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copper markedly induced myeloma cell apoptosis through activation of JNK and intrinsic and extrinsic apoptosis pathways
Biomedicine & Pharmacotherapy 126 (2020) 110048
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Disulfiram/copper markedly induced myeloma cell apoptosis through activation of JNK and intrinsic and extrinsic apoptosis pathways
T
Yaqi Xua,b, Qian Zhoua,b,c, Xiaoli Fengd, Yibo Daia,b, Yang Jianga,b, Wen Jianga,b,e, Xiaoli Liua,b, Xiangling Xinga,f, Yongjing Wanga,b, Yihong Nig,*, Chengyun Zhenga,b,* a
Department of Hematology, The Second Hospital, Institute of Biotherapy for Hematological Malignancies, Shandong University, Jinan, Shandong, China Shandong University-Karolinska Institute Collaboration Laboratory for Stem Cell Research, Jinan, Shandong, China c Haemal Internal Medicine, Linyi Central Hospital, Yishui Country, Linyi, Shandong 276400, China d Clinical Laboratory, The Second Hospital, Shandong University, Jinan, Shandong, China e Central Laboratory, The Second Hospital, Shandong University, Jinan, Shandong, China f Department of Medicine, Center for Molecular Medicine (CMM) and Bioclinicum, Karolinska Institutet and Karolinska University Hospital Solna, 17164, Solna, Sweden g Department of Endocrine, the Second Hospital, Shandong University, Jinan, Shandong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Disulfiram Myeloma Apoptosis JNK Drug repurposing
Disulfiram (DSF) is an FDA approved anti-alcoholism drug in use for more than 60 years. Recently, antitumor activity of the DSF/copper (DSF/Cu) complex has been identified. Its anti-multiple myeloma activity, however, has barely been investigated. In the present study, our results demonstrated that the DSF/Cu complex induced apoptosis of MM cells and MM primary cells. The results indicated that DSF/Cu significantly induced cell cycle arrest at the G2/M phase in MM.1S and RPMI8226 cells. Moreover, JC-1 and Western blot results showed that DSF/Cu disrupted mitochondrial membrane integrity and cleaved caspase-8 in MM cells, respectively, suggesting that it induced activation of extrinsic and intrinsic apoptosis pathways. Interestingly, DSF/Cu induced caspase-3 activation was partly blocked by Z-VAD-FMK (zVAD), a pan-caspase inhibitor, indicating at caspase-dependent and -independent paths involved in DSF/Cu induced myeloma cell apoptosis machinery. Additionally, activation of the c-Jun N-terminal kinase (JNK) signaling pathway was observed in DSF/Cu treated MM cells. More importantly, our results demonstrated that DSF/Cu significantly reduced tumor volumes and prolonged overall survival of MM bearing mice when compared with the controls. Taken together, our novel findings showed that DSF/Cu has potent anti-myeloma activity in vitro and in vivo highlighting valuable clinical potential of DSF/Cu in MM treatment.
1. Introduction Multiple myeloma (MM) is a hematological malignancy characterized by abnormal accumulation of cancerous plasma cells in the bone marrow, secretion of monoclonal immunoglobulins, development of bone lytic lesions and extramedullary organ invasion at the later stage of the disease [1]. In the past twenty years, application of new drugs such as proteasome inhibitors (e.g., bortezomib and carfizomib), immunomodulatory drugs (e.g., thalidomide and lenalidomide), hematopoietic stem cell transplantation (SCT) has improved complete remission rate and overall survival of MM patients significantly [2]. Unfortunately, MM is still an incurable malignancy [3]. Disulfiram (DSF) is a re-purposing drug that has previously been approved by the FDA and it has been clinically used as an anti-alcoholism drug for more than 60 years [4]. Recently, several studies ⁎
demonstrated that DSF was highly effective against several types of solid tumors such as breast cancer [5,6], colon cancer [7], melanoma [8,9], as well as hematological malignancies, including acute myeloid leukemia [10,11]. Copper (Cu) plays a critical role in a variety of basic biological functions in living organisms through regulation of a number of copper-dependent enzymes [12]. Eric C et al. showed that copper serum levels significantly influenced survival rates of cutaneous T cell lymphoma (CTCL) patients [13]. But in MM, no correlation between serum Cu concentration and myeloma disease activity was observed [14]. Interestingly, copper could inhibit the cellular proteasome to induce tumor cell apoptosis [15]. Bortezomib, the first Proteasome inhibitor has been used in clinic to treat MM patients for more than 20 years, which acts as a backbone drug for MM treatment. Speculatively, Cu may have anti-myeloma activity in vivo but not facilitate myeloma progress [16,17]. As a divalent metal ion chelator, DSF strongly
Corresponding authors at: Hematology Department of the Second Hospital of Shandong University, 247th of Beiyuan Rd., Jinan, Shandong, China. E-mail addresses: [email protected] (Y. Ni), [email protected] (C. Zheng).
https://doi.org/10.1016/j.biopha.2020.110048 Received 8 November 2019; Received in revised form 24 February 2020; Accepted 25 February 2020 0753-3322/ © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.3. MTT cytotoxicity assay
interacts with Cu to form the disulfiram/copper (DSF/Cu) complex which significantly enhances the DSF-induced anti-tumor cytotoxicity [18–20]. Apoptosis, referred to as type I programmed cell death, plays a critical role in development and aging, and in homeostatic mechanism to maintain cell population in tissues [21]. There are two major apoptotic signaling cascades, the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway. The former initiates apoptosis through a diverse array of non-receptormediated stimuli that produce intracellular signals which may act in either a positive or negative fashion. All of these stimuli cause changes in the inner mitochondrial membrane that result in opening of the mitochondrial permeability transition (MPT) pores, loss of the mitochondrial transmembrane potential (MMP), and release of cytochrome c from mitochondria into the cytoplasm followed by cleavage/ activation of executioner caspase-3, ultimately inducing apoptosis [22]. The latter is dependent on the activation of extracellular death signals, which are transmitted to the cell through binding of extracellular death ligands to the corresponding death receptors on the membrane, followed by recruiting and activating intracellular caspase-2 and caspase-8 [23]. The C-Jun NH2-terminal kinase (JNK), a member of the mitogenactivated protein (MAP) kinase family, is activated by a variety of other JNK kinases and MAPK kinases [24]. Recent studies showed that modulation of JNK/c-Jun activity was involved in drub induced apoptosis of acute leukemia cells [25,26]. In order to study the antitumor activity of DSF/Cu on MM, we designed this approach. In the present study, our results showed that the DSF/Cu complex significantly inhibited MM cell proliferation, induced MM cell apoptosis in vitro and inhibited tumor growth and prolonged overall survival in vivo.
The viability of MM cells and peripheral blood mononuclear cells (PBMCs) was determined by the MTT assay [27]. Briefly, the concentration of the MM cells and PBMCs was adjusted to 2 × 104 cells/ well and 200 μl cell suspension was added into each well of the 96-well plate. Various concentrations of DSF (0.05, 0.1, 0.25, and 0.5 μmol/l) combined with/without Cu (0.5 μmol/l) were added to the plates and incubated for 12 h. Only one concentration of DSF (0.25 μmol/l) was used for 24 h and 48 h experiments (very close to IC_50)). At the indicated time points, 20 μl of 5 mg/ml MTT was added to each well and incubated for 4 h at 37 °C, followed by decantation of the supernatant and addition of 0.15 ml of 10 % sodium dimethyl sulfoxide to each well. The absorbance for each well was read on a microplate reader (Sunrise, Tecan) at 570 nm. To obtain significant experimental results, we repeated each procedure for three times. Cell inhibition was calculated by the following formula: cell inhibition (%) = 100 %-(average absorbance of treated group- average absorbance of blank) / (average absorbance of untreated group - average absorbance of blank) ×100 %. 2.4. Flow cytometric analysis of apoptotic cells The apoptosis assay was performed as previously described [28]. Briefly, MM.1S and RPMI8226 cells were plated in 12-well plates (2 × 105 cells/well) with different concentrations of DSF (0.1, 0.25, and 0.5 μmol/l) combined with Cu (0.5 μmol/l) for 12 h while for 24 and 48 h experiments, the concentration of DSF was 0.25 μmol/l. Apoptosis was assessed using the Annexin V-FITC/PI kit (KeyGEN BioTECH, Jiangsu, China). The cultured cells were harvested and washed twice with PBS at 4 °C, resuspended in the binding buffer, and, followed by Annexin VFITC staining for 15 min and PI staining for another 10 min in the dark. The samples were analyzed with FACS Aria II (BD Biosciences). Primary cells were plated in 24-well plate (2 × 105 cells/well) with DSF/Cu (0.5 μM/0.5 μM) for 24 h. The cultured cells were collected, washed and stained with anti-human CD38-PE monoclonal antibody in PBS for 20 min at room temperature in dark. Afterwards, the cells were washed with PBS twice and then resuspended in 100 μl binding buffer containing 2 μl Annexin-V. The percentage of apoptosis rate was monitored by the FACS Aria II (BD Biosciences) cytometer.
2. Materials and methods 2.1. Reagents DSF and Cu were purchased from Sigma-Aldrich (St Louis, MO, USA). DSF was dissolved in DMSO at a stock concentration of 10 mM and Cu was dissolved in sterile water as 10 mM stock solution. Both stock solutions were stored at −20 °C and freshly diluted with culture medium before use. Pan-caspase inhibitor, Z-VAD-FMK (zVAD) was purchased from R&D Systems (Minneapolis, MN, USA). RPMI1640 medium, fetal bovine serum, penicillin, streptomycin, and glutamine were obtained from Gibco (Waltham, MA, USA). Fluorescent labeled anti-human monoclonal antibodies (mAb) used for flow cytometry analysis was CD38-PE (Miltenyi Biotec, BergischGladbach, Germany). Annexin V-FITC/PI kit (KeyGEN BioTECH, Jiangsu, China) was used for detect apoptosis and Mitochondrial Membrane Potential Detection kit (KeyGEN BioTECH, Jiangsu, China) was used for detect mitochondrial membrane potential.
