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Background of acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL) is the more common diseases in hematological malignancies, including T-ALL vulnerable to relapse due to its easy infiltration of of extramedullary tissue and organs, relieve , the treatment is also more difficult; Burkitt's lymphoma is an aggressive B-cell NHL, rapid disease progression, conventional chemotherapy ineffective. With the improvement of modern treatment methods as well as the strengthening of supportive care, high-dose multidrug combination chemotherapy improved complete remission rate (CR) of the high-risk lymphoid tumors, but the long-term effect is not satisfactory, the recurrence is the main reason for treatment failure Once relapse, remission again low, with poor prognosis. Refractory and relapsed remains the main obstacle to high-risk lymphoid tumor treatment and urgent problem, while the resistance is an important factor leading to refractory and relapsed. In addition, the large dose of multi-drug chemotherapy side effects lead to serious complications is affecting one of the main obstacles to further improve the high-risk lymphoid tumor efficacy, many patients die from complications of chemotherapy instead of tumor progression. Because chemotherapy-induced systemic toxicity and drug resistance is a major obstacle to high-risk lymphoid tumor treatment failure, and new molecular targeted drugs there are expensive problems, developed from the traditional cheap, and the toxic side effects of drugs The anti-tumor activity of the \Disulfiram (DSF) is anti-alcoholism drug clinical applications for more than 60 years, security is good, the price is low. The study found that the the DSF single drug can induce a range of solid tumor cell apoptosis and inhibit proliferation still controversial, but about its role in leukemia cells. As the divalent metal ion chelator, DSF strongly chelated Cu ions form DSF / Cu complexes in vitro experiments found that Cu can significantly improve the DSF-induced apoptosis of solid tumor cells. Previous studies found that the main mechanism of DSF induce tumor cell apoptosis inhibition of NF-κB expression, but the molecular mechanism is not clear on the DSF / Cu-induced apoptosis in solid tumor cells. Whether there are other mechanisms involved in the DSF / Cu induced solid tumor cell apoptosis still needs further exploration. Drugs induce tumor cell apoptosis, while also trigger the generation of anti-apoptotic factors, leading to tumor cell resistance mechanisms. NF-κB is one of the most important anti-apoptotic factors. NF-κB is a heterodimer with p50 and p65, including. P65 proportion of more regulation downstream of a series of anti-apoptotic gene expression, p65 is considered to be the key component of the regulation of NF-κB activity. Inhibit the activity of p65 can induce apoptosis of tumor cells, and can reduce the regulation of the expression of anti-apoptotic factors NF-κB. The overexpression of p65 can induce NF-κB and sustained activation caused cell drug. ROS generation is a result of cellular respiration, the ROS generation is associated with some pathological processes in many cell biological characteristics. ROS can induce the destruction of cell structures, including proteins, phospholipids and DNA, including. Studies have shown that tumor cells ROS levels slightly higher than normal cells, but because of the presence of anti-oxidation mechanism of tumor cells to relatively high levels of ROS can be tolerated. However, when a variety of factors led to tumor cells ROS levels exceed the tolerable threshold will result in apoptosis of the tumor cells, and high levels of ROS can damage the antioxidant activity of the transcription factor Nrf2, thus further enhanced ROS-mediated apoptosis. In addition, ROS and JNK pathways may exist in the process of induction of tumor cell apoptosis and promote each other. In recent decades, Disulfiram (DSF) can inhibit aldehyde dehydrogenase has been