0.00), while pretreatment with IgG had no impact on ROS accumulation induced by DS/Cu (P=0.23). TNF-a mAb and IgG alone had no effect on ROS accumulation in KG1a cells ( P>0.05). These results confirmed that TNF-a is upstream molecular of ROS induced by DS/Cu in leukemia stem-like KG1a cells.

In conclusion, our data demonstrated that DS/Cu could induce leukemia stem-like KG1a cells apoptosis, and the underlying mechanisms may be related with TNF-a/ROS pathway.