2.5. Cell cycle analysis MM.1S and RPMI8226 cells were cultured with or without DSF/Cu (DSF: 0.1, 0.25, 0.5 μM/Cu: 0.5 μM) for 12 h. Cells were harvested and washed with PBS and fixed in 75 % ethanol at 4 °C overnight, followed by incubation with cold PI solution and RNase-A for 30 min. Cell cycle arrest was also analyzed using the FACS Aria II (BD Biosciences). 2.6. Determination of mitochondrial membrane potential by JC-1 dye on a flow cytometer The loss of mitochondrial membrane potential in response to the DSF/Cu complex was examined by JC-1 dye (KeyGEN BioTECH, Jiangsu, China) on a flow cytometer. Images were taken by an inverted fluorescent microscope [29,30]. Briefly, MM.1S and RPMI8226 cells were treated with DSF (0.25 μM) with Cu (0.5 μM) for 12 h. After incubation, the cells were washed and resuspended with JC-1 working Solution and incubated for another 20 min at 37 °C in a CO2 incubator. After incubation, cells were washed with 500 μl incubation buffer and centrifuged. Finally, the samples were resuspended with 300 μl incubation buffer, processed for data acquisition, and analyzed on a Becton-Dickinson FACS Aria flow cytometer.
2.2. Cell lines and primary cells Myeloma cell line MM.1S and RPMI8226 were obtained from ATCC (Manassas, VA, USA). The cells were cultured in RPMI1640 medium with 10 % FBS and 100 U/ml penicillin and streptomycin at 37 °C in 5 % CO2. The study involving seven human patient bone marrow samples and three healthy volunteers’ blood samples were approved by the Human Ethics Research Committee of the Second Hospital of Shandong University and the patients all signed the informed consent. Bone marrow aspirates were obtained from newly diagnosed or relapsed/ refractory patients with myeloma and primary cells were isolated by Ficoll (Tianjin HY Bioscience Co. LTD) density gradient centrifugation.
2.7. Western blot analysis MM.1S 2
and
RPMI8226
cells
were
cultured
with
various
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Fig. 1. DSF and DSF/Cu inhibited myeloma cell viability in a dose-dependent and time-dependent manner and DSF and DSF/Cu induced little cytotoxicity to peripheral blood mononuclear cells (PBMCs). (A, C) MM.1S (A) and RPMI8226 cells (C) were exposed to the indicated concentrations of DSF (0.05, 0.1, 0.25, and 0.5 μM) with or without 0.5 μM Cu for 12 h, after which the anti-viability effect was determined by MTT assay. (B, D) MM.1S (B) and RPMI8226 cells (D) were treated with only one concentration of DSF (0.25 μM) with or without 0.5 μM Cu for 12, 24, and 48 h, after which the percentage of inhibition was determined by MTT assay. (E) DSF (0.1, 0.25, and 0.5 μM) with or without 0.5 μM Cu and 0.5 μM Cu alone induced little cytotoxicity to PBMCs. * P < 0.05, ** P < 0.01, *** P < 0.001 vs DSF alone or the DSF/Cu complex, respectively. Results were presented as Mean ± SEM from three independent experiments.
concentrations of DSF (0.1, 0.25, and 0.5 μmol/l) and Cu (0.5 μmol/l). Expressions of SAPK/JNK, Phospho-SAPK/JNK, Phospho-c-jun, Caspase-3, Cleaved-PARP, P38, ERK, GAPDH, and β-actin were analyzed by Western blot as described previously [31] In brief, drugtreated cells were lysed in RIPA buffer supplemented with a phosphatase inhibitor cocktail (Roche, Mannheim, Germany) and 1 mM PMSF on ice for 20 min. Protein concentrations were measured using the BCA Protein Assay kit according to the manufacturer’s instructions (Thermo Scientific Pierce). Whole protein (30 μg/lane) from each sample was resolved on 10 % SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a PVDF membrane (Millipore, UK), and blotted with various antibodies (SAPK/JNK, rabbit polyclonal, 1:1000, CST; Phospho-SAPK/JNK, rabbit polyclonal 1:1000, CST; Phospho-c-jun, rabbit polyclonal, 1:500, CST; Caspase-3, mouse polyclonal, 1:1000, CST; Cleaved-PARP, rabbit polyclonal, 1:1000, CST; Caspase-8, mouse monoclonal, 1:1000, CST; P38, rabbit monoclonal, 1:1000, CST; ERK, rabbit polyclonal, 1:1000, CST; GAPDH, mouse polyclonal, 1:1000, CST; β-actin, rabbit polyclonal, 1:1000, Abcam), and HRP-conjugated monoclonal secondary antibody (1:5000; Amersham Pharmacia Biotech, NJ). Membranes were exposed to the Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). Blots were detected on X-ray film using the ChemiDoc™ MP Imaging System (Bio-Rad).
and DSF (50 mg/kg/d) each day in the evening (18:00). DSF was dissolved in the buffer with 10 % DMSO, 10 % Cremophor EL, and 80 % normal saline. Administration of compounds was carried out as a blind experiment (all information about the expected outputs and the nature of compounds used were kept from the animal technicians). The mice were humanely euthanized by cervical dislocation when they reached the endpoint of our observation, which was defined as when the tumor size exceeded 2.0 cm in any direction or when a mouse was unable to creep for taking food and/or water. Changes in tumor volume were monitored in the control and experimental groups every 3 days until the first mouse in the control group reached the endpoint. Tumors were measured using calipers and volumes were calculated using the formula V = long diameter × (short diameter)2×π/6 (Guo et al. 2016). 2.9. Statistical analysis All results were analyzed by the Student's t test using SPSS 13.0. to compare differences between the groups. GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA, USA) was used for statistical tests. Statistical significance was indicated by a p value < 0.05. 3. Results 3.1. The DSF/Cu complex inhibited cell viability of MM cells
2.8. Xenograft experiments To investigate the effects of DSF or DSF/Cu complex on inhibition of cell viability, MM cell lines were treated with increasing doses of DSF/ Cu. To determine whether DSF or the DSF/Cu complex had an effect on cancer cells in a time-dependent manner, we only tested the toxicity of one concentration of DSF (0.25 μM) and/or Cu (0.5 μM) on the human myeloma MM.1S and RPMI8226 cells for 24 h and 48 h. As shown in Fig. 1A, in the medium without Cu, DSF was toxic to the MM.1S cell line (IC50_12 h was 0.32 ± 0.04 μM). In the Cu-supplemented medium, DSF was even more cytotoxic to the MM.1S cell lines (IC50_12 h: 0.23 ± 0.18 μM; Fig. 1A). And the cytotoxic of Cu to this cell line was only 3.4 % ± 2.0 %. To further confirm the toxicity of DSF or DSF/Cu to the myeloma cell lines, we tested the other tumor cell line RPMI8226 in vitro. As shown in Fig. 1C, DSF alone was lower toxic to RPMI8226 cells (IC50_12 h = 0.87 ± 0.03 μM). DSF/Cu was more toxic (IC50 of
All animal study procedures were approved by the Animal Ethics Research Committee of the Second Hospital of Shandong University. The NOD/SCID mice (6–8 weeks of age, non-fertile, female, and 18–20 g each), purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) were kept under specific- pathogen-free conditions. Mice were subcutaneously injected into the right foreleg with 1 × 107 human myeloma cell line RPMI8226 cells suspended in 100 μl normal saline (NS) buffer. After approximately three weeks, when tumors reached a size of approximately 200 mm3, 12 mice were randomly assigned into two groups (six mice per group). Control mice had NS contained DMSO and Cremophor EL administered by gavage and the remaining received DSF/Cu every day for 14 days. Cu (0.15 mg/kg/d) was administered each day in the morning (08:00) 3
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0.25 ± 0.02 μM) to RPMI8226 cells. And the cytotoxic of Cu to RPMI8226 cell line was only 4.5 % ± 1.2 %. Furthermore, administration of the DSF/Cu complex resulted in a time-dependent decrease in MM cell viability. The inhibitions of MM.1S cells at 12, 24, and 48 h were reported as 55.3 ± 2.1 %, 74.9 ± 1.4 %, and 96.6 ± 1.8 %, respectively (Fig. 1B), while the inhibitions of RPMI8226 cells at 12, 24, and 48 h were 43.2 ± 2.2 %, 54.9 ± 2.1 %, and 62.2 ± 0.9 %, respectively (Fig. 1D).
inhibition and apoptosis, we examined whether DSF/Cu treatment affects cell cycle of MM.1S cells. As shown in Fig. 4A-B, MM.1S cells were treated with or without DSF/Cu for 12 h. The results of flow cytometry revealed accumulation of MM.1S and RPMI8226 cell line in G2/M phase. The percentage of MM.1S cells in G2/M phase increased from 26.6 ± 2.0 % (DSF: 0.1 μM/Cu: 0.5 μM), 47.8 ± 4.1 % (DSF: 0.25 μM/Cu: 0.5 μM), and 45.4 ± 3.2 % (DSF: 0.5 μM/Cu: 0.5 μM) (P < 0.001) as compared to the control 22.5 ± 2.7 %, while the percentage of G2/M phase increased from 12.8 ± 1.2 % (DSF: 0.1 μM/Cu: 0.5 μM), 21.5 ± 2.3 % (DSF: 0.25 μM/Cu: 0.5 μM), and 23.1 ± 0.8 % (DSF: 0.5 μM/Cu: 0.5 μM) (P < 0.001) as compared to the control 1.4 ± 1.2 % in RPMI8226 cells. These results indicated that the growthinhibitory effect of DSF/Cu against MM.1S and RPMI8226 cells was correlated with a cell cycle arrest in G2/M phase.
3.2. DSF/Cu induced little cytotoxicity against peripheral blood mononuclear cells To evaluate the cytotoxicity of DSF or DSF/Cu to PBMCs from normal human peripheral blood, we used MTT assays to compared cell viability between PBMCs and MM.1S cells incubated with DSF or DSF/ Cu for 12 h. After exposure to DSF (0.1 μM, 0.25 μM, 0.5 μM) or DSF/Cu (0.1/0.5 μM, 0.25/0.5 μM, 0.5/0.5 μM) for 12 h, MTT results showed that DSF (12.9 ± 1.8 %, 15.6 ± 1.8 % and 20.5 ± 0.2 %) or DSF/Cu (15.8 ± 8.1 %, 26.3 ± 6.5 % and 34.1 ± 7.9 %) exhibited little toxicity to normal PBMCs, while a great killing effect on MM.1S cells (DSF: 11.9 ± 1.8 %, 33.6 ± 2.2 % and 52.8 ± 2.9 %; DSF/Cu 22.2 ± 2.5 %, 63.4 ± 6.5 % and 77.7 ± 3.1 %) was observed (Fig. 1E). And the cytotoxic of Cu (0.5 μM) to the PBMCs was only 13.4 % ± 2.1 %.