used for anti-alcoholism treatment, side effects are minor. Recent studies concerned about the role of the DSF anti-tumor, many studies have reported the DSF joint Cu can induce apoptosis in many solid tumors, including glioma, lung cancer, melanoma cells, including cells. About the DSF / Cu lymphoid tumor cells reported less. The purpose of this project is to investigate the DSF / Cu linkages between the ability to effectively kill people the lymphoid lineage the tumor Molt4 and Raji cell lines as well as relations with ROS, JNK, Nrf2 and NF-κB pathway and access. The purpose of this is subject to human acute T lymphoblastic leukemia cell lines Molt4 as well as B-cell lymphoma (Burkitt's) cell line Raji investigate whether DSF / Cu Molt4 and Raji cells apoptosis induced in vitro study, and further from the ROS , JNK, Nrf2, and NF-κB pathway to explore its potential molecular mechanism. Research methods, cell lines: human acute T lymphoblastic leukemia cell lines Molt4 and human B-cell lymphoma (Burkitt's) cell line Raji. MTT method concentration for 0.125,0.25,0.5,1,2 μm / ml of DSF and the above concentration DSF joint Cu ions (1μM) for 24 hours for the different tumor cells in vitro inhibition of proliferation and clear DSF and DSF of these tumor cells / Cu IC50 of. 3,1) Annexin Ⅴ / PI double staining flow cytometry 0.125,0.25,0.5,1,2 μm / ml of DSF and the concentration of DSF joint Cu ions (1μM) treatment Molt4 cell lines After 24 hours the proportion of apoptotic cells; 2) Annexin Ⅴ / PI double staining flow cytometry 0.5,1,2.5,5,7.5 μm / ml of DSF and the concentration of DSF joint Cu ion (1μM) treatment of Raji cells 24 hours the proportion of apoptotic cells; 4 by Hoechst33342 staining observed different concentrations of DSF and the DSF / Cu handle morphological changes in both tumor cells; 5, flow cytometry, respectively 0.125,0.25,0.5,1,2 μm / ml concentration of DSF and DSF joint DSF and the concentration of DSF joint Cu ions (1μM) handle Molt4 cell lines 8 child intracellular ROS levels; 6, using real-time quantitative PCR method were detected concentration 0.125,0.25,0.5,1,2 μm / ml Cu ions (1gM) and DSF / Cu / NAC, processing Nrf2 gene expression in the the Molt4 cell lines after 24 hours, using 2-ΔCT method to the PCR results, calculation, analysis of Nrf2 expression amount of the relationship between the various treatment groups. 7, Western Blotting detection 0.125,0.25,0.5,1,2 μm / ml of DSF and the concentration of DSF joint Cu ions (1μM) 24 hours handle Molt4 cell lines as well as Cu group (1 μm), DSF group (DSF: IC50-DSF / Cu), DSF / Cu (DSF: IC50-DSF/Cu, Cu: 1μm); DSF / Cu / NAC group (DSF: IC50-DSF/Cu, Cu: 1μm, NAC: 10mM) five groups of 24 hours after Molt4 cells Bcl-2, JNK, phosphorylated JNK (p-JNK), Nrf2 and p65 protein expression. 8, using SPSS13.0 software for statistical analysis, the mean between the two groups using independent samples t-test comparison; the paired dose data compared using paired samples t-test; groups were compared using one-way ANOVA statistics, variance analysis the situation multiple comparisons using the Bonferroni (homogeneity of variance); If heterogeneity of variance using approximate F-test (Welch method) instead of analysis of variance for multiple comparisons using the Dunnett T3 method; measurement data x ± s, inspection level α = 0.05, two-sided test. 1, DSF single drug and DSF / Cu, the two cell lines in vitro, significantly inhibit the proliferation. The MTT results: IC50 values ??of the DSF single drug of Molt4 and Raji cells 24 hours were: (IC50-Molt4-DSF = 1.314 ± 0.229μM/ml) (IC50-Raji-DSF = 5.064 ± 2.268μM/ml); The DSF / Cu Molt4 and Raji cells IC50 values, respectively: (IC50-Molt4-DSF/Cu = 0.435 ± 0.109μM/ml) (IC50-Raji-DSF/Cu = 0.891 ± 0.093μM/ml). DSF / Cu IC50 of Molt4 and Raji cells was significantly lower than the the DSF single drug, the difference had statistical significance (t = 5.993, P = 0.004 and t = 0.610, P = 0.033), suggesting that Cu can be significantly enhanced the DSF on Molt4 and Raji cell proliferation. 