The Influence Of Disulfiram/Cu Complex On Raji Cells And Xenograft Tumor Growth Of Raji Cell In Nude Mice And Its Mechanisms

BackgroundBurkitt’s lymphoma is an invasive B cell non Hodgkin’s lymphoma with rapid disease progression and high death rates.As the development of chemotherapy and immunotherapy progressed,the therapeutic effect for patients with malignant lymphoma has been obviously improved. 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 (DS), 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. Various studies have shown that DS have anticancer effects on several types of solid cancers,it also shows cytotoxicity to leukemia cells in our previous research.s 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.It is still unknown that the influence of DS or DS/Cu on lymphoma cells and its mechanisms.Consequently.it is necessary to make a further exploration.Mitochondria are also known to be a major source of ROS, that is, superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH), which have been implicated as mediators of apoptosis in response to several anti-cancer drugs.Not only reactive oxygen species (ROS) can induce inflammatory injury as a toxic chemical,but in recent years it has been found an important member of signal protein family with significant effect on the regulation and function of cells.ROS as an early signal effect plays important role in cell apoptosis. ROS-induced a caspase-dependent apoptosis occurred through the mitochondrial death pathway as evidenced by reduction of the mitochondrial transmembrane potential, cytochrome c release and caspase-9activation. Interaction of the released cytochrome c with dATP and APAF-1results in the activation of the initiator caspase, caspase-9, which in turn triggers activation of executor caspases of cell death.More ROS generated in cells with mitochondrial damage can further induce cell apoptosis. 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.Advancement has been made in the study of anti-tumor effects for DS in recent years,especially in cell apoptosis.The mechanism of pro-apoptotic effect probably associated with diverse signal transduaction pathways.C-Jun NH2-terminal kinase (JNK)is an important member of MAPK cascade.JNK activation exerts its proapoptotic property especially induced by various cellular stress.Under external stimulus, the up-regulation or phosphorylation of JNK could activate c-jun as its substration that is referred to nucleus leading to the cell apoptosis.However,ROS is one of the most common cell stresses.Recent studies found that sustained JNK activation caused by the increase of ROS production will lead to cell death.Thus,it is speculated that JNK signal pathway and the effect of ROS on it play important role in the mechanism of malignant lymphoma cells apoptosis induced by DS or DS/Cu.NF-κB is constitutively activated responsible for regulation of gene expression for cell survival、differentiation and apoptosis in human various tumor cells. NF-κB is typically a heterodimer that consists of p65(Rel A) and p50proteins. The most common p65is relatively abundant, controls the expression of numerous genes. The depletion of p65induces cancer cell apoptosis and decreases expression of cellular inhibitors of apoptosis which is regulated by NF-κB.Previous study has shown that DS/Cu could induce apoptosis of breast cell lines by generally controlling the expression level of NF-κB.Moreover,NF-κB activation can be inhibited by ROS.Therefore,proapoptotic induced by JNK activation may be improved by inhibition of NF-κB.Nuclear factor-E2-related factor2(Nrf2) is a key transcription factor for modulation of oxidate stress. In the absence of stimuli, Keap-1inhibits the transcriptional activity of Nrf2by retaining it in the cytoplasm. However, oxidation of redox-sensitive cysteines within Keap-1releases Nrf2, and Nrf2then translocates fromthe cytosol to the nucleus. In the nucleus, Nrf2increases gene expression by binding to the antioxidant response element (ARE) of phase Ⅱ antioxidant enzymes、 antioxidation enzymes,and are closely correlated with drug resistance of tumor cells.Significant accumulation of ROS in tumor cells can inhibit the activity of Nrf2,and in turn accelerating the apoptosis induced by ROS.Nrf2is expected to be a novel therapeutic target for anticancer therapy.In this study,Raji cells and four week female nude mice were investigated.We investigated the in vitro effect of DS/Cu on Raji cells and the relation with ROS/Nrf2、JNK and NF-κB pathways. We also investigate the in vivo influence of DS or DS/Cu on xenograft tumor growth of Raji cells in nude mice,laying a solid experimental base for future clinical application.The aims of this project are set as follows.1. To determine the in vitro chemosentisizing effect of DS/Cu on the cytotoxicity in Raji cells and the in vivo influence of DS/Cu on xenograft tumor growth of Raji cells in nude mice.2. To investigate DSF/Cu-mediated changes in the ROS-JNK, NF-κB and Nrf2pathway.Methods:a) Cell lines:human acute leukemia cell line Raji.b) Experimental animals:four week female nude mice.c) MTT analysis of cytotoxicity of serial concentrations of DS or DS plus Cu (1μM) to Raji cells after72hours.d) Flow cytometric analysis of the apoptotic Raji cells after treatment with3.3μM of DS or DS plus Cu (1μM) for6hours、12hours and24hours.e) Flow cytometric analysis of the ROS level in Raji cells after treatment with3.3μM of DS or DS plus Cu (1μM) for6hours、12hours and24hours.f)1) Western blotting analysis of p65protein expression in Raji cells after