3.6. DSF induced loss of Mitochondrial membrane potential (MMP) To determine the effects of DSF and DSF/Cu complex on the loss of the mitochondrial membrane potential, MM.1S and RPMI8226 cells were incubated with 0.25 μM DSF in combination with 0.5 μM Cu for 12 h. Data from flow cytometry demonstrated that the loss of the MMP in MM.1S cells was 91.8 % ± 3.4 % in presence of DSF/Cu when compared with the control group (24.0 ± 4.6 %) (P < 0.001). Similarly, DSF/Cu significantly induced loss of MMP in RPMI8226 cells (P < 0.001) (Fig. 5A-B). Consistently, the images taken by a fluorescent microscope showed that myeloma cells (MM.1S and RPMI8226) treated with DSF/Cu presented weaker red fluorescence, respectively (Fig. 5C). These results suggested that intrinsic apoptosis pathway was involved in the DSF/Cu induced myeloma cell apoptosis machinery.
3.3. The DSF/Cu complex enhanced apoptosis of MM cells Apoptosis of MM.1S and RPMI8226 cells was detected using Annexin-V FITC and propidium iodide (PI) staining followed by flow cytometry. To determine the combined effect of DSF and Cu on both myeloma cell lines, the cells were exposed to increasing doses of DSF with Cu for 12 h. The total number of AV+ and AV+ PI+ positive cells was counted as the number of apoptotic cells, which were used for the comparisons between groups. Compared to the control group (9.8 ± 2.9 %), apoptosis in MM.1S cells after 12 h was increased by 35.6 % ± 6.5 %, 65.4 % ± 3.5 % and 87.4 % ± 2.9 % at the dose of 0.1 μM, 0.25 μM, 0.5 μM, respectively, while apoptosis in RPMI8226 cells was also increased by 16.8 % ± 2.3 %, 54.4 % ± 3.9 % and 83.8 % ± 5.0 % at 0.1 μM, 0.25 μM, 0.5 μM, respectively (Fig. 2A-B). It was also observed that the apoptotic rate of cells treated with DSF/Cu was time-dependent with a higher apoptotic ratio at 48 h than that at 12 h and 24 h, especially in MM.1S cells (P < 0.001) (Fig. 2C-D). Apoptosis was induced in both MM.1S and RPMI8226 cells in a dose- and timedependent manner, consistent with our findings in the MTT assay.
3.7. DSF induced activation of caspase 8, caspase 3, and PARP cleavage in MM cells Caspase 8 and Caspase-3 play a central role in the initiation and execution of apoptotic program, respectively. Caspase-3 is primarily responsible for the cleavage of PARP (poly ADP-ribose polymerase) during cell apoptosis. As expected, DSF/Cu induced cleavage of caspase 8 (Fig. 6A, C), caspase-3 and PARP (Fig. 6B, C). To further confirm the involvement of caspase-3 in DSF/Cu-induced apoptosis, MM.1S and RPMI8226 cells were pre-treated with a broad-spectrum pan-caspase inhibitor (zVAD) at 20 μM concentration for 1 h and then added with DSF/Cu (DSF: 0.25 μM/Cu: 0.5 μM). Pre-treatment with zVAD significantly blocked apoptosis in MM.1S cells, 15.7 % ± 4.1 % vs. 58.4 % ± 3.6 % and in RPMI8226 cells, 15.8 % ± 1.1 % vs. 54.9 % ± 5.2 % (Fig. 6D-E). Western blotting (Fig. 6F-G) results also showed that DSF/ Cu-induced activation of caspase-3 and the cleavage of PARP was significantly inhibited by zVAD. Taken together, these results suggested that extrinsic cell apoptosis pathway is involved in the myeloma cell apoptosis induced by DSF/Cu in caspase dependent and independent manners.
3.4. The DSF/Cu complex induced apoptosis of MM primary cells The cytotoxic activity of DSF/Cu against primary MM cells obtained from bone marrow of newly diagnosed or relapsed/refractory patients was tested. Consistent with the anti-myeloma activity of DSF/Cu observed in MM cell lines, co-treatment with DSF (0.5 μM) and Cu (0.5 μM) resulted in significant increases in apoptosis of primary cells (69.1 ± 9.3 % vs 17.2 ± 9.6 %, P < 0.001, n=7; Fig. 3A-B). Since myeloma patient samples were valuable and limited in volumes, we had difficulty to apply more concentrations and time points in those experiments. To find a more suitable dose of DSF and time point, we first tested on two different MM patient samples and found that treatment by 0.25 μM DSF for 12 h and 24 h did not induce significant apoptosis when compared with untreated ones (controls), respectively. Therefore, we increased the DSF dosage from 0.25 to 0.5 μM DSF and used the combination of 0.5 μM DSF for 24 h in all the experiments on primary MM cells. These findings suggested that DSF/Cu might eliminate MM primary cells.
3.8. DSF/Cu induced activation of caspase and JNK signaling pathway C-Jun N-terminal kinase (JNK), P38 and ERK are members of the mitogen activated protein kinase (MAPK) family. JNK pathway could be activated by a variety of stimuli including UV radiation and DNA damaging agents contributing to apoptosis. To investigate potential roles of JNK pathway in DSF/Cu mediated apoptosis of myeloma cells, we analyzed JNK, c-Jun, P38 and ERK expression by western blot. As shown in Fig. 7A-C, although there was no change in JNK, P38 and ERK expression, phosphorylation of JNK and c-Jun expression were increased in the MM.1S and RPMI8226 cells treated by DSF/Cu. To further confirm whether JNK/c-Jun pathway activation contributed to DSF/Cu-triggered apoptosis, MM.1S and RPMI8226 cells were pretreated with 20 μM of JNK inhibitor SP600125 for 2 h and then exposed to DSF/Cu for 12 h. Apoptosis induced by the combination of DSF and
3.5. DSF caused cell cycle arrest in G2/ M phase To study the mechanisms of DSF/Cu-induced MM cell lines growth 4
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Fig. 2. DSF/Cu significantly induced apoptosis in MM cells (MM.1S and RPMI8226). (A, B) MM.1S and RPMI8226 cells were treated with a series of concentrations of DSF (0.1, 0.25, and 0.5 μM) with 0.5 μM Cu for 12 h, after which the percentage of apoptotic cells was determined by flow cytometry using Annexin V/PI double staining (n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs the untreated control or the DSF/Cu complex, respectively. (C, D) Apoptotic MM.1S and RPMI8226 cells were measured by flow cytometry at different time points (24 and 48 h) after being treated with DSF/Cu (DSF: 0.25 μM/Cu: 0.5 μM) (n = 3).
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Fig. 3. DSF/Cu induced apoptosis in MM primary cells. Mononuclear cells were isolated from primary bone marrow of newly diagnosed or relapsed/refractory patients with multiple myeloma. Representative plots of bone marrow sample showing the gating strategy for CD38+ MM cells. (A) Representative flow cytometry plots of Annexin-V expression in control and DSF/Cu. (B) Percentage of Annexin-V expression in control and DSF/Cu. P value is shown. Value of P＜0.001 is considered as a significant difference.
