Disulfide Inhibits Proliferation,Migration And Invasion Of Gastric Cancer By Down-regulating The Wnt And NF-?B Signaling Pathways

Background and objective:Gastric cancer?GC?is one of the leading causes of cancer-related death,according to statistics from the American Cancer Society,the incidence of gastric cancer ranks fifth in digestive system cancers.Meanwhile,in East Asia,it has been estimated that gastric cancer is the second most commonly diagnosed cancer,and it ranks third among tumor-related deaths for both genders.Disulfiram,also known as Antabuse,is applied as an anti-alcohol dependent drug due to its inhibitory effect on enzyme acetaldehyde dehydrogenase.Recently,preclinical studies have revealed its potential role in targeting malignant tumors such as breast cancer,myeloma,pancreatic cancer,and so on.In addition,it was reported that disulfiram was capable of reversing drug resistance,inhibiting DNA methylation and inducing cell apoptosis.However,its effect on gastric cancer has not yet been elucidated.Therefore,a study on this candidate for tumor chemotherapy was conducted,aiming to explore its role in the proliferation,migration,and invasion of GC cells to explore the molecular mechanism,Wnt further,and NF-?B signal was investigated as well.Materials and MethodsCell linesGES,MKN-45,and SGC-7901 cell lines were obtained from the Xiangya Medical College at Central South University.ReagentsDisulfiram was purchased from MilliporeSigma?St.Louis,MO,USA?.RPMI-1640 medium,fetal bovine serum?FBS?and Matrigel were purchased from Gibco?Gaithersburg,MD,USA?.Matrigel and Transwell chambers were purchased from Costar Inc.?NY,USA?.Rabbit antibodies against MMP2,PCNA,Wnt,?-catenin,and NF-?B were purchased from Santa Cruz Biotechnology?Dallas,TX,USA?,and secondary antibody and cell counting kit-8?CCK-8?were obtained from Beyotime Biotechnology?Haimen,Jiangsu,China?.Cell cultureCells were cultured in complete medium?RPMI-1640 medium with 10%FBS?at37°C in 5%CO₂ and 100%relative humidity.CCK-8 Proliferation assayTo investigate the effects of disulfiram on the proliferation of gastric cancer cells,a CCK-8 assay was performed.Cells in exponential growth phase were collected and processed into a single cell suspension using 0.25%trypsin.They then were seeded?1×10⁴ cells/well?in a 96-well reaction plate with culture medium containing disulfiram.After 24,48,72,and 96 hours,10?L CCK-8 solution was added,and absorbance was measured after a two-hour incubation at 37°C.Transwell assaysTo evaluate the migration and invasive abilities of cells treated by disulfiram,Transwell assays were conducted.Briefly,Transwell chambers were added to24-well-plate containing 0.1%BSA-RPMI 1640,followed by the addition of 100?L cell suspension into the chambers with disulfiram at different concentrations?0,12.5,25,50,and 100?mol/l?.After 12 hours,the chambers were removed,fixed in formalin,and stained.Subsequently,cells on the surface of the permeable membrane were removed,and samples were processed in neutral balsam.The number of cells penetrating from the chambers was counted from five different randomly selected fields of each membrane.To evaluate the invasiveness of cancer cells,5?g Matrigel was added to the surface of a permeable membrane to form a basement membrane.Subsequently,the Transwell assay was performed as described above.ELISA AnalysisELISA assays were performed following instructions supplied by the manufacturer.In short,cell culture supernatant samples and standard proteins were added to the multi-well plate coated and incubated for two hours.Plates were then washed using washing buffer?Tween 20,and phosphate-buffered saline solution,Sigma Aldrich,USA?.In the next step,a biotin-labeled antibody was added to each well and incubated for another two hours.ELISA plates were rewashed,and a streptavidin-horseradish-peroxidase solution was added.After adding tetramethylbenzidine?Sigma,USA?,a color reaction was achieved.Optical density was measured at 450 nm on an ELISA plate reader.Western blottingGastric cancer cells were first treated by disulfiram at different concentrations?12.5,25,50,and 100?mol/L?for 24 hours,and complete medium at the same volume was added into the control group.Afterwards,proteins were extracted from the cells using RIPA buffer.Subsequently,extracted samples were mixed with 5×SDS protein loading buffer at a ratio of 1:4 according to their concentration.Following denaturation,samples were transferred to a PVDF membrane by SDS-PAGE and processed in TBS-T containing 5%skim milk.Next,the membrane was incubated with primary antibody for two hours,followed by a two-hour incubation at room temperature with a horseradish peroxidase-conjugated secondary antibody.For autoradiography,we used ECL followed by X-ray exposure.Images were obtained using an ImageQuant 350 gel protein imaging system.Statistical analysisEach experiment was repeated at least three times.Acquired data were analyzed by SPSS Statistics for Windows Version 24.0?IBM Corp.,Armonk,NY,USA?and plotted using GraphPad Prism 6.0.One-way ANOVA or Student's t-tests were conducted to compare experimental and control groups.A P-value<0.05 was considered statistically significant.ResultsDisulfiram inhibits proliferation of gastric cancer cellIn order to determine the effective concentration of the drug,we tested the inhibition rate at different concentrations?0,10,25,50,75,100,and 125?mol/L?of disulfiram on GES and SGC-7901 by CCK8.The result showed that 50?mol/L is a semi-inhibition rate?IC 50?.Then,the CCK-8 assay was