The Role And Mechanism Of Cinobufotalin In Inhibiting Colon Cancer Metastasis By SPHK1/STAT3 Pathway

Objective: Cinobufotalin is widely used in adjuvant therapy after surgery and chemotherapy,but the exact mechanism is not clear,so the study will investigate the effects and mechanisms of internal Cinobufotalin on colon cancer metastasis,providing for the treatment of colon cancer metastasis new theoretical basis.Methods: 1.Cinobufotalin was applied to three colon cancer cell lines,HCT116,HCT8 and SW620,and its effect on the survival rate of colon cancer cells was detected by CCK-8 reagent,and the IC50 of three cells was obtained;2.Cloning formation experiments to observe the effect of Cinobufotalin on cell proliferation;3.Scratch migration experiment and Transwell chamber migration experiment to observe the migration ability of cells in vitro under the action of Cinobufotalin;4.The nude mouse model of PDX subcutaneous tumor and HCT116 orthotopic tumor model were successfully constructed and divided into control group and experimental group.Cinobufotalin was administered intraperitoneally as an experimental group,and the weight,tumor size and size of nude mice were recorded.Tumor weight,after the end of the experiment,the tumor and nude mouse liver were taken for HE staining and immunohistochemical analysis;5.Western blotting(WB)was used to detect the expression of SPHK1/STAT3 related proteins under different concentrations of Cinobufotalin;6.Overexpression of SPHK1 stable strain cultured in colon cancer cell line HCT116 with low expression of SPHK1;7.Transwell chamber observed the change of in vitro migration ability of HCT116 after SPHK1 overexpression,and added the effect of Cinobufotalin on its migration ability;8.Western blotting to verify the changes of STAT3 pathway and downstream metastasis-associated proteins of HCT116 after overexpression of SPHK1.Results: Cinobufotalin has a toxic effect on colon cancer cells,which inhibits the survival rate of colon cancer in a time-and concentration-dependent manner;Cinobufotalin can inhibit the proliferation and migration of colon cancer cells.Cinobufotalin can inhibit tumor growth and metastasis in two nude mouse models.Immunohistochemistry showed that Cinobufotalin could affect the protein expression of SPHK1,STAT3,MMP-9 and E-cadherin.Moreover,WB detection of Cinobufotalin can inhibit the protein expression of SPHK1,STAT3 and MMP-9 and promote the protein expression of E-cadherin.The migration ability of the stable strain HCT116 overexpressing SPHK1 was enhanced,and Cinobufotalin could inhibit its enhanced migration ability,and the overexpressed SPHK1 was also inhibited by WB,while the downstream protein STAT3 was also inhibited,MMP-9 and E-cadherin were also affected.Conclusion: Cinobufotalin has the effect of inhibiting the proliferation and migration of colon cancer cells;it can inhibit the growth and metastasis of colon cancer in nude mice;its mechanism is to inhibit the transfer of related proteins by inhibiting the SPHK1/STAT3 pathway.