cells from patients. For example, Manman Deng et al. have reported that DSF/Cu could induce apoptosis of primary cells isolated from BALL patients [34]. To evaluate activity of DSF/Cu against primary myeloma cell, mononuclear cells were isolated from bone marrow of MM patients and flow cytometry as well as apoptosis detection techniques were utilized to define myeloma cell apoptosis. As expected, DSF/Cu markedly induced apoptosis of primary MM cells. And it only had a negligible toxicity to normal PBMCs. More importantly, our results from animal studies showed that DSF/Cu significantly inhibited tumor growth and prolonged overall survival in vivo, indicating a potent anti-myeloma activity of DSF/Cu in vivo. The dosage of DSF and administration approach applied in our animal experiments were followed the previously published papers [33]. In Skrott Z’s study and their clinical trial (NCT00742911), Cu was administered each day in the morning (08:00) and DSF each day in the evening (19:00). To do the animal experiment in a way close to real clinical practice, we followed Skrott Z’s protocol and treated the myeloma bearing mice by administration of Cu in the morning and DSF in the evening. It has been shown that primary metabolite of the DSF in stomach is Diethyldithiocarbamate (DDC), and anticancer activity of DDC is dependent on complexation with Cu to form a complex Cu (DDC)2. However, this complex is highly insoluble and is difficult to be absorbed in stomach [35]. This should be the main reason why DSF and Cu were given separately. Cell cycle arrest is one of the mechanisms for cell growth inhibition induced by many anticancer drugs. For example, Runzhe Chen et al. have demonstrated that the inhibition of the proliferation of MM cells by bortezomib was due to induction of cell cycle arrest in the G2/M phase [36]. Consistently, we demonstrated that DSF/Cu inhibited the growth of MM.1S and RPMI8226 cells by inducing cell cycle arrest at G2/M phase. To reveal underlying mechanism of DSF/Cu-induced apoptosis of myeloma cells, we analyzed certain key molecules involved in cell apoptosis signaling pathways. Working as the extrinsic and intrinsic pathways [37], initiator, caspase-8 and caspase-9, respectively, discriminate to engage the same proteolytic caspase cascade. It leads to the activation of interface at the point of downstream executioner caspase3 that induces the execution phase of apoptosis after being proteolytically activated [38]. Here, we found that DSF/Cu complex treatment increased activation of caspase-8, caspase-3 and accumulation of cleaved-PARP fragment. DSF/Cu treatment-associated apoptosis of MM cells was substantially inhibited by zVAD, suggesting that caspase dependent and independent apoptosis pathways are involved in the DSF/ Cu treatment mediated myeloma cell apoptosis [39]. To determine a role of intrinsic apoptotic pathway in the apoptosis induced DSF/Cu, we assessed the MMP in DSF/Cu treated MM cells. In line with the findings
Cu complex was attenuated by adding SP600125 (MM.1S: declined from 57.9 % ± 3.6%–20.2% ± 2.4 %; RPMI8226: declined from 54.5 % ± 3.1%–18.8% ± 3.3 %) with a significant statistical significance (Fig. 7D-G). These results indicated that JNK/c-Jun activation is implicated in the DSF/Cu-mediated myeloma cell apoptosis mechanism. 3.9. DSF/Cu exhibited anti-myeloma activity in vivo To evaluate therapeutic effects of DSF/Cu on MM in vivo, a myeloma mouse model was used. DSF and Cu were administered by oral gavage for 2 weeks. As shown in Fig. 8A-C, we found that tumor weight and tumor volumes in the control group grew more rapidly than that in the DSF/Cu group, and the differences in tumor sizes from the fourth day after treatment indicated in the Fig. 8C between the two groups were statistically significant (P < 0.001). Changes in survival among the control and DSF/Cu groups were compared by using Kaplan-Meier curves. As shown in Fig. 8D, DSF/Cu treatment significantly prolonged survival of the myeloma mice when compared with the control (P＜ 0.001). Our results showed that the mean survival time of the mice treated by the control group and DSF/Cu group were 25.5 days and 31 days, respectively. Together, these results demonstrated that the DSF and Cu combination selectively inhibited tumor growth and improved survival of myeloma bearing mice in vivo. 4. Discussion DSF is a widely used anti-alcoholism drug with low toxic effects on normal tissues [32]. Interestingly, its anti-cancer activity has been recently recognized [7]. DSF used to treat high-risk breast cancer in a clinical trial with promising results, DSF has been proposed as a candidate for drug repurposing in cancer [33]. In a study by Skrott et al., groups of mice were injected with human MDA-MB-231 cancer cells, fed with a diet plus DSF or DSF/Cu. Compared to control group, tumor volume in DSF and DSF/Cu-treated groups were suppressed by 57 % and 77 %, respectively (P = 0.0038). These results indicated that DSF/ Cu is an efficient anti-cancer agent in cancer therapy. In our study, we tested DSF alone and Cu toxicity using MTT assay and found that DSF/ Cu was more cytotoxic than DSF alone to the MM cell lines and Cu alone was not toxic to the MM cell lines. Therefore, in the subsequent experiments we focused only on testing cytotoxic activity of DSF/Cu complex in order to provide acknowledge important for designing a clinical trial using DSF/Cu complex to treat recurrent / refractory myeloma patients. And our results also demonstrated that the DSF/Cu complex induced apoptosis of MM cells in a time- and dose-dependent manner. Previous studies have shown that DSF/Cu can be used not only in the treatment of tumor cell lines, but also in the treatment of primary 6
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Fig. 4. DSF/Cu induced cell cycle arrest in MM.1S and RPMI8226 cells. (A) Cell cycle analysis was performed with Flow Cytometry in MM.1S and RPMI8226 cells treated with/without DSF/Cu for 12 h. (B) Cell cycle distribution in MM.1S cells. Histogram showed the percentage of cells in the G1, S and G2 phases. Data are presented as the mean ± SD (n = 3). ***P < 0.001 vs control.
Fig. 5. DSF/Cu markedly induced loss of the Mitochondrial Membrane Potential (MMP). (A) MM.1S and RPMI8226 cells were treated with DSF/Cu (0.25 μM/0. 5 μM) for 12 h, and the level of the loss of the Mitochondrial Membrane Potential was detected by JC-1 flow cytometry (n = 3). (B) The fluorescent intensity of DSF/Cu treatment groups are indicated. Data is presented as mean ± SEM (n = 3, ** P < 0.01, *** P < 0.001, as compared with the control.). (C) Fluorescent image of MM.1S and RPMI8226 cells stained with JC-1. Photograph shows JC-1 red and JC-1 green, representing JC-1 aggregates and JC-1 monomers (n = 3). 7
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Fig. 6. DSF/Cu induced cleavage of caspase 8, caspase 3 and PARP. (A) MM.1S and RPMI8226 cells were treated with various concentrations of DSF (0.1, 0.25, and 0.5 μM) and Cu (0.5 μM) for 12 h. Total proteins were extracted from the cultured cells and subjected to Western blot analysis using antibodies against caspase-8. GAPDH was used as a loading control. (B) The cells were treated in the way described previously. The cell lysates were analyzed by western blotting to detect the expression levels of caspase-3 and PARP cleavage. (C) Shows the relative protein level of caspase-8, caspase-3 and PARP cleavage calculated by the band density of western blots using Image J software of MM cell line (*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test). (D) MM.1S and RPMI8226 cells were treated with zVAD (20 μM) for 1 h prior to adding DSF/Cu (DSF: 0.25 μM/Cu: 0.5 μM) for 12 h. Apoptosis was monitored by flow cytometry detection of AV/PI staining. Dot plots of one representative experiment are shown. Percentages of positive cells for AV and PI are indicated in the plots. (E) Apoptosis of MM.1S and RPMI8226 cells treated with DSF/Cu in the presence or absence of zVAD. The results are expressed as mean percentage ± SEM of total (AV+PI– + AV+PI+) apoptotic cells from three independent experiments. *p < 0.001 by Student’s t test. (F) Western blot analysis of caspase-3 and PARP cleavage expression in MM.1S and RPMI8226 cells treated with DSF/Cu in the presence or absence of zVAD at 12 h. Results were presented as Mean ± SEM from three independent experiments. (F) Shows the relative protein level of caspase-3 and PARP cleavage on MM cell line (*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test). 8
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Fig. 7. DSF/Cu induced JNK/c-jun activation. (A) The cells were treated as previously described. The combination of DSF and Cu markedly activated the JNK/c-jun pathway while DSF/Cu induced phosphorylation of JNK and c-Jun (n = 3). GAPDH was used as a loading control. (B) Western blot analysis of P38 and ERK expression in MM.1S and RPMI8226 cells treated with DSF/Cu. (C) Shows the relative protein level of JNK, P-JNK, P-c-Jun, P38 and ERK calculated by the band density of western blots using Image J software of MM cell line (*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test). (D) MM.1S and RPMI8226 cells were treated with SP600125 (20 μM) for 2 h prior to adding DSF/Cu (DSF: 0.25 μM/Cu: 0.5 μM) for 12 h. Apoptosis was evaluated using AV and PI staining by flow cytometry. (E) The apoptosis results are expressed as mean percentage ± SEM of total (AV+PI– + AV+PI+) apoptotic cells from three independent experiments (n = 3). *P < 0.001 by Student’s t test. (F) The expressions of P-JNK proteins in MM.1S and RPMI8226 cells treated with DSF/Cu in the presence of SP600125 were measured by Western blot. (G) Shows the relative protein level of P-JNK calculated by the band density of western blots using Image J software of MM cell line (**P < 0.01, ***P < 0.001 by Student’s t test).
reported in other type of tumors. In line with the findings reported in other type of tumors [40,41], we found that DSF/Cu treatment induced loss of MMP in myeloma cells. As a known fact, MMP decreases in early apoptosis as an indicator of mitochondrial membrane permeability, this might suggest that DSF/Cu induce intrinsic apoptosis machinery. Mitogen-activated protein kinase (MAPK) is implicated in regulating differentiation, cell growth and proliferation, and apoptosis responses of tumor cells [42]. Several studies have reported the involvement of MAPK in cancer deregulation [41,43]. MAPK family consist of various members, including JNK/stress-activated protein kinase (SAPK), p38, and growth factor-regulated ERK1/2 [44,45]. JNK, as a serine/threonine protein kinase, has the ability to interact with c-Jun which acts as a focus of transcription factor-activation protein-1, and then promote the expression of pro-apoptotic protein such as p53, Bax and Fas. They in turn activate the caspase family as a consequence of the apoptosis signal transduction pathway [46,47]. Moreover, a team of researchers led by Liu Q., reported that continuous activation of JNK was necessary for the drug-induced apoptosis in myeloma cell lines in vitro [48]. Recently, there was a study that demonstrated that the JNK/c-Jun pathway may contribute to the DSF/Cu induced apoptosis in lymphoid malignant cell lines in vitro [49]. In accordance with previous studies, our results demonstrated that DSF/Cu can markedly upregulated expression of phosphorylation of JNK and c-Jun, resulting in the induction of apoptosis. Pre-treatment with SP600125, the JNK-specific inhibitor, resulted in the reduction of P-JNK expression and protected cells from apoptosis. These findings suggested that DSF/Cu induced apoptosis in multiple myeloma cells by activating the JNK signaling pathway.
5. Conclusions Taken together, our novel findings in this study presented DSF/Cu as a potent anti-myeloma agent in vitro and in animal models. This antialcoholism drug holds immense potential to be repurposed as a potent cancer drug. Ethical approval All procedures performed in studies involving animals and human samples were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Authors’ contributions ZCY and NYH designed the project, analyzed the data and revised the manuscript. XYQ did the most experiments, analyzed the data and wrote the manuscript. Flow cytometry analysis and paper writing were together accomplished by XYQ, FXL and JW. While XYQ, DYB, ZQ and WYJ were in duty of the statistical analysis. Animal experiments were performed as a teamwork by XYQ, XXL, JY and LXL. All the authors have read and approved the manuscript. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements The present study was supported by the National Natural Science 9
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Fig. 8. DSF/Cu significantly suppressed tumor weight, tumor growth and prolonged overall survival of myeloma bearing mice. DSF/Cu and control were administered to the myeloma-bearing SCID mice by oral gavage. (A) Xenograft images were shown. (B, C) The changes in tumor weight and tumor volumes among the control and treatment groups at the observation time points are indicated (**P < 0.01, ***P < 0.001, by Student's t test). The values are shown as mean ± SEM. Six mice in each group. (D) DSF/Cu treatments significantly prolonged survival of the myeloma bearing mice as compared with the control (***P < 0.001, by Long-rank test, respectively). The values are shown as mean ± SEM. Six mice in each group.
Foundation of China (grant no.81600176, no.81602694 and no.81372545), the Science and Technology innovation project of Shandong Province (grant no.2017GSF18136 and no.2018GSF118034), the Clinical Medical Technology innovation project of Jinan (grant no.201704089), and the National Natural Science Foundation of Shandong Province (grant no. ZR2016HB71). Thanks to Dr. Edward C, Mignot, Shandong University, for linguistic advice. We would like to thank Editage (www.editage.cn) for English language editing.
