Study On Cytotoxicity Effects Of Disulfiram On Multiple Myeloma And Its Underlying Mechanisms

BackgroundMultiple myeloma(MM)is a hematological malignancy characterized by abnormal accumulation of cancerous plasma cells in the bone marrow,accounting for about 10%of all hematologic malignancies,and patients typically present with bone marrow infiltration of clonal plasma cells and monoclonal protein in the serum and/or urine.The accumulation of these immune globulin can lead to organ dysfunction,usually referred to as "CRAB",which consists of hypercalcemia,renal failure,anemia,and lytic bone lesions.Treatment for MM includes glucocorticoids,alkylating agents,anthracyclines,proteasome inhibitors,immunomodulators,histone acetylase inhibitors,and monoclonal antibodies.However,patients with MM always enter the relapse/refractory phase after one or more treatment regimens and the main reason was resistance.Taking into account the advantages of low cost,low risk and less time consumption,it is an emerging therapeutic strategy to reuse approved drugs as new anticancer drug.Disulfiram,which was approved by the American Food and Drug Administration(FDA)in 1951,has been widely used to treat alcoholism for more than 60 years.In the body,DSF is metabolized to diethyldithiocarbamate(DTC)and other metabolites,which can lead to the specific accumulation of acetaldehyde,causing unpleasant symptoms such as tachypnea,tachycardia,vomiting and hypotension when drinking alcohol.In recent years,many studies have indicated that DSF was highly effective against several types of solid tumors such as breast cancer,colon cancer and melanoma.Several studies have shown that DSF combined with Copper(Cu)could enhance its anti-tumor effect.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 and transcription factors.As a divalent metal ion chelator,DSF strongly interacts with Cu to form the disulfiram/copper(DSF/Cu)complex which can reduce the insoluble property of DSF in water,make it easier to be absorbed by cells,and significantly enhance the DSF-induced anti-tumor cytotoxicity.This study aims to investigate the anti-MM effect of DSF/Cu and its underlying anti-MM mechanism,so as to provide theoretical and experimental foundation for the clinical application of DSF/Cu in the treatment of MM.Methods1.MTT assay was used to detect the effects of DSF alone(0.05,0.1,0.25 and 0.5?M)and DSF/Cu combination(0.05/0.5,0.1/0.5,0.25/0.5 and 0.5/0.5?M)on the cell viability of MM.1S and RPMI8226 cells.Only one concentration of DSF(0.25?mol/1)(very close to IC50)was used for 24 h and 48 h experiments.The above experiments could provide evidence to observe whether the cytotoxicity of DSF alone or DSF/Cu on MM cell lines were dose-dependence and time-dependence.2.MTT assay was applied to detect the effects of DSF alone or DSF/Cu combination at the same concentration on MM cell lines and human normal peripheral blood mononuclear cells(PBMC).3.To observe whether apoptosis was induced by DSF/Cu in a dose-and time-dependent manner,Annexin V-FITC/PI staining was used to detect the effect of different concentrations of DSF/Cu(0.1/0.5,0.25/0.5 and 0.5/0.5?M)and different time points(24h and 48h)on the apoptosis in MM.1S and RPMI8226 cell lines.4.Flow cytometry was used to detect the effect of DSF/Cu on primary myeloma cells in MM patients.5.The effects of different concentrations of DSF/Cu on the cell cycle of MM cell lines was detected by PI staining.6.JC-1 mitochondrial membrane potential(MMP)detection kit was performed to observe the effects of DSF/Cu on the apoptosis of MM cell lines,and to investigate whether apoptosis induced by DSF/Cu was through mitochondria-dependent endogenous apoptosis pathway.7.Western Blot was used to detect the expression of DSF/Cu-pretreated MM cell lines on caspase-3,caspase-8 and cleaved PARP protein.The pan-caspase inhibitor Z-VAD-FMK(zVAD)was added to further observe the changes in the expression of caspase-3 and cleaved PARP proteins,in order to confirming if apoptosis induced by DSF/Cu was through the caspase-dependent apoptosis pathway.8.Western Blot was used to detect the expression of DSF/Cu-pretreated MM cell lines on JNK,P-JNK,P-c-jun,ERK and P38 protein.After the addition of JNK inhibitor SP600125,the changes of P-JNK protein expression were observed to investigate the role of MAPK pathway in DSF/Cu-induced apoptosis of MM cell lines.9.By subcutaneously injected with MM cells in NOS/SCID mice,we established a human MM cell xenograft animal model to observe the effects of DSF/Cu on the tumor weight,tumor volumes and survival of the myeloma mice.Results1.DSF or the DSF/Cu complex had the effects on inhibition of cell viability on MM cells in a dose-dependent manner.The IC50 of DSF on MM.1S and RPMI8226 cells were 0.32±0.04 ?M and 0.87±0.03 ?M,while the IC50 of DSF/Cu complex were 0.23±0.18 ?M and 0.25±0.02 ?M.In addition,the inhibition of Cu on cell viability was very low,basically no toxicity.Furthermore,when MM cell lines pretreated with DSF or the DSF/Cu complex for 12h,24h and 48h,MTT results showed that the MM cell viability was in a