| Background:Acute lymphoblastic leukemia(ALL) is one of the most common malignancy tumors in children, and also the common type of acute leukemia in adults.ALL has a bimodal distribution,the first peak age is between the ages of 2-5, which decreases during later childhood, adolescence, and young adulthood before a second, smaller peak occurs in patients older than 50 years.Acute lymphoblastic leukemia(ALL) is a biologically and clinically heterogeneous disorder of lymphoid progenitors, of which approximately 85% of cases are B-cell lineage leukemia and 15% are T-cell lineage leukemia. Recently, the application of high-dose multi-agent chemotherapy regimens, molecular-targeted therapeutic drugs and allogeneic hematopoietic stem cell transplantation(HSCT) leads to approximately 80% cure rate in children,but only about 30-40% of adults achieve long-term disease-free survival (DFS),so it is urgent to improve the overall survival (OS) and event-free survival (EFS) rate of adult ALL in the clinic. Adult ALL patients show poorer tolerance, more complication and higher mortality in response to treatment as compared with children, therefore, it is necessary to seek certain efficient and low-toxicity drugs for them.Disulfiram(DS) is an anti-alcoholism drug used in the clinic for more than 6 decades, characteristic of good safety, low toxicity and cost-effectiveness.In recent years, more and more researchers have found that DS have potent anti-tumor effect against solid tumors, including breast cancer, prostate cancer, malignant melanoma and colon cancer etc. Meanwhile, various studies indicated that DS could chelate several metal ions to potentiate its anti-tumor activity. Copper is an necessary element for all organisms, essential to many key enzymes and transcription factors. As a divalent metal ion chelator, DS strongly chelates Cu to form the Cu(deDTC)2 complex, which showed significantly enhanced cytotoxicity to solid cancers and leukemia cells than DS alone. Currently, it is well defined that DS/Cu can induce apoptosis and inhibit proliferation in many solid tumors and hematologic malignancies, however, its cytotoxicity on ALL cells is ill characterized. As 80-85% ALL originate from B cells, this study will explore the cytotoxicity of DS/Cu to B-ALL cells(including primary B-ALL cells and nalm6 cell lines) and the involved molecular mechanism.Apoptosis is an autonomic and ordered programmed cell death (PCD) controlled by series of genes, and is essential for a variety of biological events, such as tissue homeostasis, the development of multiple systems, embryonic development, and biological evolution. To date, research indicates that there are three main apoptotic pathways:the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway,the last one is endoplasmic reticulum apoptotic signaling pathway. And the mitochondrial pathway is considered as the most typical apoptotic pathway.Mitochondrium, a pivotal controller of cellular activities, is not only a center for cellular respiration and oxidative phosphorylation, but also a critical regulator of cell apoptosis.There have a variety of stimuli which include both a positive and negative fashion to initiate the intrinsic pathway. Negative signals involve the lack of some essential growth factors, hormones and cytokines that can lead to failure of suppression of death programs, thereby triggering apoptosis. And the positive stimulti include radiation, drugs, hypoxia and viral infections and so on.Mitochondria-mediated apoptosis is associated with the opening of mitochondrial permeability transition pore in response to various stimuli, which increases the permeability of mitochondrial membrane and reduces the mitochondrial membrane potential, and with the release of a large amount of pro-apoptotic agents from the intermembranous space, including cytochrome C, Smac/DIABLO,Omi/HtrA2, IAPs、AIF, endonulease G, and activated the caspases which cleaved its substrates,making this substrates dysfunction,finally triggering cell apoptosis.The substrates of caspases play a key role in the process of DNA repairs、mRNA cracking、steroidogenesis and cytoskeleton