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Disulfiram/copper markedly induced myeloma cell apoptosis through activation of JNK and intrinsic and extrinsic apoptosis pathways
T
Yaqi Xua,b, Qian Zhoua,b,c, Xiaoli Fengd, Yibo Daia,b, Yang Jianga,b, Wen Jianga,b,e, Xiaoli Liua,b, Xiangling Xinga,f, Yongjing Wanga,b, Yihong Nig,*, Chengyun Zhenga,b,* a
Department of Hematology, The Second Hospital, Institute of Biotherapy for Hematological Malignancies, Shandong University, Jinan, Shandong, China Shandong University-Karolinska Institute Collaboration Laboratory for Stem Cell Research, Jinan, Shandong, China c Haemal Internal Medicine, Linyi Central Hospital, Yishui Country, Linyi, Shandong 276400, China d Clinical Laboratory, The Second Hospital, Shandong University, Jinan, Shandong, China e Central Laboratory, The Second Hospital, Shandong University, Jinan, Shandong, China f Department of Medicine, Center for Molecular Medicine (CMM) and Bioclinicum, Karolinska Institutet and Karolinska University Hospital Solna, 17164, Solna, Sweden g Department of Endocrine, the Second Hospital, Shandong University, Jinan, Shandong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Disulfiram Myeloma Apoptosis JNK Drug repurposing
Disulfiram (DSF) is an FDA approved anti-alcoholism drug in use for more than 60 years. Recently, antitumor activity of the DSF/copper (DSF/Cu) complex has been identified. Its anti-multiple myeloma activity, however, has barely been investigated. In the present study, our results demonstrated that the DSF/Cu complex induced apoptosis of MM cells and MM primary cells. The results indicated that DSF/Cu significantly induced cell cycle arrest at the G2/M phase in MM.1S and RPMI8226 cells. Moreover, JC-1 and Western blot results showed that DSF/Cu disrupted mitochondrial membrane integrity and cleaved caspase-8 in MM cells, respectively, suggesting that it induced activation of extrinsic and intrinsic apoptosis pathways. Interestingly, DSF/Cu induced caspase-3 activation was partly blocked by Z-VAD-FMK (zVAD), a pan-caspase inhibitor, indicating at caspase-dependent and -independent paths involved in DSF/Cu induced myeloma cell apoptosis machinery. Additionally, activation of the c-Jun N-terminal kinase (JNK) signaling pathway was observed in DSF/Cu treated MM cells. More importantly, our results demonstrated that DSF/Cu significantly reduced tumor volumes and prolonged overall survival of MM bearing mice when compared with the controls. Taken together, our novel findings showed that DSF/Cu has potent anti-myeloma activity in vitro and in vivo highlighting valuable clinical potential of DSF/Cu in MM treatment.
1. Introduction Multiple myeloma (MM) is a hematological malignancy characterized by abnormal accumulation of cancerous plasma cells in the bone marrow, secretion of monoclonal immunoglobulins, development of bone lytic lesions and extramedullary organ invasion at the later stage of the disease [1]. In the past twenty years, application of new drugs such as proteasome inhibitors (e.g., bortezomib and carfizomib), immunomodulatory drugs (e.g., thalidomide and lenalidomide), hematopoietic stem cell transplantation (SCT) has improved complete remission rate and overall survival of MM patients significantly [2]. Unfortunately, MM is still an incurable malignancy [3]. Disulfiram (DSF) is a re-purposing drug that has previously been approved by the FDA and it has been clinically used as an anti-alcoholism drug for more than 60 years [4]. Recently, several studies ⁎
demonstrated that DSF was highly effective against several types of solid tumors such as breast cancer [5,6], colon cancer [7], melanoma [8,9], as well as hematological malignancies, including acute myeloid leukemia [10,11]. Copper (Cu) plays a critical role in a variety of basic biological functions in living organisms through regulation of a number of copper-dependent enzymes [12]. Eric C et al. showed that copper serum levels significantly influenced survival rates of cutaneous T cell lymphoma (CTCL) patients [13]. But in MM, no correlation between serum Cu concentration and myeloma disease activity was observed [14]. Interestingly, copper could inhibit the cellular proteasome to induce tumor cell apoptosis [15]. Bortezomib, the first Proteasome inhibitor has been used in clinic to treat MM patients for more than 20 years, which acts as a backbone drug for MM treatment. Speculatively, Cu may have anti-myeloma activity in vivo but not facilitate myeloma progress [16,17]. As a divalent metal ion chelator, DSF strongly
Corresponding authors at: Hematology Department of the Second Hospital of Shandong University, 247th of Beiyuan Rd., Jinan, Shandong, China. E-mail addresses: [email protected] (Y. Ni), [email protected] (C. Zheng).
https://doi.org/10.1016/j.biopha.2020.110048 Received 8 November 2019; Received in revised form 24 February 2020; Accepted 25 February 2020 0753-3322/ © 2020 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.3. MTT cytotoxicity assay
interacts with Cu to form the disulfiram/copper (DSF/Cu) complex which significantly enhances the DSF-induced anti-tumor cytotoxicity [18–20]. Apoptosis, referred to as type I programmed cell death, plays a critical role in development and aging, and in homeostatic mechanism to maintain cell population in tissues [21]. There are two major apoptotic signaling cascades, the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway. The former initiates apoptosis through a diverse array of non-receptormediated stimuli that produce intracellular signals which may act in either a positive or negative fashion. All of these stimuli cause changes in the inner mitochondrial membrane that result in opening of the mitochondrial permeability transition (MPT) pores, loss of the mitochondrial transmembrane potential (MMP), and release of cytochrome c from mitochondria into the cytoplasm followed by cleavage/ activation of executioner caspase-3, ultimately inducing apoptosis [22]. The latter is dependent on the activation of extracellular death signals, which are transmitted to the cell through binding of extracellular death ligands to the corresponding death receptors on the membrane, followed by recruiting and activating intracellular caspase-2 and caspase-8 [23]. The C-Jun NH2-terminal kinase (JNK), a member of the mitogenactivated protein (MAP) kinase family, is activated by a variety of other JNK kinases and MAPK kinases [24]. Recent studies showed that modulation of JNK/c-Jun activity was involved in drub induced apoptosis of acute leukemia cells [25,26]. In order to study the antitumor activity of DSF/Cu on MM, we designed this approach. In the present study, our results showed that the DSF/Cu complex significantly inhibited MM cell proliferation, induced MM cell apoptosis in vitro and inhibited tumor growth and prolonged overall survival in vivo.
The viability of MM cells and peripheral blood mononuclear cells (PBMCs) was determined by the MTT assay [27]. Briefly, the concentration of the MM cells and PBMCs was adjusted to 2 × 104 cells/ well and 200 μl cell suspension was added into each well of the 96-well plate. Various concentrations of DSF (0.05, 0.1, 0.25, and 0.5 μmol/l) combined with/without Cu (0.5 μmol/l) were added to the plates and incubated for 12 h. Only one concentration of DSF (0.25 μmol/l) was used for 24 h and 48 h experiments (very close to IC_50)). At the indicated time points, 20 μl of 5 mg/ml MTT was added to each well and incubated for 4 h at 37 °C, followed by decantation of the supernatant and addition of 0.15 ml of 10 % sodium dimethyl sulfoxide to each well. The absorbance for each well was read on a microplate reader (Sunrise, Tecan) at 570 nm. To obtain significant experimental results, we repeated each procedure for three times. Cell inhibition was calculated by the following formula: cell inhibition (%) = 100 %-(average absorbance of treated group- average absorbance of blank) / (average absorbance of untreated group - average absorbance of blank) ×100 %. 2.4. Flow cytometric analysis of apoptotic cells The apoptosis assay was performed as previously described [28]. Briefly, MM.1S and RPMI8226 cells were plated in 12-well plates (2 × 105 cells/well) with different concentrations of DSF (0.1, 0.25, and 0.5 μmol/l) combined with Cu (0.5 μmol/l) for 12 h while for 24 and 48 h experiments, the concentration of DSF was 0.25 μmol/l. Apoptosis was assessed using the Annexin V-FITC/PI kit (KeyGEN BioTECH, Jiangsu, China). The cultured cells were harvested and washed twice with PBS at 4 °C, resuspended in the binding buffer, and, followed by Annexin VFITC staining for 15 min and PI staining for another 10 min in the dark. The samples were analyzed with FACS Aria II (BD Biosciences). Primary cells were plated in 24-well plate (2 × 105 cells/well) with DSF/Cu (0.5 μM/0.5 μM) for 24 h. The cultured cells were collected, washed and stained with anti-human CD38-PE monoclonal antibody in PBS for 20 min at room temperature in dark. Afterwards, the cells were washed with PBS twice and then resuspended in 100 μl binding buffer containing 2 μl Annexin-V. The percentage of apoptosis rate was monitored by the FACS Aria II (BD Biosciences) cytometer.
2. Materials and methods 2.1. Reagents DSF and Cu were purchased from Sigma-Aldrich (St Louis, MO, USA). DSF was dissolved in DMSO at a stock concentration of 10 mM and Cu was dissolved in sterile water as 10 mM stock solution. Both stock solutions were stored at −20 °C and freshly diluted with culture medium before use. Pan-caspase inhibitor, Z-VAD-FMK (zVAD) was purchased from R&D Systems (Minneapolis, MN, USA). RPMI1640 medium, fetal bovine serum, penicillin, streptomycin, and glutamine were obtained from Gibco (Waltham, MA, USA). Fluorescent labeled anti-human monoclonal antibodies (mAb) used for flow cytometry analysis was CD38-PE (Miltenyi Biotec, BergischGladbach, Germany). Annexin V-FITC/PI kit (KeyGEN BioTECH, Jiangsu, China) was used for detect apoptosis and Mitochondrial Membrane Potential Detection kit (KeyGEN BioTECH, Jiangsu, China) was used for detect mitochondrial membrane potential.
