| Background:Acute myeloid leukemia (AML) is a hierarchical disease initiating from a rare population of cells known as leukemia stem cells (LSC), which are typically enriched in CD34+CD38" cells and presumed responsible for the relapse and refractory of AML. Moreover, current regimens may not effectively discriminate between normal and malignant cells. For this reason, it is important to identify therapies that can specifically target the LSC population without affecting normal cells. Disulfiram (DS) is an anti-alcoholism drug that has recently been indicated to show cytotoxic to multiple cancers including acute myeloid leukemia (AML) and the antineoplastic activity was enhanced in the present of copper (Cu).Nuclear factor-κB (NF-κB) is a transcription factor belonging to the Rel family, has a pivotal role in the control of the cell growth and survival. NF-κB is typically a heterodimer that consists of p65(Rel A) and p50proteins and p65is likely to be a key component in regulating NF-κB activity. More recently, NF-κB activation has been connected with multiple aspects of oncogenesis, including the control of cell proliferation, migration, cell cycle progression, and apoptosis. In addition, NF-κB is constitutively activated in several types of cancer cells, including hematological and epithelial malignancies. It also demonstrated that NF-κB is constitutively active in LSC but not in normal hematopoietic stem cells.Notably, many traditional cancer therapies induce activation of NF-κB, which has emerged as a significant impediment to effective cancer treatment. Taken together, the inhibition of NF-κB may represent a promising therapeutic tool to facilitate LSC-selective cell death as well as increase the efficacy of conventional anticancer drugs.Reactive Oxygen Species(ROS) contains free radicals (e.g. superoxide and hydroxyl) and non-radical (e.g., H2O2), is the product of normal oxygen metabolism, the physiological doses of ROS can be cleared by antioxidants while sharply increased ROS can cause peroxidation of lipid protein and DNA, resulting in cell damage. In the same time, ROS can regulate multiple molecular pathways, including the JNK/c-jun pathway, and involve in the regulation of cell proliferation, differentiation or death. Recently, Increased ROS levels were found in a variety of tumor cells, slightly increased ROS may be involved in maintaining the proliferation and survival of tumor cells, but dramatically increased ROS can produce cytotoxic so that tumor cells become more vulnerable to external damage of chemotherapy drugs. Meanwhile, under the continuous oxidation stimulation, some redox-sensitive transcription factors (NF-kb, Nrf2, c-jun, HIF-1) are activated, thereby increasing the expression of antioxidant proteins so as to maintain the oxidative balance. Nuclear factor-erythroid2-related factor2(Nrf2) is an important transcription factor in the regulation oxidative balance. It is activated by oxidative stress, but its target gene products can protect cells from damage induced by oxidative stress. Effective antineoplastic agents, besides inducing ROS accumulation, must have the ability to inhibit the activity of tumor antioxidant system or the related transcription factors.For many decades, the DS has been used for anti-alcoholism treatmen by inhibiting aldehyde dehydrogenase (ALDH), it is cheaper and have minor side effects. Recent studies have shown the antitumor activity of DS in a wide range of cancer cell lines and the joint use of Cu can enhance this activity. Our team also has discovered that DS/Cu could induce apoptosis in acute hematological malignancy cell lines (Molt4cells and Raji cells) through inhibition of NF-κB activation the induction of ROS. We also found that DS/Cu can reverse the doxorubicin resistance of acute myeloid leukemia cell line HL-60cells. However, the effect of DS/Cu on LSC and the relevant mechanism still needs further exploration,Objective:To investigate the effect of DS and DS/Cu on LSC and its relationship with NF-Kb and JNK pathway.Methods:1、CD34+CD38-leukemia