Introduction
Lymphoid malignances are tumors of the immune system. Although treatments including chemotherapy and stem cell transplantation are developing very fast, patients still suffer from relapse and treatment related complications. In order to improve the prognosis and life quality of patients with lymphoid malignances, new therapeutic strategies are urgently demanded.
Disulfiram (DS) is an anti-alcholism drug used in clinic for over 60 years [
1]. DS belongs to dithiocarbamate family which is able to strongly chelate Cu and forms disulfiram/cooper (DS/Cu) complex. DS/Cu complex is highly cytotoxic to many solid tumors while DS or Cu alone had few anti-tumor effects [
2‐
4]. However, the effect of DS/Cu on lymphoid malignancies has not been reported yet.
Reactive oxygen species (ROS) contains a group of oxygen-containing chemical species normally generated from mitochondrial respiratory chain reaction with reactive chemical properties [
2]. Cancer cells usually possess and tolerate higher ROS activity than normal cells [
3]. It has been demonstrated that further increasing ROS exposure induced by ROS-generating agents such as DS/Cu [
3] can exhaust the cellular antioxidant capacity and thus induce apoptosis in tumor cells.
Nrf2 is a transcription factor with strong antioxidant effect which protects cancer cells from the damages induced by ROS, anticancer drugs and other harmful chemicals [
3]. Previous publications have demonstrated the causal relationship between Nrf2 and chemo-resistance [
5‐
7]. So, simultaneous down-regulation of Nrf2 and induction of ROS in cancer cells can lead to more apoptosis.
ROS-induced apoptosis is also highly reliant on persistent activation of pro-apoptotic mitogen activated protein kinase (MAPK) pathways [
8]. C-Jun NH2-terminal kinase (JNK) is an important member of the MAPK family [
9]. Our previous study has already shown that activation of JNK can sensitize resistant cells to anticancer drugs indicating the important role of JNK in drug-induced apoptosis [
10].
NF-κB is one of the major chemo-resistance-related anti-apoptotic factors [
8]. Lymphoid malignant cells possess high levels of constitutive NF-κB activity, leading to resistance to apoptosis [
7]. Thus, NF-κB is an attractive molecular target for therapeutic intervention. Previous studies have also shown close interactions between ROS and NF-κB [
8].
Although ROS could trigger apoptosis in cancer cells, the ROS-induced anti-apoptotic factors, e.g. NF-κB and Nrf2, can counteract apoptotic effects of ROS. Hence, development of drugs that can simultaneously activate sustained ROS pro-apoptotic pathway and inhibit NF-κB and Nrf2 activity may improve cancer chemotherapy. This study demonstrates that DS/Cu simultaneously activates ROS-JNK pro-apoptotic pathway and down-regulates NF-κB and Nrf2 anti-apoptotic pathways. Therefore, DS/Cu shows strong anti-cancer efficacy in vitro and in vivo.
Materials and methods
Cell lines and experimental animals
Raji and Molt4 cell lines were cultured at 37°C in 5% CO2 in RPMI 1640 with 10% heat-inactivated FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. The BALB/C nude mice (4–5 weeks of age, non-fertile, female and 18–20 g each) were purchased from the Experimental Center of Southern Medical University. All animal study procedures were approved by the Southern Medical University Animal Care and Use Committee.
Establishment of non-Hodgkin lymphoma animal model and treatment
Mice were injected subcutaneously on the back with 1×107 Raji cells suspended in 0.2 mL sterile PBS. Nude mice were randomly divided into 3 groups, and with 6 mice in each group. The day mice received cell injection was counted as day 0. Mice in the control group were administrated with normal saline by oral gavage once per day for 10 days from +8 day to +12 day and +15 day to +19 day while those in DS and DS/Cu groups were given DS (2.88 mg/20 g per morning) and DS/Cu (DS: 2.88 mg/20 g per morning, Cu: 0.012 mg/20 g per afternoon) respectively. The dosage used here was determined by our preliminary experiment. Length and width of the tumor were measured with a vernier caliper from the beginning of treatment every other day with the volume being calculated using the formula V = a × b2/2 and growth curve being documented. The weight of each mouse was also recorded every other day. Mice were sacrificed on day 20, and the tumors were removed for western blotting analysis.
