| BackgroundAcute leukemia (AL) is one of the top ten malignant tumors in china and occupyies the highest incidence of malignancy among teenagers and adults younger than 35 years old.With the aggravated environmental pollution in recent years,the incidence of AL increased year by year and has severely threatened people's health.Acute myeloid leukemia (AML) is the most common type of adult AL.With the improvement of chemotherapy regimens,70% of the AML patients can achieve complete remission(CR).However, relaps would occur in most of the CR patients thereby accompanying with a poorer prognosis.So relaps and resistance to chemotherapy is currently the major obstacles for successful treatment of patients with acute myeloid leukemia.Chemotherapy remains the first treatment choice for AML patients,however, the clinical success of AL chemotherapy has been seriously hindered by de novo and acquired chemoresistance of leukemia cells and the severe non-specific toxicity of high dose chemotherapy to vital tissues and organs.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 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.c-Jun NH2-terminal kinase (JNK) is a member of the mitogen-activated protein (MAP) kinase family and is activated by a variety of extracellular stimuli through a MAP kinase cascade consisting of JNK kinases (JNKK1/MKK4/SEK1 and JNKK2/MKK7) and multiple MAP kinase kinase kinases. Activated JNK, in turn, phosphorylates and activates c-Jun, a major component of the transcription factor AP-1, as well as other targets, and play a critical role in diverse cellular processes including regulation of cell proliferation, differentiation and apoptosis. Recent studies have demonstrated that JNK inactivation is a possible mechanism of inducing anthracycline resistance in AML. Certain agents have also been shown to induce apoptosis in leukemia cells by activating the JNK pathway, thereby suggesting that JNK inactivation may be another important mechanism correlated with multidrug resistance(MDR) and may be a target for reversal of chemoresistance.Despite DS/Cu was previously reported to sensitize chemotherapeutic agents to several types of solid cancers, the exact targets and molecular mechanism are still unknown.Morever,the effect of DS/Cu to drug resistant leukemia cells has not been literated. In this study, we investigated the in vitro chemosentisizing effect of DS/Cu on the cytotoxicity of doxorubicin(Dox) in HL60/Dox cells. We also examined DS/Cu-mediated changes in the JNK pathways after treatment with Dox.The aims of this project are set as follows.1. To determine the In vitro chemosentisizing effect of DS/Cu on the cytotoxicity of Dox in HL60/Dox cells.2. To investigate DS/Cu-mediated changes in the JNK/c-Jun pathway after treatment in combination with Dox.Methods:1. Cell lines:Dox resistant human acute myeloid leukemia cell line HL60/Dox and the wild-type cell line HL60.2. MTT analysis of cytotoxicity of Dox to HL60 and HL60/Dox cells.3. MTT analysis of cytotoxicity of serial concentrations of DS plus Cu (1μM) to HL60/Dox cells at 24h and 48h.4. MTT analysis of enhancing effect of～IC20 concentration of DS/Cu complex on the cytotoxicity of Dox in HL60/Dox cells at 24h.5. Flow cytometric analysis of the apoptotic HL60/Dox cells after treatment with DS/Cu complex,Dox or DS/Cu plus Dox for 24h.To vertify whether the in vitro chemosentisizing effect of DS/Cu on the cytotoxicity of Dox in HL60/Dox cell line was correlated to JNK/c-Jun pathways,HL60/Dox cells were firstly pretreated with 20μM of JNK/c-Jun pathway inhibitor SP600125 (Sigma, USA) for 2h, and then exposed to various treatments for 24h as described previously for Flow cytometric analysis.6. Morphological observation of HL60/Dox cells after exposure to indicated treatment as described above for 24h.7. Western bloting analysis of JNK,p-JNK,c-Jun,p-c-Jun and Bcl-2 protein expression in HL60/Dox cells after different treatment as described above.8. 