| BackgroundAcute lymphoblastic leukemia and non-Hodgkin lymphoma were the common disease in malignant tumor in hematological system. Owing to infiltration, relapse was the obstacle for T-ALL to cure. Burkitt's was an invasive NHL with disappointing results correlated with chemoresistance. For the past decades even though the treatments of lymphoid neoplasma have made great progress, despite the onset of lymphoid neoplasma in patients with great availability of clinical complete remission CR, but there are still more patients remission within 1 to 2 years. Due partly to the disappointing results correlated with chemoresistance and the non-specific side effects induced by high-dose chemotherapy, current research efforts are aimed at the identification of novel chemosensitizers which would target anti-apoptotic factors and improve therapeutic index of conventional anticancer drugs without further putting on patient's medication burden.Disulfiram (DSF), a member of the dithiocarbamate family capable of binding copper (Cu), is an anti-alcoholism drug used in the clinic with high safety and low toxicity for over 60 years. Various studies have shown that DS have anticancer effects on several types of solid cancers, but it is controversial about the cytotoxicity of DS to leukemia cells. As a divalent metal ion chelator, DS strongly chelates Cu and the formed DS/Cu complex significantly enhanced the cytotoxicity of DS alone to solid cancers and leukemia cells. There exists an coordination effect between DS and Cu in killing cancer cells.Drug induced cancer cells damage and triggered the generation of anti-apoptoticproteins. NF-κB is one of the major anti-apoptotic factors. NF-κB is typically a heterodimer that consists of p65 (Rel A) and p50 proteins. The most common p65 is relatively abundant, controls the expression of numerous genes. It leads to activation of NF-κB directly through p65 pathway. P65 is likely to be a key component in regulating NF-κB activity. The depletion of p65 induces cancer cell apoptosis and decreases expression of cellular inhibitors of apoptosis which is regulated by NF-κB. Over-expression of p65 induces increased NF-κB activity and drug resistance. High NF-κB activity blocks the cytotoxic drug-induced apoptosis. The p65 subunit indicates the NF-κB activation. This activation is induced by a variety of agents:their common feature is a direct or metabolic generation of ROS.While production of reactive oxygen species (ROS) is a consequence of basal cellular respiration, increased ROS production is associated with several pathological courses in many cellular systems. ROS promotes damage to the cell structure including proteins, lipids, and DNA. Under normal conditions, ROS are eliminated by anti-oxidant enzymes. Recent studies have shown that ROS are generated within cancer cells to maintain the growth of them. Because of strong anti-apoptotic system, cancer cells characterize by insensitivity to higher levels of ROS which play a key role for cancer cell proliferation. Relying on the higher levels of ROS in cancer cells, it is suggested that long-term exposure of cells to enhanced levels of ROS will lead to the cellular antioxidant capacity exhausted and apoptotic cell death. The apoptosis induced by ROS have been viewed as relying on persistent activation of pro-apoptotic JNK pathway. As a mediator of nuclear Nrf2, high levels of ROS insult the anti-oxidative of Nrf2, thereby triggering the apoptosis induced by ROS. The results demonstrated that there could be an interaction between ROS and JNK in the apoptosis.For many decades, the dithiocarbamate drug disulfiram (DSF) has been used for the treatment of alcohol abuse owing to its ability to inhibit aldehyde dehydrogenase (ALDH). More recently, investigations have focused upon the antitumor activity of DSF on its ability to induce apoptosis in a wide range of cancer cell lines, as well as its propensity to reduce cell growth in glioma, lung carcinoma and melanoma in human. Moreover, the effect of DSF/Cu on leukemia cells has not been published. In the study described here, we examined