Disulfiram Combined With Cu²⁺ Induced Leukemia Stem Cells Apoptosis Through TNF-α/ROS Pathway

Background:Acute myelogenous Leukemia (AML) is a heterogeneous disease in morphology, genetics and epigenetics. It most characteristic is the primitive and immature myeloid leukemia cells accumulated in bone marrow and peripheral blood,and is one of the most common type of adult acute leukemia(AL),In 2014,The American cancer society published data showed that 18800 people were diagnosed with AML every year in the United States, more than 10000 people died,and mortality ranked sixth in cancer.So, AML is a big challenge for the hematologists and oncologists at present. Though, as the new antitumor drugs appeared, chemotherapy scheme optimized and improved constantly, and disease remission rate has been improved, the patient’s prognosis is not optimistic,10 year overall survival (OS) in patients with good karyotype was 55%～80%, moderate and high-risk karyotype patients are 30%～40% and 8%～40%, respectively, refractory and relapse is still the most important factors affecting the prognosis of patients with AML.Current research has found that leukemia cells are derived from leukemia stem cells (LSC), the set of cells account for only a little proportion of the leukemia cells, but characteristic with self-renewal, constant growth,ability to differentiate into mature leukemia cells, and maintain cell in primitive/immature stage, the LSC most in G0/1 phase (dormant phase), with a aberrant survival signal pathway, which makes them insensitive to tradition chemotherapy drugs which are against the rapid proliferation cell, those are the root of relapse and refractory of AML.Over the past decade, a clear understanding of the LSC biological characteristics had been made a great progress, but a specificity targeted LSC without affecting the normal hematopoietic stem cells (HSC) is still a difficult task. Therefore,developed a class of durg only targeted LSC without affecting the normal HSC is not only our challenge and ultimate goal,but also the most fundamental and most effective strategies to cure AML in future.Disulfiram(DS) is member of DDTC family,which as an anti-alcoholi-sm drug in clinical has more than 60 years.Recently reported that this durg show cytotoxic to many solid tumor including breast carcinoma, melanoma, metastasis hepatic and so on, and same to hematology malignance cell like molt4,HL60,Raji while in present of copper (Cu).Reactive Oxygen Species(ROS) contains non-radical (e.g., H2O2) and free radicals (e.g. superoxide and hydroxyl),is natural products in the process of oxidative metabolism of eukaryocyte. Due to properties of highly activity of ROS, which could cause the DNA, protein and lipid damage with peroxidation.In the physiological status,cells can take advantage of the intracellular antioxidant/defense system against the toxic products to maintain the stability of cellular redox homeostasis.However, When intracellular ROS level changes largely, broke the REDOX state of stability, a dramatic increase of ROS in a short period of time can induce the apoptosis of cells.Current research shows that the induced the accumulation of ROS in the leukemia stem cells can surely lead to apoptosis and have no effect on normal Hematopoietic stem cells.Trachootham found that the magnitude of ROS increase in tumor cells is significantly larger than non-tumor cells in presence of oxidative stress, and application of pro-oxidant agent-PEITC can promote ROS increased sharply to breakthrough survival threshold level in CLL tumor cells, thereby inducing apoptosis of tumor cells, but no effect on normal lymphocytes.Recently, Lagadinou et al.who also found a group of AML leukemia cells with lower levels of ROS have a properties of stem cell, and were highly expressed BCL-2, when application with BCL-2 activity inhibitor-ABT-263 can be reduced oxidative phosphorylation of mitochondrial pathway and significant accumulated ROS in the group of AML leukemia cells, then the apoptosis also significantly increased, while ABT-263 have no effect on normal HSC, which can explain the regulation of cellular oxidative stress may be a good site in eliminating leukemia stem cells,and ROS is undoubtedly a ideal target to control the process.Tumor necrosis factor-alpha (TNF-α) is a cytokine with strong proinflam-matory function acting on pleiotropic for regulating cellular functions like proliferation, differentiation and apoptosis and so on. Clearly known the TNF-α have a vital pathophysiology role in state of inflammation and shock, and endothelial cells is primary target cells in this process. Studies have reported that TNF-α can