Experimental Study Of Wogonoside In Regulating Tumor Progression Through TRAF6 In PDAC

Background: Pancreatic ductal adenocarcinoma(PDAC)is one of worst prognostic malignancies.Radical surgical resection is the only potentially curative option,but over 80% of PDAC patients lost their chance for surgery due to disease progression at first diagnosis.Current non-surgical treatment modalities are ineffective due to low sensitivity and the tendency to develop drug resistance.There is an urgent need for new therapeutic agents.Wogonoside(Wog),a flavonoid isolated from the root of Scutellaria baicalensis,has been reported to exert anticancer effects in many cancers through different pathways.We explored the effect of Wog on disease progression in PDAC and its underlying molecular mechanisms.Methods: We examined the cell viability of PDAC cell lines SW1990 and Panc-1 under different concentrations of Wog stimulation,and three concentrations(25,50 and 100 μM)were selected for subsequent experiments.Wog intervention was used first to assess the proliferation and apoptosis of the PDAC cell lines,and also to assess the malignant features of PDAC including Stem Cell-like Transition(SCT),Epithelial-Mesenchymal Transitions(EMT)and inflammatory factor levels.Based on the results of our experiments and literature,we hypothesize that the potential target of Wog is Tumor Necrosis Factor Receptor-Associated Factor 6(TRAF6).Using Interleukin-1β(IL-1β),an exogenous activator of TRAF6 signal,in PDAC cells and inducing TRAF6 overexpression in PDAC cells,we successfully upregulated TRAF6 signal in PDAC.100 μM Wog intervention was performed to verify whether Wog can inhibit PDAC tumor development and malignant features by modulating TRAF6 signal in vitro.We then implemented in vivo experiment with TRAF6 overexpression to test whether Wog affect the growth,EMT and SCT of xenograft tumors in nude mice via TRAF6.Finally,we performed survival analysis of PDAC transcriptomic data and clinical prognosis-related data from the TCGA database and the GTEx database using bioinformatics to clarify the potential translational value of Wog for PDAC treatment.Results: In vitro,Wog inhibited cell viability and cell proliferation,promoted apoptosis and suppressed the malignant features of PDAC tumors such as SCT and EMT.In addition,Wog inhibited the expression of inflammatory factors such as TNF-α and IL-1β.In vitro,Wog inhibited the TRAF6/Nuclear Factor Kappa B(NF-κB)p65 signal pathway in PDAC.After pre-stimulation with IL-1β or TRAF6 overexpression,expression of TRAF6/ NF-κB signal was upregulated,while the proliferative ability of PDAC cell lines and the malignant features of PDAC tumors such as SCT and EMT were significantly enhanced,while treatment of the cell lines with 100 μM Wog significantly reversed these effects.In vivo,Wog at the dose of 80 mg/Kg inhibited PDAC xenograft tumor growth,reduced the expression of the invasive marker vascular endothelial growth factor(VEGF),the SCT marker CD44 and the EMT marker matrix metalloproteinase 14(MMP14)and N-cad by modulating TRAF6.Finally,we performed survival analysis of key proteins of the TRAF6 signal(TRAF6,VCAM-1),EMT marker(MMP14),SCT marker(CD44),and found TRAF6,VCAM-1,CD44 and MMP14 expression levels were negatively correlated with survival of PDAC patients.Conclusions: 1.In vitro experiments demonstrated that Wog inhibited PDAC cell proliferation,induced apoptosis,and inhibited PDAC tumor stem cell-like transformation and epithelial mesenchymal transformation.2.In vitro experiments demonstrated that Wog regulated PDAC development through TRAF6/NF-κB pathway.3.In vivo experiments demonstrated that Wog exerted anti-cancer effects on PDAC by regulating TRAF6.