stem cell (LSC) enriched subpopulations were sorted from both KGla cell lines and primary AML bone marrow by fluoresce-activated cell sorting (FACS) analysis or magnetic cell sorting (MACS) analysis; HSCs enriched subpopulations were sorted from peripheral blood or umbilical cord blood mononuclear cells.2、MTT analysis of cytotoxicity of DS or DS plus Cu (1μM) to CD34+CD38-KG1a cells;3、Annexin-V/PI staining flow cytometric analysis of the apoptotic CD34+CD38-KGla cells after treatment with0.05、0.5、5μM of DS or DS plus Cu (1μM) or DS/Cu/NAC (DS:0.5μM, Cu:1μM, NAC (10mM) for24hours;4、Methylcellulose colony formation analysis of the colony forming ability of CD34+CD38-KG1a cells after treatment with0.01、0.1μM of DS or DS/Cu (1μM) for18h; Methylcellulose colony formation analysis of the colony forming ability of primary AML cells and normal peripheral cells were also examined after treatment with with0.01、0.05、0.5μM of DS or DS/Cu (1μM) for18h;5、Flow cytometric analysis of the ROS level in CD34+CD38-KG1a cells after treatment with0.5μM DS or DS plus Cu (1μM) for6h、12h、18h、24h;6、Western blotting analysis of the expression of proteins in SAPK/JNK、 Nrf2and NF-κB pathway in CD34+CD38-KGla cells after treatment with0.5μMDS.0.5μM DS plus Cu (1μM)、Cu(1μM)、DS/Cu/NAC (DS:0.5μM, Cu:1μM, NAC:10mM) for24h;7、The expression of Nrf2pathway downstream genes NQO1、GSR、HO-1was measured by SYBR Green real-time PCR in CD34+CD38"KGla cells after treatment with0.5μM DS、0.5μM DS plus Cu(1μM)、Cu (1μM) and the relative expression quantities was calculated by formula of2-Act;8、1) Annexin-V/CD34-APC/CD38-PE/staining flow cytometric analysis of the apoptotic CD34+CD38" primary AML cells versus CD34+CD38" normal cells after treatment with0.1、0.5μM of DS or DS plus Cu (1μM) for24hours;2) Methylcellulose colony formation assay analysis of the colony forming ability of primary AML cells versus normal cells after treatment with0.01、0.05、0.1μM of DS or DS plus Cu (1μM) for18h;9、The statistical analyses were performed with the statistical software package SPSS13.0. Student’s t-test was used to compare IC50values of two independent groups, One-Way ANOVA was used to compare the difference of apoptotic populations、ROS levels and colony formation rates in different groups, LSD was used to do multiple comparison when the variance was homogenous, if not, Dunnett’s T3was employed. A value of*P<0.05was accepted as an indication of statistical significance. Results represent the mean±SEM of at least three independent experiments.Results:1、CD34+CD38-KGla cells were exposed to serial concentrations of DS for24h. DS demonstrated toxicity to cells with IC50=(0.54±0.18)μM. The cytotoxicity have been enhanced by serial concentrations of DS plus Cu (1μM). The IC50[(0.21±0.028)μM], value of DS/Cu was significantly lower than that treated with DS alone (t=3.107, P=0.036);2、The flow cytometric AnnexinV/PI staining analysis demonstrated significant increasing of apoptosis after24hours treatment with different concentrations of DS/Cu (F=119.069, P=0.000) and5μM DS (P=0.432). In comparison with the untreated cells, low concentration of DS (0.05、0.5μM) showed slightly induced apoptosis while DS at5μM significantly enhanced apoptosis after24hours treatment on CD34+CD38-KGla cells(P=0.000). We also found that different concentrations of DS/Cu could induce cells apoptosis, compared with the control group, the apoptotic rate increased gradually in a dose dependent manner. Further, compared with5μM DS treated cells, CD34+CD38’ KGla cells treated with5μM DS/Cu showed a higher apoptotic rate (t=-7.279, P=0.002).3、The methylcellulose colony formation assay demonstrated that both DS and DS/Cu clearly inhibited the growth of AML-colony-forming units (CFUs) in CD34+CD38-KGla cells (F=5.287, P=0.047;F=58.999, P=0.000) and the effect of DS/Cu is stronger than DS alone;4、The CD34+CD38-KG1a cells were exposed to DS or DS/Cu for different time durations and analyzed the ROS level using the flow cytometric analysis. Our results demonstrated that compared with the control group, the ROS level