Fucoidan Inhibits Lymphangiogenesis By Downregulating The Expression Of VEGFR3 And PROX1 In Human Lymphatic Endothelial Cells And Mechanism Research

Objective: Lymphangiogenesis is one of the promoters of tumor lymphatic metastasis.Anti-lymphangiogenesis and anti-metastatic therapies may provide new possibilities to inhibit tumor dissemination effectively.Tumor metastasis via lymphatic vessels is a critical and early stage of metastatic dissemination of various malignant tumors.After cells invade the lymphatic system,lymphangiogenesis is induced via the production of lymphangiogenic factors and various mediators.The basic process of lymphangiogenesis includes the growth and metastasis of lymphatic endothelial cells(LECs)and the formation of lymphatic vessels.Fucoidan is found in marine brown algae and is a polysaccharide that is rich in L-fucose and acidic sulfate groups,especially in the fucoidan extracted from U.pinnatifida sporophylls.Fucoidan is one of marine medicine with the development prospects;it has the activity of anti-hypertension,anticoagulant,anti-tumor and other biological activity.Currently,more and more researches aim at the anti-tumor and anti-metastatic activity of fucoidan.Prospero homeobox protein 1(PROX1)and vascular endothelial growth factor receptor 3(VEGFR3)are the main regulators of lymphangiogenesis.The homeobox transcription factor PROX1,has a vital action in the differentiation and maintenance of the lymphatic system during embryogenesis.Activated VEGFR3 and its ligands,vascular endothelial growth factor C(VEGF-C),specifically stimulates the proliferation and migration of endothelial cells,and regulates lymphangiogenesis.Furthermore,the nuclear factor-кB/phosphoinositide 3-kinase/protein kinase B(NF-кB/PI3K/Akt)signaling pathway,which plays a significant role in cell adhesion,proliferation,migration,and extracellular matrix degradation,has an effect on tumorigenesis and metastasis.In our study,we used human lymphatic endothelial cell(HLEC)to evaluate the effects of fucoidan on anti-lymphangiogenesis by the several of biology techniques.Method: 1.MTT assay was performed to assess the effect of fucoidan on the viability and proliferation of HLECs.Flow cytometry was performed to detecte the effect of fucoidan on the cell cycle of HLECs.The protein of CDK4 and cyclin D1 were detected via western blotting.2.The anti-migration effect of fucoidan on HLECs was assessed using a Transwell migration assay and a wound scratch assay.3.To investigate the anti-lymphangiogenesis activity of fucoidan,the tube formation of HLECs in vitro was assessed.4.Stained cells with green-fluorescent phalloidin to explore the effect of fucoidan on cytoskeleton morphology in HLECs.5.Immunofluorescence and RT-PCR were performed to detect the protein and mRNA expression of PROX1 and VEGFR3,western blotting were performed to detect the expressions of PROX1 and VEGFR3 and proteins of NF-кB/PI3K/Akt signaling pathway.6.Transwell co-culture system was established with HLEC and MHCC97 H to evaluate the ability of tumor induced lymphangiogenesis using 24-well plates.7.Anti-lymphangiogenesis activity of fucoidan in an animal model was evaluated by quantifing the lymphatic vessel density(LVD)of the tumors and detected the tumor weight.Results: 1.MTT assays result demonstrated that fucoidan significantly decreased cell proliferation after fucoidan treatment.Flow cytometry was performed to show that fucoidan blocked HLEC cell cycle to inhibit cell proliferation.Western blotting results showed that fucoidan decreased the expressions of CDK4 and cyclin D1.2.Transwell and wound scratch assays results showed that fucoidan inhibited the migration ability of HLECs in a dose-dependent manner.3.Treatment with fucoidan effectively decreased the tube formation of HLECs in a dose-dependent manner.4.Non-treated cells had significant lamellipodia formation and polarity,while treated cells did not.Fucoidan depressed the formation of pseudopodia.It is possible that fucoidan regulated the cytoskeleton to inhibit cell motility.5.Immunofluorescence and RT-PCR assays result demonstrated that fucoidan significantly decreased the protein and mRNA expression of PROX1 and VEGFR3.Western blotting results showed that fucoidan significantly decreased expressions of PROX1 and VEGFR3 and proteins of NF-кB/PI3K/Akt signaling pathway.6.In co-culture system,treatment with fucoidan effectively decreased the tube formation of HLECs in a dose-dependent manner.7.Animal assay data were identified that fucoidan could inhibit lymphangiogenesis in vivo.Conclusion: The study demonstrated that Fucoidan significantly decreased cell proliferation and inhibited migration and tube-like structure formation in HLECs,and had an obvious visual inhibitor effect of tumor-induced lymphangiogenesis in vitro.Fucoidan may inhibit tumor lymphangiogenesis and lymphatic metastasis by suppressing the NF-кB/PI3K/Akt signaling pathway through reduced levels of PROX1 and VEGFR3.