Disulfiram Combined With Copper Inhibits Gastric Cancer Cell Growth By Regulating Stress Response And Wnt/?-catenin Signaling Pathway

Objective: Gastric cancer,as the malignant tumors,burden on human life and economy.Currently,there are many treatment methods,accompaning by side effects and poor sensitivity,so the clinical benifits of gastric cancer patients stays inferior.In order to improve clinical benefits,it is still necessary to find new chemotherapy drugs for gastric cancer patients.As an anti-alcoholism drug,disulfiram shows a broad spectrum of antitumor effects in various cancers,and disulfiram combined with copper enhance the anti-tumor effect.Therefore,we explored the mechanism of disulfiram combined copper inhibit gastric cancer cell growth,which maybe a novel strategy for gastric cancer patients.Methods: In MKN-45 and BGC-823 cells,the toxic activity and proliferation of disulfiram combined with copper were detected by CCK8 and clony formation;the migration and invasion were measured by caused by Transwell and scratch experiment;the apoptosis were tested by flow cytometry and western blot;the autophagy were studied by transimission electron microscopy,western blot,transient transfection and stable transfection;the reactive oxygen was explored by2,7-Dichlorodi-hydrofluorescein diacetate.Using western blot,the expression of?-catenin,Cyclin D1,C-myc,Fzd7,?-H2 AX,P21 and P53 were detected;using Seahorse method,extracellular acidification rate and the oxygen consumption rate was detected.Using male Balb/C nude mice and MKN-45 cells,the subcutaneous graft tumor model of gastric cancer was established.Tumor growth inhibition was calculated by the tumor volume,the hematoxylin-eosin staining and immunohistochemistry was carried out to evaluate the proliferation ability.By recording the body weight of nude mice,the safety of disulfiram combined copper was explored,the mechanism was detected by Tunel staining and immunohistochemistry.Results: In vitro and vivo,disulfiram combined with copper inhibited cell growth,proliferation,invasion and migration in time-dose-dependent manner,and no obvious side effect.The disulfiram combined with copper manipulated the expression of BCL2 and BAX,induced the apoptosis in gastric cancer.In treatment group,autophagosomes were observed by transimission electron microscopy.The gastric cancer cells with transient expression GFP-LC3 and stable expression m RFP-GFP-LC3 were constructed by transfection method,the disulfiram combined with copper increased the expression of GFP-LC3 and m RFP-GFP-LC3.Disulfiram combined with copper increased reactive oxygen species,inhibited glycolysis and oxidative phosphorylation.At the protein level,disulfiram combined copper increased the expression of ?-H2 AX,P53 and P21,and declined the expression of ?-catenin,Cyclin D1,C-myc and Fzd7.Conclusion: In vivo and in vitro,disulfiram combined with copper repressed the growth,invasion and migration,induced apoptosis and autophagy,and displayed anti-tumor role by regulating stress response and Wnt/?-catenin signaling pathway,without obvious toxic effect in gastric cancer.In future,disulfiram maybe a potential anticancer method for gastric cancer patients.