in DS/Cu group have been significantly enhanced (P=0.000) while in DS group only enhanced after6h treatment (P=0.000). After treated with DS/Cu for different time durations, compared with the control group, the ROS levels increased gradually and reached to a peak in the18h, while reduced in the24h. We supposed that comparing with DS, DS/Cu could enhance ROS level in CD34+CD38-cells;5、Western blot results showed a markedly increase in phosphorylation of JNK in CD34+CD38-KGla cells treated with DS and DS/Cu for24h while no obvious change was noticed in the constitutive expression of JNK. The phosphorylation of c-jun, a downstream transcription factor of JNK pathway, also saw an increase after DS and DS/Cu treatment. Meanwhile, the expression of p65and NF-κb-related gene was inhibited by DS and DS/Cu while the DS/Cu displayed a more marked inhibiting fact. Western blot also demonstrated that DS alone have slight effect on Nrf2while DS/Cu complex could significantly suppressed the expression of Nrf2.6、The CD34+CD38-KGla cells were treated with0.5μM of DS and0.5DS/Cu for24hours and analyzed the NQO1、GSR、HO-1mRNA expression using the SYBR Green-PCR. The results demonstrated that DS and DS/Cu could suppress the expression of these mRNAs and the effect of DS/Cu is more notable even with a higher concentration.7、We treated CD34+CD38-KGla cells with DS/Cu and DS/Cu plus NAC(10mM) for24hours. We found that the apoptosis rate was no different between NAC and control group, while the addition of NAC could strongly diminish the DS/Cu mediated apoptosis (F=31.774;P=0.000). As a result, we supposed that DS/Cu induced cellular ROS accumulation, and ROS was the key role of DS/Cu inducing apoptosis.8、We further tested the expression of p-JNK and Nrf2after treatment with Cu、DS、DS/Cu and DS/Cu/NAC by westen blot assay. The results showed that NAC could reverse DS/Cu mediated p-JNK up-regulation and Nrf2down-regulation.9、The Annexin-V/CD34-APC/CD38-PE/staining flow cytometric analysis demonstrated different concentrations of DS/Cu could induce significant apoptosis in CD34+CD38"primary AML cells(F=42.834, P=0.000), while DS alone only slightly induce apoptosis (F=1.728, P=0.225); Meanwhile, in the same concentration, neither DS nor DS/Cu could induce apoptosis in CD34+CD38-normal cells (F=0.562, P=0.598;F=4.293, P=0.070). These results show that DS/Cu could induce apoptosis in CD34+CD38-primary AML cells and only induce apoptosis in CD34+CD38-normal cells with a higher dose; DS could not induce apoptosis in both cells.10、The methylcellulose colony formation assay demonstrated that both DS and DS/Cu clearly inhibited the growth of AML-colony-forming units (CFUs) in CD34+CD38-primary AML cells (F=35.269, P=0.000;F=16.235, P=0.000) and the effect of DS/Cu is more pronounced. But neither DS nor DS/Cu could inhibited the colony-forming ability of CD34+CD38-normal cells at the same concentration range (F=1.141, P=0.299;F=0.441, P=0.726), these data show that DS and DS/Cu could inhibite the colony-forming ability of CD34+CD38-primary AML cells while relatively spare the CD34+CD38-normal cells.Conclusion: 1. DS and DS/Cu might selectively eradicate LSC. Both DS and DS/Cu could significantly enhance the cytotoxicity to CD34+CD38" KG la cells(with the DS/Cu more effective) by inhibiting proliferation、inducing apoptosis and decrease colony ing-forming ability; In CD34+CD38’ primary AML cells, DS/Cu could induces apoptosis while both DS and DS/Cu could inhibit colonying-forming abilities; In CD34+CD38" normal cells, neither DS nor DS/Cu could induced apoptosis or inhibit CFUs;2. The lethal effect of DS and DS/Cu may involve the induction of oxidative stress and the activated its downstream apopto sis-related SAPK/JNK pathway in association with blockade translocation of Nrf2and expression of Nrf2-regulated genes3. The blockade the NF-κb pathway might be another mechanism lies in DS and DS/Cu mediated lethality, as the expression of p65and its downstream genes was reduced by DS and DS/Cu. |
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