reconstruction.Bcl-2 family proteins include three subfamilies based on their function and number of BH domains, which are:1)anti-apoptotic proteins, such as Bcl-2 and Bcl-xL with four BH domains; 2) pro-apoptotic proteins, such as BAX and BAK with three BH domains; 3)pro-apoptotic proteins with only BH3 domains, such as BID and BIM. Proteins of these subfamilies can form homodimers or heterodimers, which play different roles in regulating mitochondrial membrane potential. The homodimers or heterodimer forms are key to the Bcl-2 family proteins to execute and modulate their function. Bcl-xL and Bcl-2 are members of the antiapoptosis subfamily, playing critical roles in apoptosis,especially mitochondria-mediated apoptosis.Both has synergistic effect in aspect of cell apoptosis,inhibited apoptosis and prolonged cell survival.Bcl-2 gene (that is B cell lymphoma/leukemia 2 gene) is a proto-oncogene,which was first found in Follicular non-hodgkin’s lymphoma by Tsujimoto Y in 1984,and caused by t(14;18)(q32;q21).Bcl-2 is one of the most important anti-apoptotic proteins, which can be located in mitochondria, endoplasmic reticulum or nulear membrane in different types of cells. Bcl-2 overexpression inhibits apoptosis and prolongs lifetime of cells. Bcl-xL(BCL2L1) is a transmembrane molecule in the mitochondria, which could modulate the open and close of voltage-dependent anion channel(VDAC) which lies in the mitochondrial outer membrance,and then affect the mitochrial membrance permeability and decrease the membrance potential,finally affect the mitochondrial apoptotic pathway.To date, the molecular mechanism of the anti-tumor effect of Ds/Cu, on which researches mainly focus on the inhibition of proteosome and activation of JNK/c-Jun pathways by Ds/Cu, is still ill understood. Our group previously demonstrated that Ds/Cu could induce the apoptosis and cell cycle arrest of acute myeloid leukemia cell line KG-la, acute lymphoid leukemia cell line Molt4 and Brukitt lymphoma cell line Raji, through inhibiting NF-kB, activating JNK/c-Jun pathways and inducing intracellular accumulation of ROS;moreover, Ds/Cu could also recover susceptibility of HL-60, a drug resistant cell line, to doxorubicin. So far, there have been rare reports about the cytotoxicity and its molecular mechanism of Ds/Cu.Since the knowledge that mitochondrial pathway takes a critical part in Ds/Cu induced apoptosis and cell cycle arrest of various cancer cells, this study used B-ALL cell line NALM6 and primary B-ALL cells to decipher the role of Ds/Cu in the cytotoxicity of B-ALL cells and its relationship with mitochondrial pathway of apoptosis.Objective:In this study, the B-lineage acute lymphoblastic leukemia cells, including nalm6 cell line and several primary B-ALL cells, are the main study object. To study the effect of DS coupled with Cu on cell proliferation and apoptosis in nalm6 and primary B-ALL cells; and to further investigation the relationship between DS/Cu and the mitochondrial apoptosis pathway.Method:1、Using the cell counting kit-8 (CCK-8)to detect the proliferation inhibition rate after treatment with various concentration(0.05、0.1、0.2、0.4、0.8、1.6、3.2、 6.4uM)of DS coupled with 0.5uM Cu for 24h on nalm6 cells, and applied graph prism5 to count the corresponding IC50 values.2、Using the Annexin-V/PI double staining assay flow cytometric analysis the percentage of apoptotic cells in nalm6 and several primary B-ALL cells, after treatment with various concentration(0.025.0.05.0.1.0.2.0.5uM) of DS coupled with 0.5uM Cu for 24h.3.Methylcellulose colony formation analysis of the colony forming ability of nalm6 cells after treatment with CON.Cu. DS and DS coupled with Cu for 6h.4. Mitochondrial membrane potential detection kit (JC-1 staining kit)to detect the level of mitochondrial membrane potential (MMP/ΔΨ) on nalm6 and several primary B-ALL cells, after treatment with various concentration(0.025.0.05.0.1. 