2.5. Cell cycle analysis MM.1S and RPMI8226 cells were cultured with or without DSF/Cu (DSF: 0.1, 0.25, 0.5 μM/Cu: 0.5 μM) for 12 h. Cells were harvested and washed with PBS and fixed in 75 % ethanol at 4 °C overnight, followed by incubation with cold PI solution and RNase-A for 30 min. Cell cycle arrest was also analyzed using the FACS Aria II (BD Biosciences). 2.6. Determination of mitochondrial membrane potential by JC-1 dye on a flow cytometer The loss of mitochondrial membrane potential in response to the DSF/Cu complex was examined by JC-1 dye (KeyGEN BioTECH, Jiangsu, China) on a flow cytometer. Images were taken by an inverted fluorescent microscope [29,30]. Briefly, MM.1S and RPMI8226 cells were treated with DSF (0.25 μM) with Cu (0.5 μM) for 12 h. After incubation, the cells were washed and resuspended with JC-1 working Solution and incubated for another 20 min at 37 °C in a CO2 incubator. After incubation, cells were washed with 500 μl incubation buffer and centrifuged. Finally, the samples were resuspended with 300 μl incubation buffer, processed for data acquisition, and analyzed on a Becton-Dickinson FACS Aria flow cytometer.
2.2. Cell lines and primary cells Myeloma cell line MM.1S and RPMI8226 were obtained from ATCC (Manassas, VA, USA). The cells were cultured in RPMI1640 medium with 10 % FBS and 100 U/ml penicillin and streptomycin at 37 °C in 5 % CO2. The study involving seven human patient bone marrow samples and three healthy volunteers’ blood samples were approved by the Human Ethics Research Committee of the Second Hospital of Shandong University and the patients all signed the informed consent. Bone marrow aspirates were obtained from newly diagnosed or relapsed/ refractory patients with myeloma and primary cells were isolated by Ficoll (Tianjin HY Bioscience Co. LTD) density gradient centrifugation.
2.7. Western blot analysis MM.1S 2
and
RPMI8226
cells
were
cultured
with
various
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Fig. 1. DSF and DSF/Cu inhibited myeloma cell viability in a dose-dependent and time-dependent manner and DSF and DSF/Cu induced little cytotoxicity to peripheral blood mononuclear cells (PBMCs). (A, C) MM.1S (A) and RPMI8226 cells (C) were exposed to the indicated concentrations of DSF (0.05, 0.1, 0.25, and 0.5 μM) with or without 0.5 μM Cu for 12 h, after which the anti-viability effect was determined by MTT assay. (B, D) MM.1S (B) and RPMI8226 cells (D) were treated with only one concentration of DSF (0.25 μM) with or without 0.5 μM Cu for 12, 24, and 48 h, after which the percentage of inhibition was determined by MTT assay. (E) DSF (0.1, 0.25, and 0.5 μM) with or without 0.5 μM Cu and 0.5 μM Cu alone induced little cytotoxicity to PBMCs. * P < 0.05, ** P < 0.01, *** P < 0.001 vs DSF alone or the DSF/Cu complex, respectively. Results were presented as Mean ± SEM from three independent experiments.
concentrations of DSF (0.1, 0.25, and 0.5 μmol/l) and Cu (0.5 μmol/l). Expressions of SAPK/JNK, Phospho-SAPK/JNK, Phospho-c-jun, Caspase-3, Cleaved-PARP, P38, ERK, GAPDH, and β-actin were analyzed by Western blot as described previously [31] In brief, drugtreated cells were lysed in RIPA buffer supplemented with a phosphatase inhibitor cocktail (Roche, Mannheim, Germany) and 1 mM PMSF on ice for 20 min. Protein concentrations were measured using the BCA Protein Assay kit according to the manufacturer’s instructions (Thermo Scientific Pierce). Whole protein (30 μg/lane) from each sample was resolved on 10 % SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a PVDF membrane (Millipore, UK), and blotted with various antibodies (SAPK/JNK, rabbit polyclonal, 1:1000, CST; Phospho-SAPK/JNK, rabbit polyclonal 1:1000, CST; Phospho-c-jun, rabbit polyclonal, 1:500, CST; Caspase-3, mouse polyclonal, 1:1000, CST; Cleaved-PARP, rabbit polyclonal, 1:1000, CST; Caspase-8, mouse monoclonal, 1:1000, CST; P38, rabbit monoclonal, 1:1000, CST; ERK, rabbit polyclonal, 1:1000, CST; GAPDH, mouse polyclonal, 1:1000, CST; β-actin, rabbit polyclonal, 1:1000, Abcam), and HRP-conjugated monoclonal secondary antibody (1:5000; Amersham Pharmacia Biotech, NJ). Membranes were exposed to the Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). Blots were detected on X-ray film using the ChemiDoc™ MP Imaging System (Bio-Rad).
and DSF (50 mg/kg/d) each day in the evening (18:00). DSF was dissolved in the buffer with 10 % DMSO, 10 % Cremophor EL, and 80 % normal saline. Administration of compounds was carried out as a blind experiment (all information about the expected outputs and the nature of compounds used were kept from the animal technicians). The mice were humanely euthanized by cervical dislocation when they reached the endpoint of our observation, which was defined as when the tumor size exceeded 2.0 cm in any direction or when a mouse was unable to creep for taking food and/or water. Changes in tumor volume were monitored in the control and experimental groups every 3 days until the first mouse in the control group reached the endpoint. Tumors were measured using calipers and volumes were calculated using the formula V = long diameter × (short diameter)2×π/6 (Guo et al. 2016). 2.9. Statistical analysis All results were analyzed by the Student's t test using SPSS 13.0. to compare differences between the groups. GraphPad Prism 5 (GraphPad Software Inc, San Diego, CA, USA) was used for statistical tests. Statistical significance was indicated by a p value < 0.05. 3. Results 3.1. The DSF/Cu complex inhibited cell viability of MM cells
2.8. Xenograft experiments To investigate the effects of DSF or DSF/Cu complex on inhibition of cell viability, MM cell lines were treated with increasing doses of DSF/ Cu. To determine whether DSF or the DSF/Cu complex had an effect on cancer cells in a time-dependent manner, we only tested the toxicity of one concentration of DSF (0.25 μM) and/or Cu (0.5 μM) on the human myeloma MM.1S and RPMI8226 cells for 24 h and 48 h. As shown in Fig. 1A, in the medium without Cu, DSF was toxic to the MM.1S cell line (IC50_12 h was 0.32 ± 0.04 μM). In the Cu-supplemented medium, DSF was even more cytotoxic to the MM.1S cell lines (IC50_12 h: 0.23 ± 0.18 μM; Fig. 1A). And the cytotoxic of Cu to this cell line was only 3.4 % ± 2.0 %. To further confirm the toxicity of DSF or DSF/Cu to the myeloma cell lines, we tested the other tumor cell line RPMI8226 in vitro. As shown in Fig. 1C, DSF alone was lower toxic to RPMI8226 cells (IC50_12 h = 0.87 ± 0.03 μM). DSF/Cu was more toxic (IC50 of
All animal study procedures were approved by the Animal Ethics Research Committee of the Second Hospital of Shandong University. The NOD/SCID mice (6–8 weeks of age, non-fertile, female, and 18–20 g each), purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) were kept under specific- pathogen-free conditions. Mice were subcutaneously injected into the right foreleg with 1 × 107 human myeloma cell line RPMI8226 cells suspended in 100 μl normal saline (NS) buffer. After approximately three weeks, when tumors reached a size of approximately 200 mm3, 12 mice were randomly assigned into two groups (six mice per group). Control mice had NS contained DMSO and Cremophor EL administered by gavage and the remaining received DSF/Cu every day for 14 days. Cu (0.15 mg/kg/d) was administered each day in the morning (08:00) 3
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0.25 ± 0.02 μM) to RPMI8226 cells. And the cytotoxic of Cu to RPMI8226 cell line was only 4.5 % ± 1.2 %. Furthermore, administration of the DSF/Cu complex resulted in a time-dependent decrease in MM cell viability. The inhibitions of MM.1S cells at 12, 24, and 48 h were reported as 55.3 ± 2.1 %, 74.9 ± 1.4 %, and 96.6 ± 1.8 %, respectively (Fig. 1B), while the inhibitions of RPMI8226 cells at 12, 24, and 48 h were 43.2 ± 2.2 %, 54.9 ± 2.1 %, and 62.2 ± 0.9 %, respectively (Fig. 1D).
inhibition and apoptosis, we examined whether DSF/Cu treatment affects cell cycle of MM.1S cells. As shown in Fig. 4A-B, MM.1S cells were treated with or without DSF/Cu for 12 h. The results of flow cytometry revealed accumulation of MM.1S and RPMI8226 cell line in G2/M phase. The percentage of MM.1S cells in G2/M phase increased from 26.6 ± 2.0 % (DSF: 0.1 μM/Cu: 0.5 μM), 47.8 ± 4.1 % (DSF: 0.25 μM/Cu: 0.5 μM), and 45.4 ± 3.2 % (DSF: 0.5 μM/Cu: 0.5 μM) (P < 0.001) as compared to the control 22.5 ± 2.7 %, while the percentage of G2/M phase increased from 12.8 ± 1.2 % (DSF: 0.1 μM/Cu: 0.5 μM), 21.5 ± 2.3 % (DSF: 0.25 μM/Cu: 0.5 μM), and 23.1 ± 0.8 % (DSF: 0.5 μM/Cu: 0.5 μM) (P < 0.001) as compared to the control 1.4 ± 1.2 % in RPMI8226 cells. These results indicated that the growthinhibitory effect of DSF/Cu against MM.1S and RPMI8226 cells was correlated with a cell cycle arrest in G2/M phase.
3.2. DSF/Cu induced little cytotoxicity against peripheral blood mononuclear cells To evaluate the cytotoxicity of DSF or DSF/Cu to PBMCs from normal human peripheral blood, we used MTT assays to compared cell viability between PBMCs and MM.1S cells incubated with DSF or DSF/ Cu for 12 h. After exposure to DSF (0.1 μM, 0.25 μM, 0.5 μM) or DSF/Cu (0.1/0.5 μM, 0.25/0.5 μM, 0.5/0.5 μM) for 12 h, MTT results showed that DSF (12.9 ± 1.8 %, 15.6 ± 1.8 % and 20.5 ± 0.2 %) or DSF/Cu (15.8 ± 8.1 %, 26.3 ± 6.5 % and 34.1 ± 7.9 %) exhibited little toxicity to normal PBMCs, while a great killing effect on MM.1S cells (DSF: 11.9 ± 1.8 %, 33.6 ± 2.2 % and 52.8 ± 2.9 %; DSF/Cu 22.2 ± 2.5 %, 63.4 ± 6.5 % and 77.7 ± 3.1 %) was observed (Fig. 1E). And the cytotoxic of Cu (0.5 μM) to the PBMCs was only 13.4 % ± 2.1 %.