2, DSF single drug and DSF / Cu, induction of apoptosis on Molt4 and Raji cells. [1] Hoechst33342 staining morphological changes of cell apoptosis: microscope different concentrations of DSF and DSF / Cu 24 hours after treatment on Molt4 and Raji cells by staining Hoechst33342 observe the morphological changes of cell apoptosis, Molt4 cells strains by low concentrations of DSF (0.125,0.25,0.5 μM) treatment characteristics of apoptosis was not obvious when DSF concentration increased (1,2 gM), cell nuclear condensation, aggregation, and apoptotic bodies , realizable as bright granular blue dye; the DSF / Cu group visible than the typical apoptotic phenomena in the low-dose group (0.125μM), but when the concentration increased the 0.25μM apoptosis phenomenon is more pronounced, and with the DSF / Cu concentration increased and increased. Raji cells treated equally by the DSF and DSF / Cu similar morphological changes Molt4 cells. [2] Annexin Ⅴ / PI staining and flow cytometry detection of apoptotic cells: According to the results of MTT and preliminary experiments, is selected 0.125,0.250.512 μm / ml of DSF and DSF joint Cu ions (1μM) treatment Molt4 cell lines was observed after 24 hours proportional change in apoptosis. The results show DSF and DSF / Cu the role differences exist in Molt4 concentration main effect was statistically significant (F = 423.317, P = 0.000); DSF and DSF / Cu role main effect statistically significant differences also exist in Molt4 cell processing method (F = 84.305, P = 0.000). Prompted with the DSF or DSF / Cu concentration increased the proportion of apoptotic cells of,, Molt4 cells gradually increased, the percentage of apoptotic performance dose dependent. Further application of analysis of variance to compare two different treatment groups apoptosis rate differences. Control group DSFs DSF / Cu natural apoptosis rate were 4.016% ± 0.930% 3.977% 0.007 between A. kaempferi and A. shimadai, and 0.609%, respectively. One-way ANOVA, different concentrations of DSF or DSF / Cu can be induced Molt4 cell apoptosis compared with the control group, there were significant differences (F = 98.573, P = 0.000; F = 199.891, P = 0.000); application of two independent samples T-test found that the same concentration of DSF and DSF / Cu the role in Molt4 cells, the proportion of apoptotic cells there was significant difference (t = -8.338, P = 0.001; t = -4.957, P = 0.008; r = -24.034, P = 0.001; t = -14.614, P = 0.001; t = -14.52, P = 0.000). Prompt the same concentration of DSF / Cu ratio DSF more effective induction Molt4 apoptosis. Similarly, we select 0.5,1,2.5,5,7.5 μm / ml of the DSF and the DSF joint Cu ions (1μM) proportion of apoptotic cells was observed in the Raji cells for 24 hours after treatment; results show, DSF and the DSF / Cu respectively acting on Raji The main effect of cell concentration differences was statistically significant (F = 356.370, P = 0.000); DSF and DSF / Cu acting on Raji cells approach the main effect of differences there are also statistically significant (F = 419.434, P = 0.000), respectively. Prompt With DSF or DSF / Cu concentration increased, the proportion of apoptotic cells in Raji cells gradually increased, the percentage of apoptotic performance dose-dependent manner. The proportion of apoptosis induced by the application of two independent samples T-test detected the same concentration, DSF and DSF / Cu, Raji cells after treatment whether there are differences. The results show DSF and DSF / Cu concentration of proportion compared to the induction of apoptosis, there are significant difference (t = -12.277, P = 0.000; t = -10.748, P = 0.000; t = -9.014, P = 0.001; t = -8.334, P = 0.001; t = -11.321, P = 0.000). According to the statistical results, we believe that the DSF and DSF / Cu Raji cells the ability to induce apoptosis, but the DSF / Cu-induced apoptosis was significantly stronger than DSF. [3] Western blotting anti-apoptotic protein expression: we choose a different concentration the DSF (0.125,0.25,0.5,1,2 μM / ml) and the above concentration the DSF joint Cu 1μM role in Molt4 cells 24 hours after Western Blotting method Detection of anti-apoptotic protein Bcl-2 protein expression. The results show that: the DSF the / Cu group Molt4 cells within the Bcl-2 protein expression than the control group was inhibited in a dose-dependent manner. DSF monotherapy group only at very high concentrations, can cut the Bcl-2 protein expression. 