increase intracellular ROS content, and the expression level of NADPH oxidase and lipoxygenase in the plasma membrane, and TNF-α could trigger ROS production mainly from mitochondria that associated with cytotoxicity and cell death in these studies. In acute leukemia cells, TNF-a induced apoptosis by inhibiting NF-kB pathway, which also involves the generation of ROS and activation of JNK pathway. Thus, TNF-α may be a important cytokine of anticancer drugs in killing tumor cells by inducing ROS accumulation.In recent decades,the development of new drugs has been a hot research in the process of research and treatment of cancer, however, the development of new drugs have a low chance of success, and will spend a lot of manpower, funding, and application of new drugs in clinical may actually take years in clinical trial, which will cost a lot of time. Therefore, there are various shortcomings in the development of new drugs,and a large number of researchers to look into a new idea-the "new using for old drug ", especially the old drug has the characteristics of cheap, easily available, safe, non-toxic or low toxicity in treatment of cancer, which is a feasibility shortcut of drug development in present. DS can inhibit aldehyde dehydrogenase as an anti-alcoholism drug used in clinical more than 60 years which is inexpensive, easy to get and little side effects,and DS also is a class of metal chelating agents which has a ability of binding copper (Cu) and transport it into the cell. Recently, scholars are concerned about DS applied in cancer treatment, for example,research shows DS joint with Cu (DS/Cu) have a cytotoxic effect on leukemia cells and solid tumor like melanoma, stromal tumors, and lung cancer,and DS can enhanced tumor cells sensitivity to traditional chemotherapy drugs, at the some time.researcher found that DS also have cytotoxic effect on cancer stem cells in glioma, breast. Our previous work have found that DS can enhance the sensitivity of AML drug-resistant strains to chemotherapy drug, we also found that DS/Cu can induced acute myeloid leukemia (AML) stem cell apoptosis through increasing ROS levels, then activating JUK and MAPK pathway and inhibiting NF-κB pathway.In this experiment,we will discuss whether DS/Cu can induce AML stem cells apoptosis and explore its molecular mechanism,which is aimed at providing more scientific evidence for DS/Cu treating leukemia.Objective:Make clear of DS and DS/Cu whether can induced AML stem cells apoptosis in vitro, and further explore the upstream molecular mechanisms of ROS production.Methods:1.Sorting human acute myeloid leukemia cell lines KG1α cells by MACS;2.Staining apoptosis cell with Annexin-V, flow cytometry analysis CD34+CD38-KGla cells apoptosis induced by DS (5μM) and DS/Cu (5μM/0.5μM),Compare cell apoptosis rate between the groups;3.Incubating cell with DCFA-DA, flow cytometry analysis CD34+CD38-KG1α cells ROS content induced by DS and DS/Cu,Compare ROS change ratio between the groups(ROS change ratio=ROStest/ROScontrol);4.Staining apoptosis cell with Annexin-V, flow cytometry analysis CD34+CD38-KGla cells apoptosis induced by DS/Cu and DS/Cu/NAC(NAC=10mM),Compare cell apoptosis rate between the groups;5.qRT-PCR detected gene related with apoptosis including TNF-α,CD40,HRK, TNFRSF11B/TNFRSF1B after treating with Control,DS,DS/Cu and DS/Cu/NAC 24 hours,compare genes expressing level in each groups;6.Western Blot detected of TNF-α protein levels in CD34+CD38-KG1α cells after treating with Control,DS,DS/Cu and DS/Cu/NAC 24 hours,compare TNF-α protein level in each groups;7.Staining apoptosis cell with Annexin-V, flow cytometry analysis CD34+CD38-KGla cells apoptosis induced by Control, DS/Cu, TNF-α mAb(2μg/mL), TNF-α mAb(2μg/mL)+DS/Cu, IgG(2μg/mL) and IgG(2μg/mL)+DS/Cu,compare cell apoptosis among Control,TNF-α mAb.IgG and among DS/Cu,TNF-α mAb+DS/Cu,IgG+DS/Cu;8.1ncubating cell with DCFA-DA, flow cytometry analysis CD34+CD38-KG1α cells ROS content induced by Control, DS/Cu, TNF-α mAb(2μg/mL),TNF-α mAb(2μg/mL)+DS/Cu, IgG(2μg/mL) and IgG(2μg/mL)+DS/Cu,compare ROS change ratio between each groups;9.The statistical software SPSS 13.0 was used for statistical analysis, compared of two independent samples using Student’s t-test; compare ROS level.apoptotic in different groups using One-Way ANOVA,multiple comparison using LSD when homogeneity of variance,if not, Dunnett’s T3 is used.