the effects of DSF or DSF/Cu on Molt4 cells and showed that DSF/Cu potently induced apoptosis in leukemic cells. Our investigating indicated the mechanisms of DSF/Cu-induced apoptosis depend on the modulating ROS-JNK pathway and NF-κB and Nrf2 pathway.The aims of this project are set as follows.1. To determine the in vitro chemosentisizing effect of DSF/Cu on the cytotoxicity in Molt4 and Raji cells.2. To investigate DSF/Cu-mediated changes in the ROS-JNK, NF-κB and Nrf2 pathway.Methods:a) Cell lines:human acute leukemia cell line Molt4 and Raji.b) MTT analysis of cytotoxicity of serial concentrations of DSF or DSF plus Cu (1μM) to Molt4 cells and Raji cells at 24 hours.c) 1) Flow cytometric analysis of the apoptotic Molt4 cells after treatment with 0.125,0.25,0.5,1,2μM/ml of DSF or DSF plus Cu (1μM) for 24 hours. 2) Flow cytometric analysis of the apoptotic Raji cells after treatment with 0.5,1,2.5,5,7.5μM/ml of DSF or DSF plus Cu (1μM) for 24 hours.d) Hoechst33342 staining detect cellular morphology of the serial concentrations of DSF or DSF plus Cu (1μM) to Molt4 cells and Raji cells.e) Flow cytometric analysis of the ROS level in Molt4 cells after treatment with 0.125,0.25,0.5,1,2μM/ml of DSF or DSF plus Cu (1μM) for 24 hours. At the same time, the cells ROS which treated by ROS inhibitor NAC plus DSF/Cu complex are determined by flow cytometric analysis.f) The expression of Nrf2 was measured by SYBR Green real-time PCR in DSF, DSF/Cu complex and DSF/Cu/NAC and the relative expression quantities of Nrf2 was calculated by formula of 2-Act.g) Western blotting analysis of JNK, p-JNK, Bcl-2, Nrf2 and p65 protein expression in Molt4 cells after treatment with serial concentrations of DSF or DSF plus Cu (1μM) and Cu(1μM), DSF (DSF:IC50-DSF/Cu), DSF/Cu (DSF:IC50-DSF/Cu, Cu:1μm), DSF/Cu/NAC (DSF:IC50-DSF/Cu, Cu:1μm, NAC:10mM) for 24hours.h) The statistical analyses were performed with the statistical software package SPSS 13.0. Student's t-test was used to compare IC50 values of two independent groups, Paried-Samples T Test was used to compare the apoptotic population of Molt4 and Raji cells between groups. One-Way ANOVA was used to compare the difference of apoptotic population of Molt4 and Raji cells and Bonferroni 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. Results1. Molt4 cells were exposed to serial concentrations of DSF(0.125,0.25,0.5,1,2,4μm/ml)for 24h. DSF demonstrated toxicity to cells with IC50(1.314±0.229μM/ml). The cytotoxicity have been enhanced by serial concentrations of DSF plus Cu (1μm). The IC50 value (0.435±0.109μM/ml) of DSF/Cu was significantly lower than that treated with DSF alone. Raji cells were exposed to serial concentrations of DSF(0.5,1,2.5,5,7.5,10μm/ml) for 24h. The IC50 of DSF in Raji cells was (5.064±2.268μM/ml), and IC50 of DSF/Cu complex was (0.891±0.929μM/ml). The cytotoxicity of DSF/Cu in Raji cells was seen to be very significantly enhanced, compared with DSF alone (p=0.004 and p=0.033).2. The flow cytometric annexinⅤ/PI staining analysis demonstrated significant incrementing of apoptosis after 24 hours in DSF/Cu treated but not other groups. In contrast to the massively induced apoptosis by DSF/Cu, the apoptotic population was not or slightly induced by DSF alone. In comparison with the untreated cells, low concentration of DSF (0.125μm/ml) showed slightly induced apoptosis. DSF at 0.25,0.5,1,2μm/ml significantly enhanced apoptosis after 24 hours treatment on all tested cancer cells. We found that different concentrations of DSF/Cu could induce cells apoptosis, compared with the control group, the apoptotic rate increased gradually in a dose dependent manner. Raji cells were treated with 0,0.5,1,2.5,5 and 7.5μm/ml DSF and DSF/Cu for 24 hours. The results showed that low concentration of (0.5,1,2.5μm/ml) slightly induced apoptosis comparing with control group. DSF at 5,7.5μm/ml significantly enhanced apoptosis after 24 hours treatment on all tested cancer cells. However, we found that different concentrations of DSF/Cu could induce Raji cells apoptosis, compared with the control group. Bcl-2 expression