| Objective:The aim of this study is to synthesize and observe the effects of a novel conjugate of ursolic acid and aspirin on anti-metastasis of human breast carcinoma in vitro and in vivo.We also detected the mechanism about the corresponding influence on the expression of crucial molecules in related signaling pathways from the perspective of molecules,cells,tissues and integral animals on gene and protein levels.Methods:We use conjugation reaction to synthesize the novel conjugate Asp-UA.The structure characterization was comfirmed by IR、1H NMR and MS.The cell proliferation viabilities were determined using MTT assay.After the human breast cancer cells and normal human cells were treated with UA/Asp/Asp-UA for 24h,high efficient compound concentration with low toxicity was selected.The cell adhesion,wound healing and transwell invasion assays in vitro were performed to evaluate the effects of Asp-UA on anti-metastasis of breast cancer cells in vitro.4T1 cells experimental lung metastasis model was used to evaluate the anti-metastatic activity of Asp-UA in vivo.Expression of genes and proteins were determined by flow cytometry,western blotting,qRT-PCR,immunohistochemistry and immuno-fluorescence.Results:Compared with control group,UA and Asp,at secure and effective concentrations,Asp-UA significantly inhibits MCF-7 and MDA-MB-231 cells of their adhesion,migration and invasion abilities in a dose-dependent manner in vitro.After oral administration of the three drugs for 80 mg/kg and consecutive 28 days,lung tumor nodules reduced in the 4T1/BALB/c mouse breast cancer lung metastasis model and the inhibition efficiency of Asp-UA was much higher.We did not detect side effects with Asp-UA in mice such as weight loss and viscera tissues toxicity.Further studies showed that Asp-UA significantly inhibited the expression of MMP-2,MMP-9 and COX-2 at both the protein and gene levels.In addition,this effect was accompanied by regulation of integrin-mediated related signaling pathway including alterations in integrin α6,β1,CD44,EGFR,ERK,PTEN and EMT pathway including E-cadherin and β-catenin.Conclusion:Low dose range of Asp-UA is highly effective in suppressing breast cancer metastasis,including cell adhesion,invasion and migration,without apparent side effects both in vitro and in vivo.The mechanism is probably attributed to its down-regulation of cell invasion crucial molecules(MMP-2/MMP-9/COX-2)expression,modulating expression of integrin α6、β1、CD44、EGFR、ERK and PTEN within integrin-mediated related signaling pathway and E-cadherin、β-catenin within EMT pathway in breast cancer cells.Thus Asp-UA intervenes the metastasis cascade of breast carcinoma and exerts significant anti-metastatic activity.It exhibits a greater prospect for ideal anti-cancer metastasis drug development. |