MTT cytotoxicity assay
Drug cytotoxicity was determined using the colorimetric MTT assay. Briefly, cells (1 × 105 cells/well) were plated into 96-well plates containing 100 μl of the growth medium in the absence or presence of increasing concentrations of drugs at 37°C in 5% CO2 for 24 h and 48 h, MTT (50 μl/well, 5 mg/ml in PBS) was then added and incubated for 4 h at 37°C. The cells were further treated with 100 μl DMSO to dissolve the dark blue crystals of formazan and the absorbance was measured at 570 nm in a microplate reader (ELX800, BioTEK, USA). All experiments were repeated at least three times with triplicate in each experiment. The cytotoxicity of DS or DS/Cu was analyzed and concentration-effect curves were generated as a plot of the fraction of affected cells versus drug concentration. Growth inhibition was expressed as a percentage of the untreated controls that were processed simultaneously. The IC50 was defined as the concentration that inhibited cell growth by 50% (50% reduction of absorbance) compared with untreated controls.
Flow cytometric analysis of apoptotic cells
Cells (3 × 105) cultured in 25 cm2 flasks and exposed to different treatments for 6 h, 12 h, or 24 h were harvested respectively, washed twice with ice-cold PBS and then re-suspended in 500 μl binding buffer. The cells were further incubated with Annexin V-FITC and Propidium Iodide for 15 min at room temperature in the dark according to the manufacturer’s instructions. The stained cells were analyzed by flow cytometry using FACS Calibur (BD Biosciences, Oxford, UK) and Cell Quest (BD Biosciences) software.
Determination of ROS production
ROS production in cells was determined utilizing 2’,7’- dichlorodi hydro fluorescein diacetate (DCFDA). Briefly, 4 × 105/ml cells were taken in a culture dish and treated with DS or DS/Cu for 6 h, 12 h or 24 h. After the treatment, cells were collected and DCFDA (Sigma-Aldrich, Dorset, UK) was added to the cell suspension at a final concentration of 10 μM. After 30 minutes of incubation in the dark at 37°C, cells were centrifuged and the pellet was washed twice with ice-cold PBS. The pellet was then resuspended in FACS buffer and the fluorescence was analyzed with FACS Calibur (BD Biosciences, Oxford, UK) and CellQuest (BD Biosciences) software. DCFDA fluorescence intensity was measured in FL-1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The percentage of ROS producing cells was calculated by counting only those cells, which produced high levels of ROS.
Western blot analysis
Whole protein (50 μg/lane) from each sample was resolved in 10% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a PVDF membrane (Millipore, UK) and blotted with various antibodies. Non-specific binding was avoided by blocking the nitrocellulose membrane with 5% skimmed milk in TBS-T for 1 h. The 5% skimmed milk in TBS-T was also used to dilute primary (SAPK/JNK, rabbit polyclonal, 1:1000, CST; Phospho-SAPK/JNK, rabbit polyclonal 1:1000, Cell Signaling techonology; Phospho-c-jun, rabbit polyclonal, 1:500, Bioworld Technology Co., Ltd., c-jun, rabbit polyclonal, 1:1000, Santa cruz; P65, rabbit polyclonal, 1:1000, Santa Cruz; Nrf2, rabbit polyclonal, 1:1000) and HRP-conjugated monoclonal secondary (1:5000; Amersham Pharmacia Biotech, NJ) antibodies. The membranes were incubated with the primary antibodies overnight at 4°C and in the secondary antibody for 1 h at room temperature. The quantity of protein loaded was verified by staining the same membranes with anti-β actin antibody (1:2000, Sigma-Aldrich, Dorset, UK). The signals were detected on X-ray films using an ECL Western blotting detection kit (Amersham, Pharmacia Biotech).
Statistical analysis
All results were analyzed by Student’s t-test and ANOVA using SPSS 13.0. The statistical significance was indicated by a p value < 0.05.
Discussion
Disulfiram is a Food and Drug Administration-approved anti-alcoholism drug used in clinic for more than 6 decades with numerous available pre-clinical and clinical data [
1]. Previous studies have already demonstrated its low toxicity to normal tissues [
1]. Recently, the anti-cancer ability of disulfiram has been reported [
11]. Moreover, DS can facilitate intracellular Cu uptake in cancer cells and potentiate the cytotoxicity of anticancer drugs in drug resistant and sensitive breast, colon cancer and leukemia cell lines [
12]. We have reported that DS/Cu is cytotoxic and able to reverse chemoresistance in drug resistant HL60/DOX leukemia cell line
in vitro[
10]. However, there was no report about the cytotoxicity and related molecular mechanisms of DS/Cu in lymphoid malignant cell lines.
