Fucoxanthin Inhibits Tumor-induced Lymphangiogenesis 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.Fucoxanthin,which a kind of promising marine drug material from brown algae,belongs to a carotenoid with variety of pharmacological functions such as anti-tumor,anti-inflammatory,fat acid decomposition.However,fucoxanthin directly effects on lymphatic endothelial cell to explore the effects of anti-metastasis are unclear.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-?B/phosphoinositide 3-kinase/protein kinase B(NF-?B/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 evaluate the effects of fucoxanthin on anti-lymphangiogenesis by the several of biology techniques with human lymphatic endothelial cell(HLEC)Method: 1.An MTT assay was performed to assess the effect of fucoxanthin on the viability and proliferation of HLECs.Flow cytometry was performed to detecte the effect of fucoidan on the cell cycle of HLECs.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.Immunofluorescence and RT-PCR were performed to detect the protein and mRNA expression of VEGFR3,western blotting was performed to detect the expressions of VEGFR3 and VEGF-C.An ELISA kit assay was performed to assess the effect fucoxanthin on p-VEGFR-3 in HELCs(http://www.immunoway.com).4.Effects of of fucoxanthin on HLECs NF-?B/PI3K/Akt signaling pathway were detected by western blot.5.An MTT assay was performed to assess the effect of fucoxanthin on the viability in MDA-MB-231 cells.6.The anti-migration effect of fucoxanthin on MDA-MB-231 cells was assessed using a Transwell migration assay.7.The anti-invision effect of fucoidan on MDA-MB-231 cells was assessed using a Transwell assay.Western blotting and ELISA were performed to detect the expressions of MMP-2,MMP-9,TIMP-1.8.The effects of fucoxanthin on the secretion of VEGF-C and the expression of mRNA in MDA-MB-231 cells were detected by ELISA and RT-PCR methods.9.Effects on the activity of HLECs,the ability,migration and the formation of lymphatic vessels and the expression of p-VEGFR-3 were detected by conditional medium and MTT,Transwell,ELISA assay.10.Anti-lymphangiogenesis activity of fucoxanthin in animal model was evaluated by quantifing the lymphatic vessel density of tumors.Results: 1.MTT assays result demonstrated that fucoxanthin significantly decreased HLECs proliferation after fucoxanthin treatment.Flow cytometry was performed to show that fucoidan blocked HLEC cell cycle to inhibit cell proliferation.2.Transwell and wound scratch assays results showed that fucoxanthin inhibited the migration ability of HLECs in a dose-dependent manner.3.Treatment with fucoxanthin effectively decreased the tube formation of HLECs in a dose-dependent manner.RT-PCR assays and western blotting results showed that fucoxanthin significantly decreased the protein and mRNA expression of VEGF-C and VEGFR-3.ELISA kit assay results showed that fucoxanthin inhibited the expression of p-VEGFR-3.4.Western blot results show that the p-Akt,p-PI3 K and NF-?B signaling pathway were passivated with fucoxanthin treatment.5.Immunofluorescence and result demonstrated that.fucoidan significantly decreased expressions of VEGFR3 and PROX1 and proteins of NF-?B/PI3K/Akt signaling pathway.5.MTT assays result demonstrated that fucoxanthin significantly decreased MDA-MB-231 cells viability after fucoxanthin treatment.6.A Transwell migration assay data were identified that fucoxanthin could inhibit migration on MDA-MB-231 cells.7.Fucoxanthin inhibited MDA-MB-231 cells invision using a Transwell invision assay.Western blotting and ELISA assay results suggested that expressions of MMP-2,MMP-9,TIMP-1 were significantly inhibited and showed a dose dependent relationship.8.Results with ELISA and RT-PCR method showed that fucoxanthin had significant inhibitory effects on VEGF-C secretion and mRNA expression levels in MDA-MB-231 cells,and showed a dose dependent relationship.9.The conditional medium combined with MTT,Transwell,and ELISA assay were used to detect,under the conditional condition,the results showed that HLECs viability,migration and tube formation ability of lymph with fucoxanthin treatment were significantly inhibited,and the expression level of p-VEGFR-3 was significantly reduced and showed a dose dependent relationship.10.Animal assay data were identified that fucoxanthin inhibited lymphangiogenesis in vivo.Conclusion The results showed that fucoxanthin inhibited the proliferation,migration and tumor induced lymphangiogenesis of HLECs.The detection expression of VEGF-C and VEGFR-3 and inhibition of PI3K/Akt/NF-?B signaling pathway with fucoxanthin treated were highly associated with inhibition of tumor induced lymphangiogenesis and tumor metasis.