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 HL60/Dox cells between groups with or without Sp600125.One-Way ANOVA was used to compare the difference of apoptotic population of HL60/Dox 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. MTT analysis showed the acquired Dox-resistant cell line (HL60/Dox; IC50=7.69±1.87) was highly resistant to Dox (71.87-fold) compared to the wild-type HL60 cells (IC50=0.107±0.017) (t=7.000,p=0.002)2. The HL60/Dox cells were exposed to serial concentrations of DS in combination with CuCl2 (1μM) for 24 h and 48 h and analysed using the MTT technique. DS/Cu complex demonstrated high toxicity to HL60/Dox cell line with IC50=1.11±0.025μM and 0.439±0.108μM at 24 h and 48 h, respectively.The cytotoxicity of DS/Cu complex in HL60/Dox cell line enhanced accompanying with prolonged time and increased concentration. The IC50 value of DS/Cu at 48 h was significantly lower than that at 24h incubation(t=14.114,p=0.005).3. Furthermore, to examine if DS/Cu complex can enhance the cytotoxicity of Dox in the HL60/Dox cell line, the cells were exposed to increasing concentrations of Dox in combination with the IC20 concentration of DS/Cu complex (0.63μM) in 1μM solution of CuCl2, to simulate the in vivo cell environment, for 24 h. The effect of exposing the HL60/Dox leukaemia cells to various concentrations of Dox in combination with DS/Cu complex is represented on the dose-effect curves.When combined with DS/Cu complex, the cytotoxicity of Dox in HL60/Dox cells (IC50=0.48±0.21μg/ml) was seen to be very significantly enhanced (15.95-fold;t=6.654,p=0.003), compared with Dox alone (IC50=7.69±1.87μg/ml)4. To investigate the effect of apoptosis induction with combination of Dox and DS/Cu complex in HL60/Dox leukaemic cells, the cells were exposed to Dox (1.25μg/ml),DS/Cu(DS:0.63μM, CuCl2:1μM) alone and Dox plus DS/Cu for 24 h. The cells were harvested and their DNA contents stained with PI and Annexin V are analysed by the flow cytometry technique.Treatment of the cell line with DS/Cu or Dox alone showed no significant increase in apoptotic population compared with the control cells(F=92.326, p>0.05). When treated with the combination of Dox and DS/Cu complex, a 11.46-fold increase in the population of apoptotic cells was observed compared with Dox alone.(F=92.326, p<0.05). For pharmacological inhibition study, HL60/Dox cells were pretreated with the JNK inhibitor SP600125 (20μM, sigma, USA) for 2 hour, and subsequently exposed to different treatment for 24 hours for for Flow cytometric analysis.SP600125 obviously decrease DS/Cu + Dox induced apoptosis of HL60/Dox cells from 73.24±10.58% to 40.70±6:03%(t=6.218,p=0.025).5. HL60/Dox Cells treated with DS/Cu or Dox alone did not show any visible apoptosis characters after 24 hours treatment. Whereas, apoptotic morphology appeared under DS/Cu plus Dox treatment. Apoptosis is characterized by marked changes in cellular morphology, including chromatin condensation, membrane blebbing, nuclear breakdown, and the appearance of membrane-associated apoptotic bodies, internucleosomal DNA fragmentation.Notabley,the apoptosis characters of HL60/Dox cell exposed to DS/Cu plus Dox were inbited with addition of JNK inhibitor SP600125.6. Western blot results showed markedly increase in phosphorylation of JNK and c-Jun in HL60/Dox cells treated with a combination of Dox and DS/Cu complex for 24h, while no obvious increase was noticed with Dox alone and DS/Cu complex alone. Although there was no change in the constitutive expression of JNK, expression of c-Jun was also noticeably increased with the combination therapy. As a control, HL60/Dox cells pretreated with 20μM of JNK inhibitor SP600125 for 2h were exposed to various treatments for 24 h and analysed by Western blotting. As expected, SP600125 inhibited the Dox plus DS/Cu complex-induced phosphorylation of JNK, and c-Jun and the increased expression of c-Jun.7. HL60/Dox cells were were exposed to Dox (1.25μg/ml),DS/Cu(DS:0.63μM, CuCl2:1μM) alone and Dox plus DS/Cu for 24 h.Western blotting analysis demonstrated Bcl-2 expression increased in HL60/Dox cells treated with Dox alone and decreased significantly in the presence of DS/Cu complex compared with control cells.Conclution1. DS/Cu alone was cytotoxicity to HL60/Dox cells,and-IC20 concentration of DS/Cu can significantly enhance the cytotoxicity of Dox to HL60/Dox cells by inhibiting proliferation and inducing apoptosis.2. DS/Cu plus Dox can sigificantly induced phosphorylation of JNK, c-Jun and the expression of c-Jun in HL60/Dox cells, while JNK inhibitor Sp600125 not only decreased the apoptotic population of HL60/Dox but also markedly inhitbited the protein expression of JNK/c-Jun pathway,indicating JNK/c-Jun pathway is responsible for the chemosentisizing effect of DS/Cu on the cytotoxicity of Dox in HL60/Dox cells.3. Treatment of the HL60/Dox cells with the combination of Dox and DS/Cu resulted in the inhibition of the constitutive expression of Bcl-2.Inhibition of Bcl-2 activity may be another important mechanism through which DS/Cu can enhance the cytotoxicity of Dox to HL60/Dox cells.
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