(Test level a=0.05, and tested by two-sided, NS represents statistically nonsignificant,* represents P<0.05,** represents P≤0.01,*** represents P≤0.001).Results:1.Making AML stem cell model. Using anti-human CD34-APC, CD38-PE labeled KG1α cell, flow cytometry detected the proportion of CD34+CD38-KG1α cell, the proportion of CD34+CD38- KG1β cells is (64.73±2.01)% before sorting, after sorting by MACS is(95.37±1.84)%;2.DS and DS/Cu could induce AML stem cells apoptosis. Treating CD34+CD38-KG1α 24h with Control, DS (5μM), DS/Cu (5μM/0.5μM), the percentage of apoptotic cells analysised by flow cytometry.The results showed that the proportion of apoptotic in group of control, DS and DS/Cu were (6.65±0.64)%, (11.87±1.30)% and (27.43±1.65)%. respectively. DS and DS/Cu induced AML stem cells significantly (P=0.008 and P=0.001), in addition. DS/Cu induced AML stem cells apoptosis was also higher than the DS (P<0.001).So, we think that DS and DS/Cu can induce AML stem cells apoptosis. and Cu can significantly enhance the abilityof DS to induce apoptosis;3.DS and DS/Cu make ROS accumulation within the AML stem cells. Treating CD34+CD38-KG1α 24h with Control, DS and DS/Cu,and flow cytometry detected ROS level. The results showed that ROS levels in group of control, DS and DS/Cu were (359.67±32.50), (497.38±36.85), (1008.93±83.72), respectively. Compare to Control,ROS content in DS and DS/Cu group increased by (1.39±0.12)fold, (2.81 ±0.11) fold (P=0.028 and P=0.001):and DS/Cu accumulated ROS more significant than DS (P<0.001).4.NAC clear ROS accumulation may inhibit DS/Cu-induced AML stem cells. Treating CD34+CD38-KG1α 24h with DS/Cu and DS/Cu/NAC (NAC=10mM), flow cytometry analysis the percentage of apoptotic and ROS levels. The results showed that the content of ROS in DS/Cu and DS/Cu/NAC were (1008.93±83.72) and (413.95±70.90),and the proportion of apoptotic were (27.43±1.65)% and (12.37 ±0.85)%, respectively. These results showed that NAC can scavenge ROS in AML stem cell (P=0.001),in consistent with inhibiting apoptosis induced by DS/Cu(P= 0.001);5.TNF-α is a upstream genes of ROS accumulation induced by DS/Cu in AML stem cells. Treating CD34+CD38·KG1α 24h with Control, DS, DS/Cu and DS/Cu/NAC (NAC=10mM), qRT-PCR detected expression of gene inciuding TNF-α, CD40, TNFRSF11B, TNFRSF1B, HRK which related to apoptosis. The results showed both DS and DS/Cu can upregulated these genes (P <0.05), and DS/Cu more significant than DS (P<0.05), NAC suppressed ROS accumulation and rescue apoptosis induced by DS/Cu, in consistent with inhibiting expression of CD40, TNFRSF11B, TNFRSF1B, HRK(P<0.05),but not for TNF-α(P=0.726);We further verificated expression of TNF-α protein by Western blot, the results were same to qRT-PCR;6.Neutralizing TNF-α can suppress ROS accumulation induced by DS/Cu in AML stem cells. First,we pretreated CD34+CD38·KG1α cells 2h with Neutralization antibody TNF-α mAb (2μg/ml) and control antibody IgG (2μg/ml),then we set the group of control, DS/Cu, TNF-α mAb, TNF-α mAb+DS/Cu, IgG, IgG+DS/Cu (DS=5μM, Cu=0.5μM),flow cytometry to detect the ROS level, The results showed that ROS levels of control. DS/Cu. TNF-α mAb, TNF-α mAb+DS/Cu, IgG, IgG+DS/Cu were (355.33±35.23), (985.33±120.97), (280.00±23.07), (451.33±44.66), (328.00± 30.27), (1145.33±59.50), respectively, and the ROS change ratio is 2.78±0.25,0.82 ±0.14,1.28±0.18.0.93±0.16,3.25±0.49. respectively. Data display that TNF-a mAb and 1gG would not effect on ROS producted in cell(P>0.05).TNF-α mAb will inhibit ROS accumulation induced by DS/Cu in CD34+CD38-KG1α cells(P <0.001),while IgG have no effect on it(P=0.23);7.Neutralizing TNF-a can recue apoptosis induced by DS/Cu in AML stem cells. CD34+CD38-KG1α cells were treated as previously described, apoptosis detected by flow cytometry, The results showed that proportion of apoptosis of control, DS/Cu, TNF-α mAb, TNF-α mAb+DS/Cu, IgG, IgG+DS/Cu were (5.74±0.91)%, (27.0± 2.33)%, (6.08±1.01)%, (12.17±0.91)%, (7.57±0.84)%, (24.17±2.03)%, respectively.Compare to control,TNF-a mAb and IgG have no effect on apoptosis in CD34(CD38-KG1α cells(P=0.69, P=0.063),Compare to DS/Cu, neutralization antibody TNF-α mAb can recue cell apoptosis induced by DS/Cu (P=0.004), while control antibody IgG have no effect on apoptosis induced by DS/Cu (P=0.188).Conclusion:1.Both of DS and DS/Cu can kill AML stem cells, and the ability to induce apoptosis of DS/Cu more powerful than DS;2.DS/Cu can make accumulation of ROS significantly in the AML stem cells,and pretreated with ROS scavenger NAC can significantly recue cell apoptosis induced by DS/Cu in AML stem cells.which indicated that the accumulation of ROS is an important factor for apoptosis induce by DS/Cu;3.DS/Cu by upregulating the expression of TNF-α,then induced accumulation of ROS, leading to apoptosis in AML stem cells finally, and Neutralizing TNF-α can inhibit the accumulation of ROS follow by recue apoptosis induced by DS/Cu.