Experimental Evidence Of Immunogenic Cell Death On Colon Cancer Induced By Apigenin

Objective:To investigate the inhibitory effectss of natural flavonoid Apigenin(API)on colon cancer,along with the effects and mechanism on immunogenic cell death(ICD),providing experimental evidence for API as an immunomodulator for anti-tumor therapy.Methods:(1)Human liver cell line Lo2 was treated with 6.25μM～200μM API,and the cell viability was detected by MTT assay.Human colon cancer cell line Lo Vo and mouse colon cancer cell line CT26 were treated with 3.12μM～200μM and 4.68μM～300μM API respectively,and the survival rate was detected by MTT assay.The effects of API on proliferation of cancer cells was assayed by cell colony formation experiment.Apoptosis rate and apoptosis-related molecules were analyzed by flow cytometry and Western blot,respectively.(2)The expression of ICD-related molecules HMGB1,CALR,HSP70 and HSP90 as well as MHC-Ⅰrelated to antigen-presenting,were simultaneously examined by immunofluorescence and Western blot in Lo Vo and CT26 cells treated with 25μM～100μM and 50μM～200μM,respectively.The expression of MAPK-p38,NF-κB-p65 and their phosphorylated proteins were also detected by Western blot.MAPK-p38inhibitor treated on CT26 cells and Western blot detected the expression of ICD-related molecules.Lo Vo cells pretreated with API were co-cultured with Jurkat T cells,and the survival rate of Lo Vo cells was detected by MTT assay.(3)Two kinds of subcutaneous transplanted tumor models in mice and nude mice were randomly divided into saline blank control group,API low dose(50 mg/kg),API medium dose(100 mg/kg),API high dose(200 mg/kg)group and 5-Fu positive control group.The body weight,tumor growth and immune organ index were observed and the effects of API intervention on the pathology of transplanted tumor was detected by H&E staining.(4)The expression of CALR,MHC-Ⅰ,p-MAPK-p38 and p-NF-κB-p65 in transplanted tumor were investigated by immunohistochemistry.The expression of ICD-related molecules and MHC-Ⅰproteins was examined by Western blot in mouse transplanted tumor,along with the expression of MAPK-p38 and NF-κB p65 and their phosphorylated protein.CD8~+T lymphocytes in mouse transplanted tumor were performed by immunohistochemistry method.Results:(1)The effects of API on the proliferation and apoptosis of colon cancer cellsAPI had no cytotoxicity to Lo2 in the concentration range from 6.25μM to 200μM.However,3.12μM～200μM and 4.68μM～300μM API inhibited the proliferation of Lo Vo and CT26 cells,respectively.and the IC50 at 48 h was 45.65μM and 83.64μM.Compared with the control group,the apoptosis rate of Lo Vo and CT26 cells treated with API increased,the expression of apoptotic protein Bax and Cleaved caspase-3increased,while the expression of anti-apoptotic protein Bcl-2 decreased.(2)ICD induced by API in colon cancer cells is associated with MAPK-p38 and NF-κB p65 signalCompared with the control group,API enhanced the fluorescence signal of ICD-related molecules and MHC-Ⅰin Lo Vo and CT26 cells,and the expression of proteins was significantly increased.The expression of p-MAPK-p38 protein was higher than that of the control group,while that of p-NF-κB-p65 protein was decreased.Compared with the group treated with API,the expression of MAPK-p38 and its phosphorylation were inhibited and the expression of ICD-related molecules protein was significantly decreased in the group treated with MAPK-p38 inhibitor and API.The survival rate of cancer cells co-cultured with Jurkat T cells was further decreased compared with Lo Vo cells treated with API.(3)The effects of API intervention on transplanted tumor of colon cancerAPI intervention had no significant effects on the body weight of mice and nude mice.Compared with the control group,API intervention made the growth of the transplanted tumor slower and the volume relatively smaller.The indexes of spleen and thymus were higher than those in the control group.The apoptotic and necrotic areas of transplanted tumor expanded gradually.(4)ICD induced by API in colon cancer is associated with MAPK-p38 and NF-κB-p65 signalThe immunohistochemistry experiment of transplanted tumor showed that treated with API increased the expression of CALR,MHC-Ⅰ,p-MAPK-p38 and decreased the expression of p-NF-κB-p65.That’s more,the Western blot showed that transplanted tumor treated with API induced the expression of ICD-related molecules and MHC-Ⅰproteins significantly increased,the expression of p-MAPK-p38 protein up-regulated,and the expression of p-NF-κB-p65 protein down-regulated.Compared with the control group,the number of CD8~+T lymphocytes in the transplanted tumor of mice treated with API increased.Conclusion:API inhibits the proliferation of colon cancer and promotes its apoptosis,induces the expression of ICD-related molecules such as HMGB1,CALR,HSP70 and HSP90,as well as MHC-Ⅰ,and promotes the proliferation of CD8~+T lymphocytes in tumor.MAPK-p38 and NF-κB-p65 signal pathways may play an important role in the occurrence of ICD.It is suggested that API may be a new immunomodulator for the treatment of colon cancer.