Study On The Mechanism Of Usnic Acid Inhibits Colorectal Cancer Progression By Targeting HSP90β

Background Usnic acid is one of the most abundant characteristic secondary metabolites in lichens,with antibacterial,antiviral,anti-tumor,and other biological activities,and it can inhibit the development of various tumors.Previous studies have found that usnic acid has certain cytotoxicity to a variety of colorectal cancer cell lines,and can inhibit the metastasis of colorectal cancer.Usnic acid is expected to be used as a lead compound against colorectal cancer.It is very important to study and clarify the molecular mechanism of usnic acid against colorectal cancer for its development and application.Objective This study is aimed to screen out the molecular target of usnic acid against colorectal cancer and explore the molecular mechanism of usnic acid targeting HSP90β to inhibit the progression of colorectal cancer.Methods(1)Bioinformatics and network pharmacology were combined to screen the potential molecular target of usnic acid against colorectal cancer.(2)Molecular docking predicted the binding ability between usnic acid and the potential molecular target(HSP90β).The cellular thermal shift assay(CETSA)was used to verify the binding ability and efficiency between usnic acid and HSP90β.(3)The malachite green phosphate assay was used to detect the effect of usnic acid on the ATPase activity of the target protein HSP90β.(4)Real-time PCR was used to detect the mRNA expression level of genes in Wnt/β-catenin pathway and HSP90β-related genes.Western blot was used to detect the protein expression level.And nucleocytoplasmic separation was used to analyze the changes of protein distribution of β-catenin in nucleus and cytoplasm.(5)Co-IP was used to detect the effect of usnic acid on the interaction between HSP90β and its chaperones.Results(1)HSP90β was screened out as a potential target of usnic acid against colorectal cancer.(2)Molecular docking showed that usnic acid had a good combination with HSP90β.Cellular thermal shift assay proved that usnic acid could combine with HSP90β,and the higher concentration of usnic acid showed much higher binding efficiency between usnic acid and HSP90.(3)Usnic acid did not significantly inhibit the activity of HSP90β ATPase.(4)Usnic acid could decrease the mRNA expression level of some downstream target genes of Wnt/β-catenin signaling pathway.Usnic acid did not change the protein expression level and nucleoplasmic distribution of β-catenin in colorectal cancer.And the expression level of PPAR-δ protein did not change significantly.The protein expression level of HSP90β client proteins like AKT,CDK4,CDK6,TAU,and ERK1/2 was down-regulated,while the protein expression level of HSP90β chaperone proteins HSP70,HSC70,STIP1,and CDC37 did not change significantly.(5)The results of Co-IP showed that usnic acid could significantly inhibit the interaction between HSP90β and its chaperones STIP1 and HSP70.Conclusion Usnic acid could inhibit the development of colorectal cancer by targeting HSP90β with no effect on its ATPase activity.The mechanism by which usnic acid inhibits the progression of colorectal cancer may be through its effect on the protein complex formation of HSP90β with STIP1 or HSC70,or by inducing HSP90β client protein degradation.