In this study we first demonstrated that DS has a high cytotoxicity in lymphoid malignant cell lines in a Cu-dependent manner both
in vitro and
in vivo. DS/Cu can significantly induce more apoptosis than DS or Cu alone
in vitro while inhibiting the growth of xenograft lymphoma in mice. Furthermore, there has been a long history of using Cu to treat cancer [
13]. However, the intracellular transport of Cu remains to be a major hurdle for its clinical use. N,N-diethyldithiocarbamte (deDTC), one of the DS derivatives, could bind to Cu forming a Cu (deDTC)
2 complex which improves the intracellular trafficking of Cu [
5]. Previous publication has already reported that children bearing lymphoma have higher serum Cu level than healthy ones. The Cu concentrations in the serum are associated with the disease stages [
14]. Herein, DS can target cancer cells to treat lymphoma with little harm to normal tissues.
However, the mechanism of DS/Cu induced apoptosis remains unclear. Previous publications demonstrate that in combination with Cu, DS induces ROS activity in cancer cell lines [
11]. Cancer cells are under higher ROS stress than normal tissues since they usually possess high proliferative rate [
2]. However, high levels of ROS can damage DNA, mitochondrial inner membrane and membrane phospholipids leading to apoptosis [
2]. Thus, generation of ROS might be a novel method to treat cancers. Recent studies have also demonstrated that generation of ROS by drugs can induce apoptosis in lymphoid malignant cell lines [
15]. In consistence with these results, our study confirmed that DS/Cu induces ROS in Raji cell lines which might contribute to DS/Cu induced apoptosis. When ROS inhibitor NAC was added, the cytotoxicity of DS/Cu to Raji cell lines was significantly alleviated.
The c-Jun N-terminal kinase (JNK) signaling pathway is known to play a critical role in diverse cellular processes including regulation of proliferation, differentiation and apoptosis [
9]. Moreover, previous studies indicated that the sustained activation of JNK is essential for drug-induced apoptosis in lymphoma cell lines
in vitro[
16]. Furthermore, ROS are potent activators of JNK through oxidative inactivation of endogenous JNK inhibitors, such as JNK phosphatases and glutathione
S-transferase π [
4]. In accordance with previous studies, we found that JNK pathway was persistently (over 24 h) activated (phosphorylation of cJun) by DS/Cu both
in vitro and
in vivo and blocked by ROS inhibitor NAC
in vitro, confirming the important role of ROS in DS/Cu induced apoptosis.
As a double-edged sword, apart from induction of apoptotic factors, ROS also induces expression of anti-apoptotic factors. The effect of ROS on cancer cells depends on the balance between ROS-induced pro- and anti-apoptotic factors. Lymphoid malignant cell lines constitutively express high levels of NF-κB, an important anti-apoptotic factor [
16]. However, previous study has demonstrated that certain NF-κB-regulated genes play a major role in regulating the amount of ROS in the cells [
16]. ROS have various inhibitory or stimulatory roles in NF-κB signaling [
11]. Owing to the sustained ROS generation by DS/Cu in Raji cells, p65, the vital component of NF-κB, was significantly inhibited by DS/Cu
in vitro and
in vivo, and this inhibition can be blocked by NAC.
Nrf2 is another important anti-apoptotic factor induced by ROS [
3]. Nrf2 is a transcription factor that plays a vital role in activating antioxidant response which abolishes ROS activity, detoxifies harmful chemicals and eventually protects cancer cells from chemo- and radiotherapy-induced damage [
3]. It can regulate various downstream genes with a wide variety of functions, such as cellular redox homeostasis, cell growth and apoptosis, DNA repair, inflammatory response, and the ubiquitin-mediated degradation pathway [
3]. It has already been demonstrated that high background nuclear levels of Nrf2 in leukemia cells reduce the sensitivity of cancer cells to proteasome inhibitors [
14]. Thus targeting Nrf2 might be an effective way to induce cancer cell apoptosis. Our study showed that DS/Cu induces ROS generation while inhibiting Nrf2 level in lymphoma Raji cells. Figure
4A showed that Nrf2 level might increase at low levels of ROS activity but decrease when ROS activity increases. We also observed the decrease of Nrf2
in vivo. Therefore, increase in ROS activity might cause the level of Nfr2 to decrease and thus lose its ability to protect lymphoma cells from oxidative stress and thus induce cell apoptosis. This phenomenon might due to the destruction effect of ROS on cells. Since cells from normal tissue usually have lower basal ROS level and complete protective mechanism, therefore the level of ROS will remain relatively low and high levels of Nrf2 will prevent the cells from ROS induced damage.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BX and WGW designed the experiments and wrote the manuscript. FLC performed the MTT assay and participated in the writing process. HJD did the flow cytometric analysis and participated in the writing process. PCHSH did the western blot assay. YY,Zh did the ROS assay, RWL did the statistical analysis, SYW ,PL, YY and JZ performed animal experiments and wrote the manuscript. All the authors have read and approved the manuscript.