3.6. DSF induced loss of Mitochondrial membrane potential (MMP) To determine the effects of DSF and DSF/Cu complex on the loss of the mitochondrial membrane potential, MM.1S and RPMI8226 cells were incubated with 0.25 μM DSF in combination with 0.5 μM Cu for 12 h. Data from flow cytometry demonstrated that the loss of the MMP in MM.1S cells was 91.8 % ± 3.4 % in presence of DSF/Cu when compared with the control group (24.0 ± 4.6 %) (P < 0.001). Similarly, DSF/Cu significantly induced loss of MMP in RPMI8226 cells (P < 0.001) (Fig. 5A-B). Consistently, the images taken by a fluorescent microscope showed that myeloma cells (MM.1S and RPMI8226) treated with DSF/Cu presented weaker red fluorescence, respectively (Fig. 5C). These results suggested that intrinsic apoptosis pathway was involved in the DSF/Cu induced myeloma cell apoptosis machinery.
3.3. The DSF/Cu complex enhanced apoptosis of MM cells Apoptosis of MM.1S and RPMI8226 cells was detected using Annexin-V FITC and propidium iodide (PI) staining followed by flow cytometry. To determine the combined effect of DSF and Cu on both myeloma cell lines, the cells were exposed to increasing doses of DSF with Cu for 12 h. The total number of AV+ and AV+ PI+ positive cells was counted as the number of apoptotic cells, which were used for the comparisons between groups. Compared to the control group (9.8 ± 2.9 %), apoptosis in MM.1S cells after 12 h was increased by 35.6 % ± 6.5 %, 65.4 % ± 3.5 % and 87.4 % ± 2.9 % at the dose of 0.1 μM, 0.25 μM, 0.5 μM, respectively, while apoptosis in RPMI8226 cells was also increased by 16.8 % ± 2.3 %, 54.4 % ± 3.9 % and 83.8 % ± 5.0 % at 0.1 μM, 0.25 μM, 0.5 μM, respectively (Fig. 2A-B). It was also observed that the apoptotic rate of cells treated with DSF/Cu was time-dependent with a higher apoptotic ratio at 48 h than that at 12 h and 24 h, especially in MM.1S cells (P < 0.001) (Fig. 2C-D). Apoptosis was induced in both MM.1S and RPMI8226 cells in a dose- and timedependent manner, consistent with our findings in the MTT assay.
3.7. DSF induced activation of caspase 8, caspase 3, and PARP cleavage in MM cells Caspase 8 and Caspase-3 play a central role in the initiation and execution of apoptotic program, respectively. Caspase-3 is primarily responsible for the cleavage of PARP (poly ADP-ribose polymerase) during cell apoptosis. As expected, DSF/Cu induced cleavage of caspase 8 (Fig. 6A, C), caspase-3 and PARP (Fig. 6B, C). To further confirm the involvement of caspase-3 in DSF/Cu-induced apoptosis, MM.1S and RPMI8226 cells were pre-treated with a broad-spectrum pan-caspase inhibitor (zVAD) at 20 μM concentration for 1 h and then added with DSF/Cu (DSF: 0.25 μM/Cu: 0.5 μM). Pre-treatment with zVAD significantly blocked apoptosis in MM.1S cells, 15.7 % ± 4.1 % vs. 58.4 % ± 3.6 % and in RPMI8226 cells, 15.8 % ± 1.1 % vs. 54.9 % ± 5.2 % (Fig. 6D-E). Western blotting (Fig. 6F-G) results also showed that DSF/ Cu-induced activation of caspase-3 and the cleavage of PARP was significantly inhibited by zVAD. Taken together, these results suggested that extrinsic cell apoptosis pathway is involved in the myeloma cell apoptosis induced by DSF/Cu in caspase dependent and independent manners.
3.4. The DSF/Cu complex induced apoptosis of MM primary cells The cytotoxic activity of DSF/Cu against primary MM cells obtained from bone marrow of newly diagnosed or relapsed/refractory patients was tested. Consistent with the anti-myeloma activity of DSF/Cu observed in MM cell lines, co-treatment with DSF (0.5 μM) and Cu (0.5 μM) resulted in significant increases in apoptosis of primary cells (69.1 ± 9.3 % vs 17.2 ± 9.6 %, P < 0.001, n=7; Fig. 3A-B). Since myeloma patient samples were valuable and limited in volumes, we had difficulty to apply more concentrations and time points in those experiments. To find a more suitable dose of DSF and time point, we first tested on two different MM patient samples and found that treatment by 0.25 μM DSF for 12 h and 24 h did not induce significant apoptosis when compared with untreated ones (controls), respectively. Therefore, we increased the DSF dosage from 0.25 to 0.5 μM DSF and used the combination of 0.5 μM DSF for 24 h in all the experiments on primary MM cells. These findings suggested that DSF/Cu might eliminate MM primary cells.
3.8. DSF/Cu induced activation of caspase and JNK signaling pathway C-Jun N-terminal kinase (JNK), P38 and ERK are members of the mitogen activated protein kinase (MAPK) family. JNK pathway could be activated by a variety of stimuli including UV radiation and DNA damaging agents contributing to apoptosis. To investigate potential roles of JNK pathway in DSF/Cu mediated apoptosis of myeloma cells, we analyzed JNK, c-Jun, P38 and ERK expression by western blot. As shown in Fig. 7A-C, although there was no change in JNK, P38 and ERK expression, phosphorylation of JNK and c-Jun expression were increased in the MM.1S and RPMI8226 cells treated by DSF/Cu. To further confirm whether JNK/c-Jun pathway activation contributed to DSF/Cu-triggered apoptosis, MM.1S and RPMI8226 cells were pretreated with 20 μM of JNK inhibitor SP600125 for 2 h and then exposed to DSF/Cu for 12 h. Apoptosis induced by the combination of DSF and
3.5. DSF caused cell cycle arrest in G2/ M phase To study the mechanisms of DSF/Cu-induced MM cell lines growth 4
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Fig. 2. DSF/Cu significantly induced apoptosis in MM cells (MM.1S and RPMI8226). (A, B) MM.1S and RPMI8226 cells were treated with a series of concentrations of DSF (0.1, 0.25, and 0.5 μM) with 0.5 μM Cu for 12 h, after which the percentage of apoptotic cells was determined by flow cytometry using Annexin V/PI double staining (n=3). * P < 0.05, ** P < 0.01, *** P < 0.001 vs the untreated control or the DSF/Cu complex, respectively. (C, D) Apoptotic MM.1S and RPMI8226 cells were measured by flow cytometry at different time points (24 and 48 h) after being treated with DSF/Cu (DSF: 0.25 μM/Cu: 0.5 μM) (n = 3).
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Fig. 3. DSF/Cu induced apoptosis in MM primary cells. Mononuclear cells were isolated from primary bone marrow of newly diagnosed or relapsed/refractory patients with multiple myeloma. Representative plots of bone marrow sample showing the gating strategy for CD38+ MM cells. (A) Representative flow cytometry plots of Annexin-V expression in control and DSF/Cu. (B) Percentage of Annexin-V expression in control and DSF/Cu. P value is shown. Value of P＜0.001 is considered as a significant difference.
cells from patients. For example, Manman Deng et al. have reported that DSF/Cu could induce apoptosis of primary cells isolated from BALL patients [34]. To evaluate activity of DSF/Cu against primary myeloma cell, mononuclear cells were isolated from bone marrow of MM patients and flow cytometry as well as apoptosis detection techniques were utilized to define myeloma cell apoptosis. As expected, DSF/Cu markedly induced apoptosis of primary MM cells. And it only had a negligible toxicity to normal PBMCs. More importantly, our results from animal studies showed that DSF/Cu significantly inhibited tumor growth and prolonged overall survival in vivo, indicating a potent anti-myeloma activity of DSF/Cu in vivo. The dosage of DSF and administration approach applied in our animal experiments were followed the previously published papers [33]. In Skrott Z’s study and their clinical trial (NCT00742911), Cu was administered each day in the morning (08:00) and DSF each day in the evening (19:00). To do the animal experiment in a way close to real clinical practice, we followed Skrott Z’s protocol and treated the myeloma bearing mice by administration of Cu in the morning and DSF in the evening. It has been shown that primary metabolite of the DSF in stomach is Diethyldithiocarbamate (DDC), and anticancer activity of DDC is dependent on complexation with Cu to form a complex Cu (DDC)2. However, this complex is highly insoluble and is difficult to be absorbed in stomach [35]. This should be the main reason why DSF and Cu were given separately. Cell cycle arrest is one of the mechanisms for cell growth inhibition induced by many anticancer drugs. For example, Runzhe Chen et al. have demonstrated that the inhibition of the proliferation of MM cells by bortezomib was due to induction of cell cycle arrest in the G2/M phase [36]. Consistently, we demonstrated that DSF/Cu inhibited the growth of MM.1S and RPMI8226 cells by inducing cell cycle arrest at G2/M phase. To reveal underlying mechanism of DSF/Cu-induced apoptosis of myeloma cells, we analyzed certain key molecules involved in cell apoptosis signaling pathways. Working as the extrinsic and intrinsic pathways [37], initiator, caspase-8 and caspase-9, respectively, discriminate to engage the same proteolytic caspase cascade. It leads to the activation of interface at the point of downstream executioner caspase3 that induces the execution phase of apoptosis after being proteolytically activated [38]. Here, we found that DSF/Cu complex treatment increased activation of caspase-8, caspase-3 and accumulation of cleaved-PARP fragment. DSF/Cu treatment-associated apoptosis of MM cells was substantially inhibited by zVAD, suggesting that caspase dependent and independent apoptosis pathways are involved in the DSF/ Cu treatment mediated myeloma cell apoptosis [39]. To determine a role of intrinsic apoptotic pathway in the apoptosis induced DSF/Cu, we assessed the MMP in DSF/Cu treated MM cells. In line with the findings
Cu complex was attenuated by adding SP600125 (MM.1S: declined from 57.9 % ± 3.6%–20.2% ± 2.4 %; RPMI8226: declined from 54.5 % ± 3.1%–18.8% ± 3.3 %) with a significant statistical significance (Fig. 7D-G). These results indicated that JNK/c-Jun activation is implicated in the DSF/Cu-mediated myeloma cell apoptosis mechanism. 3.9. DSF/Cu exhibited anti-myeloma activity in vivo To evaluate therapeutic effects of DSF/Cu on MM in vivo, a myeloma mouse model was used. DSF and Cu were administered by oral gavage for 2 weeks. As shown in Fig. 8A-C, we found that tumor weight and tumor volumes in the control group grew more rapidly than that in the DSF/Cu group, and the differences in tumor sizes from the fourth day after treatment indicated in the Fig. 8C between the two groups were statistically significant (P < 0.001). Changes in survival among the control and DSF/Cu groups were compared by using Kaplan-Meier curves. As shown in Fig. 8D, DSF/Cu treatment significantly prolonged survival of the myeloma mice when compared with the control (P＜ 0.001). Our results showed that the mean survival time of the mice treated by the control group and DSF/Cu group were 25.5 days and 31 days, respectively. Together, these results demonstrated that the DSF and Cu combination selectively inhibited tumor growth and improved survival of myeloma bearing mice in vivo. 4. Discussion DSF is a widely used anti-alcoholism drug with low toxic effects on normal tissues [32]. Interestingly, its anti-cancer activity has been recently recognized [7]. DSF used to treat high-risk breast cancer in a clinical trial with promising results, DSF has been proposed as a candidate for drug repurposing in cancer [33]. In a study by Skrott et al., groups of mice were injected with human MDA-MB-231 cancer cells, fed with a diet plus DSF or DSF/Cu. Compared to control group, tumor volume in DSF and DSF/Cu-treated groups were suppressed by 57 % and 77 %, respectively (P = 0.0038). These results indicated that DSF/ Cu is an efficient anti-cancer agent in cancer therapy. In our study, we tested DSF alone and Cu toxicity using MTT assay and found that DSF/ Cu was more cytotoxic than DSF alone to the MM cell lines and Cu alone was not toxic to the MM cell lines. Therefore, in the subsequent experiments we focused only on testing cytotoxic activity of DSF/Cu complex in order to provide acknowledge important for designing a clinical trial using DSF/Cu complex to treat recurrent / refractory myeloma patients. And our results also demonstrated that the DSF/Cu complex induced apoptosis of MM cells in a time- and dose-dependent manner. Previous studies have shown that DSF/Cu can be used not only in the treatment of tumor cell lines, but also in the treatment of primary 6
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Fig. 4. DSF/Cu induced cell cycle arrest in MM.1S and RPMI8226 cells. (A) Cell cycle analysis was performed with Flow Cytometry in MM.1S and RPMI8226 cells treated with/without DSF/Cu for 12 h. (B) Cell cycle distribution in MM.1S cells. Histogram showed the percentage of cells in the G1, S and G2 phases. Data are presented as the mean ± SD (n = 3). ***P < 0.001 vs control.
