Effect And Mechanism Of Immunogenic Cell Death Of Thiolidazine On Colon Cancer CT26.WT Cells

Background : The traditional treatment of colon cancer is mainly surgery combined with chemotherapy.Because the early symptoms of colorectal cancer are not obvious,about 1/3 to 1/4 of the patients are found in the advanced stage and lose the opportunity for surgery.Although chemotherapy drugs such as 5-FU,irinotecan,and oxaliplatin have greatly improved the efficacy of colon cancer treatment,they are prone to recurrence,metastasis,drug resistance,and obvious side effects.After the successful application of immunotherapy in the treatment of melanoma,various studies have actively explored its application in different types of solid tumors,including colon cancer.However,only some subtypes of colorectal cancer（d MMR/MSI-H patients）have a good immune response to immunotherapy.In recent years,immunogenic cell death has become a new research hotspot.When some specific drugs act on tumor cells,they can trigger the endoplasmic reticulum stress response and release immune signaling molecules from the cells to improve the immunogenicity of tumor cells.Tumor cells are more easily recognized and killed by the body.In addition to anti-psychiatric diseases,thioridazine can also inhibit the growth of tumor cells by inducing apoptosis and regulating the activity of anti-apoptotic signaling pathways,suggesting that it has a certain anti-tumor effect.Purpose: In this experiment,the colon cancer CT26.WT cell line was used as the research object to investigate the immunogenic cell death induced by thioridazine and its possible mechanism.Material and methods: After thioridazine was treated with different concentration gradients of colorectal cancer CT26.WT cells for 24 hours,the changes in cell morphology were observed under a light microscope.The inhibition of cell proliferation was detected by MTT method.The concentration-inhibition rate curve was drawn,and the median inhibitory concentration（IC50）value of thiolidazine on CT26.WT cells was calculated.The effects of different drug concentrations on the apoptosis rate of CT26.WT and the calreticulin（CRT）on the cell surface were detected by flow cytometry.Western blot was used to explore the expression levels of mitochondrial apoptosis-related proteins（Cleaved Caspase-3,Cleaved Caspase-9,Cleaved PARP）and endoplasmic reticulum stress-related proteins（PERK,Phospho-e IF2α,ATF-4,CHOP,BIP and HMGB1）.Results: With the increase of drug concentration,normal fibrous adherent cells decreased and rounded floating cells increased.MTT results showed that within a certain range,the higher thiolidazine concentration,the greater the inhibition of CT26.WT.And the IC50 value was 25.373μmol/L.Flow cytometry showed that thiolidazine could increase cell apoptosis after 24 h treatment.The expression of CRT on cell surface increased with the increase of thiolidazine concentration.The results of Western blot indicated that within a certain concentration range,thioridazine could up-regulate the expression of mitochondrial apoptosis-related proteins and endoplasmic reticulum stress-related proteins.Compared with the control group,the differences were statistically significant（P<0.01）.Conclusions: Thioridazine has a proliferation inhibitory effect on colon cancer cell CT26.WT and induces immunogenic cell death.The possible mechanism is related to mitochondria-mediated Caspase activation and endoplasmic reticulum stress.