Inhibitory Effect Of Artesunate-5-FU Combination On 5-FU Sensitive And Resistant HCT-15 Colorectal Cancer Cells And The Potential Mechanism Investigation

Background:Colorectal cancer(CRC)is a common malignant tumor of the digestive system in clinical practice,ranking second in mortality among common new cancers worldwide.Chemotherapy based on 5-fluorouracil(5-FU)is an important component of the comprehensive treatment plan for colorectal cancer.However,most colorectal cancer patients have resistance to 5-FU chemotherapy and the treatment effect is poor.Therefore,exploring new anti-tumor active drugs to improve 5-FU chemotherapy resistance in colorectal cancer patients is of great significance.Artesunate(ARS)has been shown to restore the sensitivity of various cancers to chemotherapy drugs by regulating various signaling pathways.The aim of this study is to investigate the effects of artesunate and its combination with 5-FU on sensitive cell lines HCT-15 and 5-FU resistant HCT-15/5FU cell lines,and their possible mechanisms of action.To provide new evidence for the search for drugs that can simultaneously treat sensitive and drug-resistant colorectal cancer,and to provide new strategies for the clinical treatment of colorectal cancer.Methods:1.Different concentrations of artesunate and 5-FU were used to treat colorectal cancer HCT-15 cells respectively.MTT methods,flow cytometry,cell scratch test,Transwell migration and invasion test were used to explore the effects of artesunate on colorectal cancer cell proliferation,cell apoptosis and cycle,cell migration and invasion ability.2.Flow cytometry was used to analyze the changes in reactive oxygen species(ROS)of cells after artesunate monotherapy and combination with 5-FU.RT qPCR and Western Blot were used to detect the mRNA and protein expression changes of PI3K and MAPK pathway related genes after administration.The expression of PI3K signaling pathway related molecules in stage D human colorectal cancer tissue was detected using tissue immunochemistry and tissue immunofluorescence methods.3.MTT assay was used to detect the effect of artesunate alone and in combination with 5-FU on the proliferation of 5-FU resistant colorectal cancer cells HCT-15/5FU;Western blot was used to detect the protein expression changes of Bcl-xl,Bcl-2,MDR1(P-gp),MRP1,and ABCG2(BCRP)in two types of cells.Flow cytometry,cell scratch test,Transwell migration and invasion test were used to analyze the effects of drugs on apoptosis,cycle,cell migration and invasion of drug-resistant cells.4.Flow cytometry assay was used to detect the changes in reactive oxygen species(ROS)of drug-resistant cells after administration.RT qPCR and Western Blot detection of PI3K and NF-κB in drug-resistant cells after administration.MAPK and WNT/β-catenin in mRNA and protein expression of key factors in the catenin signaling pathway.Western blot was used to detect the influence of related multiple drug resistance proteins in drug resistant cells after administration.Results:(1)Artesunate and 5-FU inhibit the proliferation,cell migration and invasion of colorectal cancer cells in a time and concentration dependent manner,promote cell apoptosis and affect G1 and S phases of cells,respectively.And the effect is more significant after combined use.(2)After artesunate intervention,the ROS of HCT-15 cells significantly increased,and the expression of key factors in PI3K related pathways decreased.The effect was more significant when combined with 5-FU.But for MAPK and NF-κB pathway protein has no effect.(3)Artesunate alone or in combination with 5-FU can significantly inhibit the proliferation,migration,and invasion of HCT-15/5FU cells,promote cell apoptosis,and affect cell cycle,with significant differences in the efficacy of the combination group.(4)Artesunate alone or in combination with 5-FU can increase reactive oxygen species in drug-resistant cells and downregulate the expression of key factors in PI3K related pathways,with no significant difference in effect compared to the combination of 5-FU.And for MAPK and NF-κB pathway protein has no effect.In addition,after drug intervention,the expression of c-Myc protein and drug-resistant protein in the WNT pathway also decreased.Conclusion:The combination of artesunate monotherapy and 5-FU significantly inhibits the proliferation,migration,and invasion of sensitive and drug-resistant colorectal cancer cells,promotes cell apoptosis,and affects cell cycle.Compared with the monotherapy group,the combined therapy had a more significant effect on both types of cells.The mechanism may be through ROS regulation of the PI3K signaling pathway,which exerts a killing effect on colorectal cancer.The mechanism of action on drug-resistant cells may also be related to drug-resistant proteins such as c-Myc protein and MRP1.