time-dependent decrease,and DSF/Cu complex was even more cytotoxic to the MM cell lines.2.After exposure to the same concentration of DSF or DSF/Cu for 12h,MTT results suggested DSF or DSF/Cu showed little toxicity to normal PBMCs,while a great killing effect on MM.1S cells was observed.3.Since the previous MTT results showed DSF/Cu complex was more toxic to the MM cell lines,only the DSF/Cu complex was used as the treatment group for the subsequent experiments.The apoptosis detected by flow cytometry was in a dose-and time-dependent manner,consistent with our findings in the MTT assay.4.We tested the cytotoxic activity of DSF/Cu against primary MM cells obtained from bone marrow of seven newly diagnosed or relapsed/refractory patients.After co-treatment with DSF(0.5 ?M)and Cu(0.5 ?M),the apoptosis of primary MM cells was 69.1 ± 9.3%.These findings suggested that DSF/Cu might eliminate MM primary cells.5.After the treatment of different doses of DSF/Cu(0.1/0.5,0.25/0.5 and 0.5/0.5?M),the results of flow cytometry revealed the percentage of MM cells in G2/M phase was higher compared with the control group.These results indicated that the growth-inhibitory effect of DSF/Cu against MM.1S and RPMI8226 cells could arrest a cell cycle in G2/M phase in a dose-dependent manner.6.Incubated with DSF/Cu(0.25/0.5?M)on the MM.1S an RPMI8226 cells for 12h,data from flow cytometry demonstrated that the monomer ratio of JC-1 increased obviously.Observing under fluorescence microscope,the color of normal MM.1S and RPMI8226 cells was yellow-green due to the preponderance of JC-1 polymers in the cytoplasm.After the addition of DSF/Cu,a large number of early and late apoptotic cells appeared,MMP depolarized,mitochondria released and red fluorescence became weak.And the MM cells presented green fluorescence because of the increasing JC-1 monomers.These results suggested that mitochondria-dependent apoptosis pathway was involved in the DSF/Cu induced myeloma cell apoptosis machinery.7.Western Blot results showed that MM.1S an RPMI8226 cells treated with different doses of DSF/Cu(0.1/0.5,0.25/0.5 and 0.5/0.5?M)for 12h induced cleavage of caspase 8,caspase-3 and PARP.Pre-treatment with pan-caspase inhibitor zVAD at 20 ?M concentration for 1 h significantly blocked apoptosis in MM cells.The expression of pro-caspase-3 was increased while the expression of cleaved caspase-3 and cleaved PARP was decreased,confirmed by the Western Blot.These results indicated that caspase-dependent manner was one of the pathways of DSF/Cu induced apoptosis in MM cell lines.8.Although there was no change in JNK,P38 and ERK expression,P-JNK and P-c-jun expression were increased in MM cells after the treatment of different doses of DSF/Cu(0.1/0.5,0.25/0.5 and 0.5/0.5?M)for 12h.Pretreated with 20?M of JNK inhibitor SP600125 for 2h,apoptosis induced by DSF/Cu was significantly attenuated and P-JNK expression was decreased.These results indicated that MAPK activation was implicated in the DSF/Cu-mediated myeloma cell apoptosis mechanism.9.NOD/SCID myeloma bearing mouse model was successfully established by subcutaneously injected with 1×107 human myeloma cell line RPMI8226 cells.When tumors reached a size of approximately 200 mm3,the treatment group was provided DSF/Cu every day for 14 days by gavage.The mice were humanely euthanized by cervical dislocation when they reached the endpoint of our observation.Tumor weight and tumor volumes in the treatment group grew much slower comparing to in the control group,and the differences in tumor sizes from the fourth day were statistically significant(P<0.001).For humanitarian reasons,the mice were euthanized by cervical dislocation 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.The results showed that DSF/Cu treatment significantly prolonged survival of the myeloma mice.And the mean survival time of the mice treated by the control group and DSF/Cu group were 25.5 days and 31 days,respectively(P<0.001).Together,these results demonstrated that DSF/Cu selectively inhibited tumor growth and prolong the survival of myeloma bearing mice in vivo.Conclusion1.DSF or the DSF/Cu complex had the effects on inhibition of cell viability on MM cells in a dose-dependent and time-dependent manner.And DSF/Cu complex was more cytotoxic to the MM cell lines.2.DSF or DSF/Cu exhibited little toxicity to normal PBMCs,while a great killing effect on MM.1S cells was observed at the same concentration of DSF or DSF/Cu.3.The apoptosis induced by DSF/Cu in MM cells was in a dose-and time-dependent manner.And DSF/Cu could also induce apoptosis in primary myeloma cells from MM patients.4.DSF/Cu against MM.1S and RPMI8226 cells could arrest a cell cycle in G2/M phase.5.DSF/Cu could induce MMP and it might suggest that mitochondria-dependent endogenous apoptosis pathway was involved in the DSF/Cu induced myeloma cell apoptosis machinery.6.Caspase-dependent apoptosis pathway and MAPK signaling pathway were involved in the myeloma cell apoptosis induced by DSF/Cu.7.DSF/Cu selectively inhibited tumor growth and prolong the survival of myeloma bearing mice in vivo.