Experimental Study On The Immunogenic Death Of Tumor Cells Induced By Disulfiram Combined With Copper Ions

Objective:To explore the anti-lung cancer immune activity of disulfiram combined with copper ion（DSF/Cu）and the effect of DSF/Cu on the immunogenic death of lung cancer and colon cancer cells,and provide experimental basis for the application of DSF/Cu in the treatment of lung cancer and colon cancer.Method:（1）The human lung cancer cell lines NCI-H460 and A549 cells were treated with 0.05μM～1.6μM DSF/Cu,and MTT,Western blot and flow cytometry were used to detect the expression of tumor cell cytotoxicity,apoptosis and immunogenicity-related molecules CRT,HSP70,HSP90and MHC-I.（2）By constructing a mouse lung cancer subcutaneously transplanted tumor animal model,the effect of DSF/Cu on the body weight and tumor growth of lung cancer transplanted mice was detected.Mice were randomly divided into five groups:blank control group,low-dose（50 mg/kg）DSF/Cu,medium-dose（75 mg/kg）DSF/Cu,high-dose（100 mg/kg）DSF/Cu and positive control group.Immunofluorescence and flow cytometry were used to detect the effect of DSF/Cu on the expression of ICD-related molecules CRT and HSP70,the classification of T cells and the release of cytokines by T cells by DSF/Cu in lung cancer tissues.（3）The human colon cancer cell lines HCT-8,SW620 and HCT-116cells were treated with 0.05μM～1.6μM DSF/Cu,and MTT and flow cytometry were used to detect the expression of tumor cell cytotoxicity,apoptosis and immunogenicity-related molecules CRT and HSP70.（4）By constructing a mouse colon cancer subcutaneously transplanted tumor animal model,the effect of DSF/Cu on the body weight and tumor growth of lung cancer transplanted mice was detected.Mice were randomly divided into five groups:blank control group,low-dose（50 mg/kg）DSF/Cu,medium-dose（75 mg/kg）DSF/Cu,high-dose（100 mg/kg）DSF/Cu and positive control group.Flow cytometry was used to detect the effect of DSF/Cu on the expression of ICD-related molecules CRT and HSP70 in colon cancer tissues.Results:（1）Treated human lung cancer cell lines NCI-H460 and A549 cells with DSF/Cu for 48 h,we found that the cell viability gradually decreases,and the IC₅₀ was 0.44μM and 0.38μM,respectively;the apoptosis rate was significantly increased（P<0.05）,the expression of apoptosis-related proteins Caspase-3 and Caspase-9 were up-regulated,and the ratio of Bax/Bcl-2 was higher than that of the control group（P<0.05）;the expression of CRT,HSP70,HSP90 and MHC-I molecules on the surface of lung cancer cells increased（P<0.05）.（2）We found that in the lung cancer transplantation experiment,the body weight of the mice did not change significantly,the growth rate of the tumor decreased,the tumor volume was significantly reduced,and the overall survival of the tumor-bearing mice was significantly increased in the DSF/Cu treatment group compared with the control group（P<0.05）;The results of immunofluorescence and flow cytometry showed that the expression of CRT and HSP70 in solid tumor tissues increased（P<0.05）;The ratio of CD4⁺and CD8⁺T cells in tumor tissue of mice in the DSF/Cu treatment group and the level of IFN-γreleased by CD4⁺and CD8⁺T cells were significantly increased（P<0.05）.（3）Treated human colon cancer cell lines HCT-8,SW620 and HCT-116 with DSF/Cu for 48 h,we found that the cell viability gradually decreased,and the IC₅₀ was 0.307μM,0.295μM and 0.271μM,respectively;Apoptosis rate was significantly increased（P<0.05）;The expression of CRT and HSP70 on the surface of colon cancer cells was up-regulated（P<0.05）.（4）We found that in the Colon cancer transplantation experiments,the body weight of the mice in the DSF/Cu treatment group did not change significantly,the growth rate of the tumor decreased,and the tumor volume was significantly reduced compared with the control group（P<0.01）;The results of flow cytometry showed that the expression of CRT and HSP70 in solid tumor tissues increased（P<0.05）.Conclusion:DSF/Cu could induce immunogenic death of lung and colon cancer.In detail,DSF/Cu promoted tumor cell apoptosis,increased the expression of CRT,HSP70,HSP90 and MHC-I and other molecules,and enhanced T lymphocyte immunity by targeting NPL4 and IFN-γexpression.