Huaier Induces Autophagy In Colorectal Cancer Cells Through The AMPK/mTOR/ULK1 Signaling Pathway

Objective The objective of this study is to investigate the effect of Huaier on the autophagy and apoptosis of human colorectal cancer cells,in order to provide new theoretical and practical insights for the clinical adjunctive treatment of colorectal cancer.Methods Biological informatics analysis was used to investigate the biological function of Huaier.The CCK-8 assay was used to evaluate the effect of different concentrations of Huaier on the viability of LOVO and HCT116 cancer cells.The morphological changes of colorectal cancer cells after treatment with different concentrations of Huaier for 48 hours were observed under a microscope.Western blot experiments were performed to detect the relative expression of autophagy protein LC3,P62,apoptosis protein Bax,Bcl-2,and signaling pathway proteins AMPK,mTOR,and ULK1 in cells treated with Huaier and AMPK inhibitor.Transmission electron microscopy was used to observe the effect of Huaier on cancer cell autophagy.Fluorescence microscopy（Annexin V-FITC/PI staining）was used to observe the apoptosis of cancer cells after 48 hours of treatment with Huaier.Cell autophagy staining（MDC method）was used to observe the effect of Huaier and inhibitors on cancer cell autophagy changes.Results The biological function analysis of Huaier suggests that it may exert anticancer effects through 39 target genes.These genes are involved in various biological processes,including the Ephrin receptor signaling pathway,DNA damage response,apoptosis regulation,autophagy process,cell oxidative stress damage,and protein self-phosphorylation.The related pathway enrichment analysis results show that they involve PI3K-Ak,JAK-STAT,IL-17 signaling pathways,as well as autophagy,apoptosis,axon guidance,and other processes.Among these genes,MAPK1,SLC29A1,SETD2,PRKACA,IL2,MMP3,MPO,EPHA2,EPHB2,and EPHB4 targets may be key regulatory molecules for the development and prognosis of colon cancer.CCK-8 experiment results show that with the increase of Huaier concentration and culture time,the survival rate of cancer cells gradually decreases.The IC50 of LOVO and HCT116 cancer cells after treatment with different concentrations of Huaier for 48 hours were 3.78 and 3.59 mg·ml^-1,respectively.According to the results of microscopic observation,compared with the group without Huaier,the number of LOVO and HCT116 cancer cells in the group treated with 4mg·ml^-1 Huaier concentration was significantly reduced,and the cell morphology also changed significantly.Western Blot results show that LC3Ⅱ protein in LOVO and HCT116 cancer cells in the Huaier treatment group significantly increased,while P62 protein decreased;Bax protein expression increased,and Bcl-2 expression decreased;signal pathway proteins p-AMPK and p-ULK1 were up-regulated,and p-mTOR expression was down-regulated.In the Huaier+AMPK inhibitor treatment group,the expression of LC3Ⅱ,Bax,p-AMPK,and p-ULK1 proteins decreased,while the expression of P62,Bcl-2,and p-mTOR proteins increased.These results reveal the ability of Huaier to induce cancer cell autophagy and apoptosis and its possible regulatory mechanisms.Transmission electron microscopy observation found that compared with the control group,the number of autophagosomes in cancer cells in the Huaier treatment group significantly increased,further indicating the ability of Huaier to induce cancer cell autophagy.Through fluorescence staining（Annexin V-FITC/PI）,we observed that there were many green fluorescence and few red fluorescence spots in the LOVO and HCT116 cancer cells in the control group.In the 4mg·ml^-1 Huaier treatment group,the number of red fluorescence spots significantly increased,indicating that Huaier induced colon cancer cell apoptosis.MDC fluorescence staining results show that cancer cells treated with Huaier exhibit higher fluorescence intensity and more bright blue MDC-labeled particles,while the fluorescence intensity of cancer cells in the Huaier+inhibitor group is much lower than that in the Huaier treatment group.These results indicate that Huaier induces cancer cell autophagy through the AMPK-mTOR-ULK1 signaling pathway..Conclusions Huaier participates in the biological processes of human colorectal cancer cells LOVO and HCT116,promoting cancer cell apoptosis and inducing autophagy by activating the AMPK-mTOR-ULK1 signaling pathway.