0.2.0.5uM) of DS coupled with 0.5uM Cu for 24h.5. Western blotting analysis of the expression level of proteins related to the mitochondrial apoptosis pathway,such as Bcl-2. Bcl-xL. BAX. caspase-3 and its substrate PARP, after treatment with CON、Cu、DS and DS plus Cu for 24 h on nalm6 cells.6.The statistical analyses were performed with the statistical software SPSS 19.0. Student’s t-test was used to compare the proliferate inhibition rate in different treatment group(CON.Cu and various concentration of DS plus 0.5uM Cu), One-Way ANOVA was used to compare the difference of apoptotic populations、 colony formation rates in different groups,LSD was used to do multiple comparison when the variance was homogenous, if not, Dunnett’s T3 was employed. A value of *P<0.05 was accepted as an indication of statistical significance. Results represent the mean±SEM of at least three independent experiments.Results:1、The cell counting kit-8 cytotoxicity experiment results indicated that various concentration DS/Cu could significantly inhibit the Nalm6 cells proliferation: exposed to different drug groups for 24h, the inhibiton rate of Cu(0.5uM) was (6.39±4.93)%, compared with control group, there was no statistical difference between two groups(t=-2.244, P=0.088). While various concentrations of DS coupled with 0.5uM Cu had significantly inhibited nalm6 cells proliferation, in a dose-dependent mode. The inhibition rate of (0.05、0.1、0.2、0.4、0.8、1.6、3.2、 6.4uM)DS coupled with 0.5uM Cu on nalm6 cells were respectively (22.29±6.69)%、(48.66±11.58)%、(50.83±12.61)%、(59.24±9.43)%、 (62.74±9.17)%、(66.7±7.64)%、(72.13±7.94)%、(77.86±5.43)%,through the ANVOA statistical analysis demonstrated that there had significantly statistical differences between various concentrations DS/Cu and control group(P<0.05); the graph prism5 software calculated that the IC50 for 24h was (0.18±0.08)uM.2, The results of Annexin-V/PI double staining assay detected by flow cytometer indicated that DS/Cu could significantly induce nalm6 cells apoptosis:after treatment with different drug groups on nalm6 cells for 24h, there was no statistical difference between Cu(0.5uM) and CON group(t=0.112,P=0.916), and the apoptotic ratio was separately (7.82±5.13)% and(8.34±6.23)%. However, various concentration DS plus Cu(0.5uM) could remarkably induce apoptosis, in a dose-dependent mode, specific results were as follows:the apoptotic ratio of 0.025, 0.05、0.1、0.2、0.5uM DS plus Cu(0.5uM) were respectively (17.84±7.68)%、 (31.39±5.86)%、(60.41±13.87)%、(69.26±13.29)%、(81.37±12.72)%,the ANOVA stastical analysis indicated that DS/Cu induced significantly Nalm6 cell apoptosis,compared with CON group,there was significantly stastical differences(F=28.471, P=0.000).Future using LSD method to compared with the differences between any two of various concentrations(0.025、0.05、0.1、0.2、0.5uM) DS plus 0.5uM Cu indicated that there were stastical differences among 0.025uM group and 0.1、0.2、0.5uM groups with P value less than 0.01;and there had significantly differences among 0.05uM group and 0.1、0.2、0.5uM groups with P value less than 0.01; and between 0.1uM and 0.5uM group with P value less than 0.05;and among the rest groups,there were no stastical differences.3、DS/Cu could also has the ability to induce primary B-ALL cells apoptosis: using 16 cases different patients origin of primary B-ALL cells to study the cytotoxicity of DS/Cu on B-ALL cells, we found that DS/Cu could also induce primary B-ALL cells apoptosis, and in a dose-dependent mode. After dealing with different drug groups for 24h, there was no statistical difference between Cu(0.5uM) and CON group(t=0.146, P=0.885),and the apoptosis percent was separately (34.66±16.99)% and(33.83±15.25)%;however, the apoptosis percent of various concentrations(0.025、0.05、0.1、0.2、0.5uM)DS plus Cu(0.5uM)were respectively (43.0±15.93)%、(57.02±23.8)%、(64.81±24.03)%±(70.22±22.2)%、 (68.09±22.72)%,compared with CON group,there were obviously statistical differences (F=14.716,P=0.000). Using LSD method to compared with the differences between any two of various concentrations(0.025、0.05、0.1、0.2、0.5uM) DS/Cu indicated that there were stastical differences among 0.025uM group and 0.1、 0.2、0.5uM groups with P value less than 0.001;and there had significantly differences among 0.05uM group and 0.1、0.2uM groups with P value less than 0.05;and among