determined by Western Blotting did not significantly change after treatment with serial concentrations of DSF alone for 24h. In contrast, following treatment of cells for 24 hours with the DSF/Cu complex, a marked decrease in Bcl-2 expression was observed.3. We used Hoechst33342 to observed morphological changes, was found in low concentration of DSF(0.125,0.25,0.5μM), uniform nuclear diffuse blue fluorescence. Whereas, apoptotic morphology appeared under high concentration of DSF (1,2μM) treatment. However, DSF/Cu groups (0.125,0.25,0.5,1,2μM) were nuclear condensation, aggregation, and apoptotic bodies, showing bright blue staining granular. The similar apoptotic morphology of Raji cells appeared under DSF and DSF/Cu treatment.4. The Molt4 cells were exposed to DSF or DSF/Cu for 8 hours and analyzed the ROS level using the flow cytometric annexin. Our results demonstrated that compared with the control group, the ROS level in DSF group and DSF/Cu complex group have been enhanced. However, the ROS have been significantly enhanced by DSF/Cu complex compared with DSF alone. We supposed that comparing with DSF, DSF/Cu could enhance ROS level in cells.5. The Molt4 cells were treated with a series concentrations of DSF and DSF/Cu for 24 hours and analyzed the Nrf2 expression using the SYBR Green-PCR. The results demonstrated that DSF alone have no effect on Nrf2 in cells; but DSF/Cu complex suppressed the expression of Nrf2.6. Western blot results showed markedly increase in phosphorylation of JNK in Molt4 cells treated with serial combinations of DSF/Cu complex for 24h, while no obvious increase was noticed with DSF alone. There was no change in the constitutive expression of JNK. The expression of p65 also analysis by western blot indicated that DSF alone could inhibit the expression of p65. But the DSF/Cu group inhibited the p65 expression markedly. Western blot demonstrated that DSF alone have no effect on Nrf2 in cells; but DSF/Cu complex suppressed the expression of Nrf2.7. We treated Molt4 cells with different concentrations of DSF/Cu plus NAC for 24 hours and analyzed the apoptosis using the flow cytometric annexin. The results demonstrated that low concentrations (0.125,0.25,0.5μM) slightly induced apoptosis. But the DSF/Cu plus NAC at 1 or 2μM could significantly induced cell apoptosis. Meanwhile, we treated Molt4 cells with Cu (1μM),DSF (0.5μM),DSF/Cu (DSF:0.5μM, Cu:1μM) and DSF/Cu/NAC (DSF:0.5μM, Cu:1μM, NAC:10mM) for 24 hours. We found that the apoptosis was no different between Cu and control group as same as DSF and DSF/Cu/NAC. DSF, DSF/Cu or DSF/Cu/NAC could significantly induced apoptosis comparing with control group. However, the apoptosis induced by DSF/Cu was more than DSF/Cu/NAC. We supposed that DSF/Cu induced cellular ROS accumulation, and ROS was the key role of DSF/Cu inducing apoptosis.8. The Molt4 cells were exposed to several groups [Cu:1μM,DSF(0.5μM),DSF/Cu(DSF:0.5μM, Cu:1μM),DSF/Cu/NAC (DSF:0.5μM, Cu:1μM, NAC:lOmM/ML)] for 24h. The western blotting results showed that DSF/Cu increased the expression of JNK protein, while the no obvious increase was noticed with Cu or DSF alone. However, the expressions of p65 and Nrf2 have been inhibited by DSF/Cu, but the NAC could reversed them.Conclusion1. DSF alone was cytotoxicity to neopasia of the lymphoid system, and DSF plus Cu could significantly enhance the cytotoxicity to these cells by inhibiting proliferation and inducing apoptosis.2. DSF and DSF/Cu complex promoted the ROS accumulation in cells, but the DSF/Cu are more effective.3. DSF/Cu complex could significantly induce phosphorylation of JNK in Molt4 cells, while suppressed the expression of p65 and Nrf2. DSF/Cu effectually suppressed the expression of p65, but there was no influence in cellular p-JNK which treated with DSF alone.4. DSF/Cu plus ROS inhibitor NAC not only decreased the expression of phosphorylation of JNK but also increased the expression of p65 and Nrf2. These results indicated that ROS was the criminal role to modulate the JNK, p65 and Nrf2 pathway. |
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