Study On The Effect And Mechanism Of Eupalinolide J Inhibiting Cancer Metastasis By Promoting STAT3 Ubiquitin-dependent Degradation

Eupatorium lindleyanum DC.(EL)is a dried above-ground part of Eupatorium lindleyanum,a plant in the Asteraceae family,which has various effects such as clearing heat and detoxifying,resolving phlegm and relieving asthma,and lowering blood pressure.The team’s previous study found that some of the Eupalinolides(Eups)contained in EL inhibited STAT3 and PI3K/Akt signalling pathways,which play a key role in promoting cancer metastasis.Currently,there are no studies on the effect of the active ingredients of EL on cancer metastasis.Therefore,we hope to discover potential drugs from natural Eups that can inhibit cancer metastasis for the application of EL in cancer therapy.[Objective]In this study,we used bioinformatics,molecular biology and experimental zoology to discover the anti-tumour metastatic compounds in the Eups constituents and investigate their effects and mechanisms:(1)virtual screening of Eups constituents for anti-tumour metastasis;(2)verification that Eupalinolide J(EJ)has anti-metastatic activity in vitro and in vivo;(3)investigation of the mechanism by which EJ inhibits tumour cell metastasis.[Methods](1)Molecular docking was used to analyze the binding affinity between Eupalinolide J and cancer metastasis-related targets.A network analysis was conducted to screen the compounds with anti-metastatic activity.(2)Wound healing,Transwell migration,and invasion assays were used to investigate the effect of Eupalinolide J on cancer cell metastasis in vitro.(3)The MDA-MB-231-Luc cell nude mouse lung metastasis model was used to explore the in vivo effect of Eupalinolide J on cancer cell metastasis.(4)Western blotting experiments were performed to detect the impact of Eupalinolide J on the expression of related targets such as STAT3,MMP-2,MMP-9,PI3K,and AKT.(5)Detection of the effect of EJ on the activity of MMP-2 and MMP-9 in cancer cells through gelatinase zymography experiment.(6)Activation of STAT3 by IL-6 and detection of MMP-2 and MMP-9 expression.(7)Investigation of the impact of sh-RNA knockdown of STAT3 on the anti-metastatic effect of EJ using Transwell assay.(8)Detection of the impact of EJ on the expression of genes involved in the STAT3 signaling pathway using RT-PCR.(9)Evaluation of the effect of EJ on the protein stability of STAT3 through CHX assay.(10)Examination of the impact of EJ on gene expression in cancer cells through transcriptomics.(11)Investigation of the effect of MG-132 inhibition of proteasomes on EJ-induced STAT3 degradation using Western blotting.(12)Assessment of the impact of EJ on the level of STAT3 protein ubiquitination using immunoprecipitation technology.(13)Exploration of the mechanism underlying EJ-induced STAT3 ubiquitination through protein-protein molecular docking.[Results](1)Virtual screening revealed that EJ had potential anti-tumor metastasis activity.(2)Experiments confirmed that EJ could inhibit cancer cell metastasis in vitro and in vivo.(3)EJ significantly suppressed the activity of STAT3 signaling pathway-related proteins,including STAT3,MMP-2,and MMP-9,but had no significant effect on the PI3K/AKT signaling pathway.(4)Activation of STAT3 induced the expression of MMP-2 and MMP-9.(5)Knockdown of STAT3 significantly reduced the inhibitory effect of EJ on cancer cell metastasis.(6)EJ reduced the stability of STAT3 protein.(7)EJ affected the regulation of RNA processing,chromatin organization,RNA metabolism,and proteasome-mediated ubiquitin-dependent protein degradation in cancer cells.(8)Inhibition of proteasomes reversed the inhibitory effect of EJ on STAT3 protein.(9)EJ promoted STAT3 ubiquitination.(10)EJ binded to the DNA binding domain of STAT3.[Conclusion](1)Virtual screening identified EJ as a potential anti-tumor metastasis agent,and experiments confirmed its ability to inhibit cancer cell metastasis in vitro and in vivo.(2)EJ suppresses the expression of MMP-2 and MMP-9 by mediating the STAT3 signaling pathway.(3)EJ promotes the ubiquitination and degradation of STAT3,leading to the inhibition of the STAT3 signaling pathway.These findings shed light on the molecular mechanisms underlying EJ’s anti-metastatic activity and suggest its potential as a therapeutic agent for cancer metastasis.