treatment with Cu(1μM), DS(DS:IC50-DS/Cu), DS/Cu(DS:IC50-DS/Cu, Cu:1μm) for24hours and P-JNK、C-jun protein expression after treatment with Cu (1μM), DS (DS:IC50-DS/Cu), DS/Cu (DS:IC50-DS/Cu, Cu:1μm) respectively for6hours and1hour.2) Western blotting analysis of Nrf2protein expression in Raji cells after treatment with Cu(1μM), DS(DS:IC50-DS/Cu), DS/Cu (DS:IC50-DS/Cu, Cu:1μm) for6hours、12hours and24hours.3) Western blotting analysis of Nrf2, C-jun and P65protein expression in Raji cells after treatment with DS (DS:IC50-DS/Cu), DS/Cu (DS: IC50-DS/Cu, Cu:1μm), DS/Cu/NAC (DS:IC50-DS/Cu, Cu:1μm, NAC:10mM) for24hours.g)1) Establish animal model:Burkitt’s lymphoma xenograft was established by subcutaneous injection of Raji cell into nude mice(3×107Raji cells were resuspended in200μl saline).2)18bearing tumor mice were randomly grouped into control group、DS group and DS/Cu group.Nude mice were administrated intragastrically with3mg/20g DS and DS plus with0.17mg/20g Cu for+1～+5day and+8～+12day after Raji cells injection.Drugs were resuspended in200μl PBS.Control mice were given equal volumes of normal saline instead of the drug.3) Tumor volume was measured by vernier caliper and weight was made every two days.At+21days after Raji cells injection,tumor and tissues were peeled off and fixed in10%buffered formalin. The typical apoptotic morphological changes were observed under the light microscope.h) 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, Paried-Samples T Test was used to compare the apoptotic population of Molt4and Raji cells between groups. One-Way ANOVA was used to compare the difference of apoptotic population of Molt4and Raji cells and Bonferroni 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.Results1. Raji cells were exposed to serial concentrations of DS (0.125、0.25、0.5、1、2、4μM)for72h. DS demonstrated toxicity to cells with IC50(0.793±0.0.08μM). The cytotoxicity have been enhanced by serial concentrations of DS plus Cu (1μm). The IC50value (0.085±0.015μM/ml) of DS/Cu was significantly lower than that treated with DS alone(t=15.110,P=0.000).2. The Raji cells were exposed to DS or DS/Cu for6hours、12hours and24hours and the apoptotic cells were analyzed using the flow cytometric annexin. Our results demonstrated that the apoptotic proportion in DS group and DS/Cu complex group have been enhanced compared with the control group. However, the apoptosis have been significantly enhanced by DS/Cu complex compared with DS alone. The apoptotic cells proportion showed a time-dependent effct and reached its peak after24h.3. The Raji cells were exposed to DS or DS/Cu for6hours、12hours and24hours and the ROS level was analyzed using the flow cytometric annexin. Our results demonstrated that the ROS level in DS group and DS/Cu complex group have been enhanced compared with the control group. However, the ROS have been significantly enhanced by DS/Cu complex compared with DS alone. ROS level showed a time-dependent effct and reached its peak after24h.4. Western blotting results showed significant increase in phosphorylation of JNK and C-jun protein expression in Raji cells treated with3.3u M of DS or DS/Cu complex respectively for1h and6h. The expression of P65also analyzed by western blotting indicated that DS alone could inhibit the expression of P65,but the DS/Cu group inhibited the P65expression markedly.5. We treated Raji cells with DS or DS/Cu for6hours、12hours and24hours,the western blotting results showed that Nrf2protein expression in Raji cells were inhibited by DS alone afer6hours or12hours,but it showed increases after24hours.This role was increased by treatment with DS/Cu complex. 6. Meanwhile, we treated Raji cells withDS (3.3μM)、DS/Cu (DS:3.3μM, Cu:1μM) and DS/Cu/NAC (DS:3.3μM, Cu:1μM, NAC:10mM) for24hours. The C-jun protein expression were inhibited by DS/Cu/NAC treatment,but the Nrf2and P65protein expression were increased by DS/Cu/NAC.These results suggest that the NAC could reversed the effect induced by DS/Cu.7. The xenograft of Raji cells were developed in75%nude mice.The median time for tumor formation was6days(5-8days),there was no significant differences among three groups.Subsequent tumor size and weight in DS or DS/Cu-treated animals was reduced significantly relative to tumors in vehicle-treated animals(p=0.028andp=0.011;P=0.019andP=0.005).There was no significant differences between DS or DS/Cu-treated animals(P>0.05). We found increases in the tissue infiltration of lymphoma cells in control mice,compared with DS or DS/Cu treated mice.The Nrf2and P65protein expression in the tumors were inhibited by DS or DS/Cu treatment.Conclusion1. DS alone was cytotoxicity to neopasia of the lymphoma cell, and DS plus Cu could significantly enhance the cytotoxicity to Raji cells by inhibiting proliferation and inducing apoptosis.2. DS and DS/Cu complex promoted the ROS accumulation in cells, but the DS/Cu are more effective.3. DS/Cu complex could significantly increase phosphorylation of JNK and C-Jun in Raji cells, while suppressed the expression of P65.4. Significant accumulation of ROS in cells could inhibit Nrf2protein expression.5. DS/Cu plus ROS inhibitor NAC not only decreased the expression of phosphorylation of JNK but also increased the expression of P65and Nrf2. These results indicated that ROS was the criminal role to modulate the JNK, P65and Nrf2pathway.6. In vivo of nude mice,the growth and proliferation of Raji cells can also be inhibited by DS alone,this role could be improved by treated with DS/Cu.DS or DS/Cu treatment could inhibit Nrf2and P65protein expression.