3. DSF / Cu can increase the level of ROS in tumor cells: we select 0,0.125,0.25,0.5,1,2 μm / ml of DSF, and DSF United Cu ions (1μM) treatment Molt4 cells after 8 hours of apoptotic cells was observed within ROS level change; DSF and DSF / Cu, respectively, of acting on Molt4 cell concentration main effect differences were statistically significant (F = 123.070, P = 0.000); With DSF or DSF / Cu concentration increased ROS level of, Molt4 cells gradually increased, the proportion of the the DSF / Cu group increased more significantly of ROS level proportional performance dose-dependent. After the application of two independent samples T-test detected the same concentration, DSF and DSF / Cu handle Molt4 cells, induced cells, ROS accumulation of whether there are differences. The results show DSF and DSF / Cu concentration of proportion compared to the induction of apoptosis, there are statistically significant difference (t = -8.338, P = 0.001; t = -4.957, P = 0.008; t = -24.034, P = 0.0001; t = -14.614, P = 0.001; t = -14.529, P = 0.000). According to the statistical results, we believe that the DSF and DSF / Cu Molt4 cells induced intracellular ROS accumulation capacity the DSF / Cu induced significantly stronger than the induction of intracellular ROS accumulation ability DSF. 4, DSF / Cu can downregulate Nrf2 expression levels in tumor cells: role of different concentrations of DSF and DSF / Cu, respectively Molt4 cells after 24 hours, real-time PCR method to detect cell Nrf2 expression levels. The results show DSF and DSF / Cu respectively acting on Molt4 concentration main effect differences were statistically significant (F = 34.909, P = 0.000); DSF and DSF / Cu main effect differences also exist significant role in Molt4 cell processing method (F = 159.371, ρ = 0.000). As the DSF / Cu concentration increased, Molt4 cells Nrf2 expression levels gradually decreased in a dose-dependent manner. Further analysis comparing two different cells treated with Nrf2 expression level differences in the application of two independent samples T-test detected the same concentration, DSF and DSF / Cu after the processing Molt4 cells of Nrf2 level of change there was significant difference. The results show DSF and DSF / Cu concentration induced cell the Nrf2 level of change compared with a statistical difference (t = 3.263, P = 0.031; t = 11.668, P = 0.000; t = 11.563, P = 0.000; t = 10.612, P = 0.000; t = 13.142, P = 0.000). The results show that the DSF, DSF / Cu inhibited intracellular Nrf2 expression the DSF / Cu group of Nrf2 inhibition effect is more pronounced. 5, DSF / Cu can upregulate the JNK pathway expression down p65 and Nrf2 pathway expression: different concentrations of DSF and DSF / Cu acting on Molt4 cells after 24 hours, respectively, and Western blotting results show that total JNK protein expression of the two treatment groups had no significant change, DSF / Cu group phosphorylation of JNK (p-JNK) expression with the DSF / Cu concentration increased significantly up-regulated the expression of p-JNK DSF monotherapy group did not change significantly. The same changes in the p65 protein was detected by Western blotting, and results the show the DSF single drug and the DSF / Cu group with the higher concentration of the drug, p65 expression gradually lowered, but the the DSF / Cu group of p65 expression lowered significantly higher than DSF monotherapy group. By Western blotting to detect the Nrf2 expression of DSF group of low concentration seems to be down Nrf2 expression, but