performed to evaluate the effect of disulfiram on the proliferation of gastric cancer at different concentrations?0,25,50,and 75?mol/L?.disulfiram significantly inhibited the proliferation of gastric cancer cells in vitro?p<0.05?and exhibited a corresponding concentration-dependent inhibition in cell growth as disulfiram concentrations increased.In addition,we evaluated the level of PCNA,a biomarker of proliferation,using ELISA assay.The result showed that the disulfiram significantly inhibited the expression of PCNA.Disulfiram inhibits migrating and invasive potential of gastric cancer cellTo determine the effect of disulfiram on migration and invasion,Transwell assays were performed.Consequently,we observed that disulfiram showed a significant inhibitory effect on the migration and invasion of cells in disulfiram groups.the migration potential was more significantly weakened at a higher concentration of disulfiram?P<0.05?.Furthermore,the invasion ability was also significantly inhibited at higher concentration of disulfiram?P<0.05?.In addition,we evaluated the expression of MMP2,a biomarker for migration and invasion,using ELISA assay.Results showed that the disulfiram significantly inhibited the expression of MMP2.Wnt and NF-?B signaling pathways were regulated by disulfiramTo investigate whether disulfiram regulates Wnt and NF-?B signal in GC cells,the protein was extracted from cells treated by disulfiram at different concentrations and those in the control group.The level of related proteins was measured by ELISA.As a result,compared with the results in control groups,we found that disulfiram inhibited the expression of Wnt,?-catenin,and NF-?B in both cell lines.Expression of target proteins in the Wnt and NF-?B signaling pathwaysProtein was extracted from cells in the control groups and from those treated with disulfiram at different concentrations?12.5,25,50,and 100?mol/L?,and subjected to western blot analysis.The expression levels of drug resistance associated protein of the Wnt and NF-?B signaling pathways were investigated.Compared with the results in control groups,it was revealed that disulfiram inhibited both pathways.DiscussionIn this in vitro study,inhibition of proliferation was observed in GC cells treated by disulfiram,SGC 7901 cell lines showed decreased growth,its potential in inhibiting was supported by ELISA:PCNA,which is a marker for proliferation,was attenuated after disulfiram treatment.We observed inhibitory effects of disulfiram on the migration and invasion of GC cells,down-regulation of MMP-2,which is correlated with migrating and invasive potential,was identified by ELISA.As we further explored the molecular mechanism,we found that Wnt and NF-?B signaling pathways were regulated by disulfiram.Disulfiram is clinically used in the treatment of alcoholism due to its inhibitory effect on the enzyme acetaldehyde dehydrogenase,causing strong and unpleasant symptoms such as flushing,tachycardia,headache,and so on after drinking alcohol.It is found to be effective in regulating a wide range of malignancies.Cong et al reported that disulfiram was effective in inhibiting the growth of pancreatic ductal adenocarcinoma both in vitro and in vivo.In the human body,disulfiram is catalyzed into diethyldithiocarbamate,and this metabolite has been shown to chelate into complexes in combination with Cu²⁺.These complexes are reported to inhibit proteasome activity and elevate radical oxygen species,therefore influence the activities of cancer cells.Abnormal activation of the canonical Wnt/?-catenin signal was observed in carcinoma of the breast,prostate,lung,etc.In this pathway,the Wnt ligand is a secreted glycoprotein that binds to membrane receptors.?-catenin translocates to the nucleus and forms a transcriptional complex,which subsequently activates the target genes of the Wnt signal,consequently facilitates proliferation or metastasis.In the absence of Wnt,?-catenin is degraded by a destruction complex containing Axin,glycogen synthase kinase 3?GSK3?,adenomatosis polyposis coli?APC?and so on.As Wnt ligands bind to the membrane receptors,proteins such as Dishevelled?Dvl?will be recruited and activated,then the polymers inactivate the destruction complex,leading to stabilization and accumulation of?-catenin.It was also observed in breast cancer cells that the epithelial to mesenchymal transition?EMT?related protein SNAI2 was stabilized by Wnt ligands in the same way.ASPP2 is a metastasis-related protein,Wang et al reported that ASPP2 directly bound to?-catenin-E-cadherin complex and,in addition,they observed ASPP2 inhibited?-catenin's N-terminal phosphorylation,thus stabilize?-catenin,preventing it from degradation.NF-?B pathway participates in cell apoptosis and is closely related to the initiation,growth,and metastasis of the tumor.NF-?B controls the transcription of cellular DNA and participates in the response of cells to noxious stimuli.In cancer cells,it is usually overexpressed.Aberrant activation of NF-?B signaling reduced the eradication of cancer cells by the immune system and promoted cancer cell metastasis.In addition,NF-?B expression resulted in decreased apoptosis by regulating anti-apoptotic gene TRAF?tumor necrosis factor receptor-associated factor?.We found that both the Wnt and NF-?B pathways,which involve in the proliferation,apoptosis and progression of cancer,were inhibited by disulfiram.Taken together,we infer that the inhibitory effects of disulfiram on gastric cancer cells were associated with the regulation of Wnt and NF-?B pathways.