Fig. 5. DSF/Cu markedly induced loss of the Mitochondrial Membrane Potential (MMP). (A) MM.1S and RPMI8226 cells were treated with DSF/Cu (0.25 μM/0. 5 μM) for 12 h, and the level of the loss of the Mitochondrial Membrane Potential was detected by JC-1 flow cytometry (n = 3). (B) The fluorescent intensity of DSF/Cu treatment groups are indicated. Data is presented as mean ± SEM (n = 3, ** P < 0.01, *** P < 0.001, as compared with the control.). (C) Fluorescent image of MM.1S and RPMI8226 cells stained with JC-1. Photograph shows JC-1 red and JC-1 green, representing JC-1 aggregates and JC-1 monomers (n = 3). 7
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Fig. 6. DSF/Cu induced cleavage of caspase 8, caspase 3 and PARP. (A) MM.1S and RPMI8226 cells were treated with various concentrations of DSF (0.1, 0.25, and 0.5 μM) and Cu (0.5 μM) for 12 h. Total proteins were extracted from the cultured cells and subjected to Western blot analysis using antibodies against caspase-8. GAPDH was used as a loading control. (B) The cells were treated in the way described previously. The cell lysates were analyzed by western blotting to detect the expression levels of caspase-3 and PARP cleavage. (C) Shows the relative protein level of caspase-8, caspase-3 and PARP cleavage calculated by the band density of western blots using Image J software of MM cell line (*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test). (D) MM.1S and RPMI8226 cells were treated with zVAD (20 μM) for 1 h prior to adding DSF/Cu (DSF: 0.25 μM/Cu: 0.5 μM) for 12 h. Apoptosis was monitored by flow cytometry detection of AV/PI staining. Dot plots of one representative experiment are shown. Percentages of positive cells for AV and PI are indicated in the plots. (E) Apoptosis of MM.1S and RPMI8226 cells treated with DSF/Cu in the presence or absence of zVAD. The results are expressed as mean percentage ± SEM of total (AV+PI– + AV+PI+) apoptotic cells from three independent experiments. *p < 0.001 by Student’s t test. (F) Western blot analysis of caspase-3 and PARP cleavage expression in MM.1S and RPMI8226 cells treated with DSF/Cu in the presence or absence of zVAD at 12 h. Results were presented as Mean ± SEM from three independent experiments. (F) Shows the relative protein level of caspase-3 and PARP cleavage on MM cell line (*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test). 8
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Fig. 7. DSF/Cu induced JNK/c-jun activation. (A) The cells were treated as previously described. The combination of DSF and Cu markedly activated the JNK/c-jun pathway while DSF/Cu induced phosphorylation of JNK and c-Jun (n = 3). GAPDH was used as a loading control. (B) Western blot analysis of P38 and ERK expression in MM.1S and RPMI8226 cells treated with DSF/Cu. (C) Shows the relative protein level of JNK, P-JNK, P-c-Jun, P38 and ERK calculated by the band density of western blots using Image J software of MM cell line (*P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test). (D) MM.1S and RPMI8226 cells were treated with SP600125 (20 μM) for 2 h prior to adding DSF/Cu (DSF: 0.25 μM/Cu: 0.5 μM) for 12 h. Apoptosis was evaluated using AV and PI staining by flow cytometry. (E) The apoptosis results are expressed as mean percentage ± SEM of total (AV+PI– + AV+PI+) apoptotic cells from three independent experiments (n = 3). *P < 0.001 by Student’s t test. (F) The expressions of P-JNK proteins in MM.1S and RPMI8226 cells treated with DSF/Cu in the presence of SP600125 were measured by Western blot. (G) Shows the relative protein level of P-JNK calculated by the band density of western blots using Image J software of MM cell line (**P < 0.01, ***P < 0.001 by Student’s t test).
reported in other type of tumors. In line with the findings reported in other type of tumors [40,41], we found that DSF/Cu treatment induced loss of MMP in myeloma cells. As a known fact, MMP decreases in early apoptosis as an indicator of mitochondrial membrane permeability, this might suggest that DSF/Cu induce intrinsic apoptosis machinery. Mitogen-activated protein kinase (MAPK) is implicated in regulating differentiation, cell growth and proliferation, and apoptosis responses of tumor cells [42]. Several studies have reported the involvement of MAPK in cancer deregulation [41,43]. MAPK family consist of various members, including JNK/stress-activated protein kinase (SAPK), p38, and growth factor-regulated ERK1/2 [44,45]. JNK, as a serine/threonine protein kinase, has the ability to interact with c-Jun which acts as a focus of transcription factor-activation protein-1, and then promote the expression of pro-apoptotic protein such as p53, Bax and Fas. They in turn activate the caspase family as a consequence of the apoptosis signal transduction pathway [46,47]. Moreover, a team of researchers led by Liu Q., reported that continuous activation of JNK was necessary for the drug-induced apoptosis in myeloma cell lines in vitro [48]. Recently, there was a study that demonstrated that the JNK/c-Jun pathway may contribute to the DSF/Cu induced apoptosis in lymphoid malignant cell lines in vitro [49]. In accordance with previous studies, our results demonstrated that DSF/Cu can markedly upregulated expression of phosphorylation of JNK and c-Jun, resulting in the induction of apoptosis. Pre-treatment with SP600125, the JNK-specific inhibitor, resulted in the reduction of P-JNK expression and protected cells from apoptosis. These findings suggested that DSF/Cu induced apoptosis in multiple myeloma cells by activating the JNK signaling pathway.
5. Conclusions Taken together, our novel findings in this study presented DSF/Cu as a potent anti-myeloma agent in vitro and in animal models. This antialcoholism drug holds immense potential to be repurposed as a potent cancer drug. Ethical approval All procedures performed in studies involving animals and human samples were in accordance with the ethical standards of the institution or practice at which the studies were conducted. Authors’ contributions ZCY and NYH designed the project, analyzed the data and revised the manuscript. XYQ did the most experiments, analyzed the data and wrote the manuscript. Flow cytometry analysis and paper writing were together accomplished by XYQ, FXL and JW. While XYQ, DYB, ZQ and WYJ were in duty of the statistical analysis. Animal experiments were performed as a teamwork by XYQ, XXL, JY and LXL. All the authors have read and approved the manuscript. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements The present study was supported by the National Natural Science 9
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Fig. 8. DSF/Cu significantly suppressed tumor weight, tumor growth and prolonged overall survival of myeloma bearing mice. DSF/Cu and control were administered to the myeloma-bearing SCID mice by oral gavage. (A) Xenograft images were shown. (B, C) The changes in tumor weight and tumor volumes among the control and treatment groups at the observation time points are indicated (**P < 0.01, ***P < 0.001, by Student's t test). The values are shown as mean ± SEM. Six mice in each group. (D) DSF/Cu treatments significantly prolonged survival of the myeloma bearing mice as compared with the control (***P < 0.001, by Long-rank test, respectively). The values are shown as mean ± SEM. Six mice in each group.
Foundation of China (grant no.81600176, no.81602694 and no.81372545), the Science and Technology innovation project of Shandong Province (grant no.2017GSF18136 and no.2018GSF118034), the Clinical Medical Technology innovation project of Jinan (grant no.201704089), and the National Natural Science Foundation of Shandong Province (grant no. ZR2016HB71). Thanks to Dr. Edward C, Mignot, Shandong University, for linguistic advice. We would like to thank Editage (www.editage.cn) for English language editing.
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