the rest groups,there were no stastical differences.4、The methylcellulose colony formation assay demonstrated that DS/Cu groups could inhibit the growth of BALL-colony-forming units (CFUs) in nalm6 cells:the percentage of colony-forming units (CFUs) in CON、Cu、DS and DS/Cu groups were respectively(33.63±4.15)%、(30.93±4.22)%、(10.83±2.63)%、(0.9±0.66)%. Compared with CON group,there was no statistical difference between Cu and CON (t=0.791, P=0.473);however, compared with CON groups, DS and DS/Cu groups could remarkably inhibit the growth of BALL-colony-forming units (CFUs), through ANOVA method analysis demonstrated that there were significantly statistical difference (F=70.949, P<0.001).Using the LSD method to further analysis the difference between DS/Cu and DS groups, the result indicated that there was obviously statistical difference with P value equaled to 0.006.5、Using Mitochondrial membrane potential detection kit (JC-1) to detect the level of mitochondrial membrane potential (MMP/A/ΔΨ) after treatment with DS/Cu for 12h:Our study indicated that after treatment with Cu(0.5uM) for 12h on nalm6 cells, the ratio of JC-1 aggregate/monomer mean fluorescence intensity was (1.01±0.09), compared with control group whose JC-1 aggregate/monomer mean fluorescence intensity ratio was (1.21±0.36), there was no statistical difference between two groups(t=-0.996, P=0.375). And various concentrations (0.025、0.05、 0.1、0.2、0.5uM)DS plus 0.5uM Cu caused gradually the MMP decreasing, the specific JC-1 aggregate/monomer mean fluorescence intensity ratio were as follow: (1.01±0.15)、(0.64±0.22)、(0.39±0.25)、(0.21±0.11)、(0.11±0.07), the statistics analysis demonstrated that various concentrations DS plus 0.5uM Cu, there was significantly statistical differences (F=14.535,P=0.000). Future using LSD method to compared with the differences between any two of various concentrations(0.025、 0.05、0.1、0.2、0.5uM) DS/Cu indicated that there were statistical differences among 0.025uM group and 0.05、0.1、0.2、0.5uM groups with P value less than 0.05;and there had significantly differences among 0.05uM group and 0.2、0.5uM groups with P value less than 0.05;and among the rest groups,there were no statistical differences.And our study found that DS/Cu affected the mitochondrial membrane potential of 7 cases primary B-ALL cells similar to nalm6 cells, JC-1 aggregate/monomer mean fluorescence intensity ratio of single-agent Cu(0.5uM) was(1.75±1.3) compared with control group whose JC-1 aggregate/monomer mean fluorescence intensity ratio was (1.69±1.04), there was no statistical difference between two groups (t=-0.099, P=0.923); while the ratio of various concentration DS/Cu were respectively (1.3±0.78)、(1.02±0.71)、(0.68±0.64)、(0.36±0.34)、 (0.15±0.12),through the ANOVA method analysis indicated that various concentrations DS/Cu could decrease the mitochondrial membrance potential in primary B-ALL cells, compared with CON group, there were significantly statistical differences (F=7.33, P<0.01)6、Western blot results showed that DS/Cu affected the protein expression related to the mitochondrial pathway:after treatment with CON、Cu、DS and DS/Cu for 24h, the results indicated that DS and DS/Cu markedly decreased the expression of anti-apoptotic protein,such as Bcl-2、Bcl-xL; and then activated Caspase-3 and its substrate PARP protein, finally caused cell apoptosis.Conclusion:1、Various concentrations of DS plus Cu markedly inhibited nalm6 cells proliferation,in dose-dependent mode, the IC50 for 24h was (0.18±0.08) uM.2、Various concentrations of DS plus Cu significantly induced B-ALL cells(including nalm6 and primary B-ALL cells) apoptosis, in dose-dependent mode; and DS/Cu could obviously inhibit nalm6-colony-forming unit.3、DS/Cu significantly decreased the mitochondrial membrane potential in B-ALL cells(including nalm6 and primary B-ALL cells); and then western blot results showed that DS/Cu groups decreased the expression of Bcl-2、Bcl-xL, activated Caspase-3 and its substrate PARP protein, finally caused B-ALL cells apoptosis.And the effect of DS/Cu was stronger than DS group. |
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