this role but not obvious with increasing concentration the DSF / Cu group with the concentration of the drug increased Nrf2 expression gradually lowered. ROS inhibitor-NAC of DSF / Cu induced the impact of Molt4 apoptosis: application of flow cytometry concentration of DSF / Cu and join ROS inhibitors NAC the role Molt4 cells 24 hours after the changes in the proportion of apoptosis. Joined NAC the DSF / Cu proportion of apoptotic cells induced Molt4 cells are different degrees of decline, the application of two independent samples T-test show the same concentration of DSF / Cu and DSF / Cu / NAC, the proportion of apoptotic cells after two treatments there are statistics learning differences (t = -1.719, P = 0.161; t = 5.090, P = 0.007; t = -4.941, P = 0.008; t = 4.814, P = 0.009; t = 4.870, P = 0.008), DSF / Cu group The percentage of apoptotic cells was significantly higher than the DSF / Cu / NAC group. Tip DSF / Cu percentage of apoptotic cells induced Molt4 cells can be inhibited by NAC. 7, ROS is the DSF / Cu regulation JNK.p65 well Nrf2 expression: different concentration the DSF / Cu, and DSF / Cu / NAC processing Molt4 cells 24 hours after application of quantitative PCR method to detect intracellular Nrf2 expression levels. Results are shown joined NAC DSF / Cu Molt4 cells induced Nrf2 expression levels of different degrees rise, the application of two independent samples T-test detected the same concentration, the two methods of DSF / Cu with DSF / Cu / NAC, cells were treated 24 hours after The observed differences in the expression levels of intracellular Nrf2. Statistics show that at low concentration group (0.125μM). Nrf2 level of the two treatment no difference, but from 0.25μM cases, DSF / Cu and DSF / Cu / NAC, cells treated Nrf2 water average statistical difference exists, DSF / Cu group significantly lower than the expression level of DSF / Cu / NAC group, t = -2.622, P = 0.059; t = -3.473, P = 0.026; t = -4.651, P = 0.010; t = -5.245, P = 0.006; t = -8.693, P = 0.001). Prompted the NAC inhibits DSF / Cu of Nrf2 expression level of the reduction in capacity. Select Cu (1μM) of DSFs (0.5μM), the DSF / Cu (DSF: 0.5 μm, and Cu: 1μM) and DSFs / Cu / NAC (DSF: 0.5 μm, and Cu: 1 μM, NAC: 10mM) acting on the Molt4 cell 24 hours after application Western Blotting, observed changes in the intracellular pathway. Tip: Cu group, DSF group compared with the blank control group, p-JNK protein expression was no significant change in the the DSF / Cu group of p-JNK protein expression was significantly higher, but can be NAC inhibited. Cu group no significant changes in p65 expression with blank control group, the DSF group with the DSF / Cu group p65 expression compared with blank control group was significantly lower, while the the DSF / Cu group of p65 downregulation is more obvious, DSF / Cu downward effect on p65 expression NAC can also be suppressed. Same DSF / Cu group of Nrf2 protein downregulation NAC inhibited. These results suggest that DSF / Cu is likely via ROS regulate JNK, p65 and Nrf2 expression. Conclusion 1 DSF single drug lymphoid tumor cells with a certain degree of inhibition of proliferation and induction of apoptosis; However, Cu can significantly increase the inhibition of proliferation and induction of apoptosis. 2, DSF single drug and DSF / Cu, can significantly improve Molt4 intracellular ROS level, the DSF / Cu effect is more significant. 3, DSF / Cu can increase the expression levels Molt4 intracellular phosphorylation of JNK (p-JNK), lowered p65 and Nrf2 expression in a dose-dependent manner. DSF single drug lowered p65 levels were significantly lower than the DSF / Cu, and no significant effect on the expression of p-JNK. ROS inhibitors NAC can significantly inhibit the DSF / Cu Molt4 apoptosis induced capacity, and can inhibit the DSF / Cu regulation of p-JNK, p65 and Nrf2 protein expression, suggesting that ROS regulation of JNK, p65 and Nrf2 The passage of the key.
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