Fucoidan Inhibits Lymphangiogenesis Induced By SGC7901 Cells In Human Gastric Adenocarcinoma

Objective: Gastric cancer is one of the malignant tumors that seriously threate n human health.Lymphatic metastasis is one of the main ways of cancer metastasis,and it is also the reason why cancer is so difficult to treat.Lymphangiogenesis is an important process of lymphatic metastasis including the proliferation,migration and tube formation of lymphatic endothelial cells.Tumor induced lymphangiogenesis is a complex process involving many factors involved.Tumor cells interact with the interstitial cells and induce lymphangiogenesis by secreting some factors in a tumor microenvironment.Fucoidan,a sulfated polysaccharide,is abundant in marine brown algae and composed of L-fucose and sulfate.It has anti-cancer activity and much less side effects compared to traditional anti-cancer chemotherapy agents.Fucoidan induces cells death within tumor cells and increases the survival rate of tumor nude animal models by suppressing metastasis and angiogenesis.It has an effect on against hepatocellular carcinoma and inducing apoptosis of human breast cancer and colon carcinoma.In addition,fucoidan plays an important role in inhibiting the growth of non-small-cell bronchopulmonary carcinoma cells and human lymphoma HS-Sultan cells.However,the mechanisms of fucoidan inhibited lymphangiogenesis and lymphatic metastasis induced by gastric cancer are not completely understood.Vascular endothelial growth factor C(VEGF-C)promotes the lymphangiogenesis by combining with vascular endothelial cell growth factor receptor 3(VEGFR-3).Matrix metalloproteinase-2,matrix metalloproteinase-9(MMP-2,MMP-9)and tissue inhibitor of metalloproteinase-1(TIMP-1)can lead to metastasis of malignant tumors.LYVE-1 is a receptor of hyaluronic acid,which is homologous to CD44 and may induce the metastasis of tumor cells via lymph nodes by transferring peripheral hyaluronic acid to lymphatic metastasis.Expression of lymphatic endothelial marker Podoplanin in lymphatic endothelial is selective.CXCL1,a member of the CXC chemokine family,is associated with angiogenesis and tumorigenesiss.Furthermore,the PI3K/Akt/NF-?B signaling pathway is a signal pathway for cell proliferation,migration,and molecular mechanisms in LECs.In our research,we take some biological methods to detect the influence of fucoidan on gastric cancer and tumor-induced lymphangiogenesis.Methhods:1.MTT assay explored the effects of fucoidan on SGC7901 cells growth and proliferation.The influence of fucoidan on SGC7901 cells cycle was detecte d by Flow cytometry.2.Transwell assay was used to observe the migration and invasion of SGC7901 cells with fucoidan.3.ELISA assay was performed to detect the expression of MMP-2,MMP-9 and TIMP-1 cultured with fucoidan in SGC7901.RT-PCR evaluated the mRNA expression of vegf-c.4.Western blotting was performed to assess the efforts of fucoidan on PI3K/Akt/NF-?B signaling pathway in SGC7901 cells.5.Co-culture system was performed to evaluate the ability of tumor-induced lymphangiogenesis and the efforts of fucoidan on the expression of VEGFR-3 and p-AKT.6.The invasion of SGC7901 cells cultured with recombinat CXCL1 was detected by Transwell assay.ELISA assay evaluated the vital protein MMP-2 which was from SGC7901 cells cultured with recombinat CXCL1.Immunofluorescence assay was performed to testify the expression of CXCL1 in vivo.7.Quantifing the lymphatic vessel density of tumors tested anti-lymphangiogenesis activity of fucoidan in animal model.Results:1.MTT assays result showed that fucoidan inhibited SGC7901 cells viability.Fucoidan blocked SGC7901 cell cycle to inhibit cell proliferation was showed by the result of Flow cytometry.2.The results that fucoidan inhibited migration and invasion of SGC7901 cells were performed by Transwell assays.3.The results suggested fucoidan down-regulated the expression quantity of MMP-2,MMP-9,VEGF-C and up-regulated the expression quantity of TIMP-1 by ELISA assays.RT-PCR indicated that fucoidan also inhibited the mRNA expression of vegf-c.4.To testify that whether fucoidan affect PI3K/Akt/NF-?B signaling pathway in SGC7901 cells.As shown in western blotting,the protein levels of phopho-PI3 K,phopho-AKT and NF-?B were all reduced by fucoidan in concentration-dependent manner.5.Co-culture system for evaluating the interactions between SGC7901 cells and HLEC with fucoidan showed that fucoidan inhibited tube formation of HLEC co-cultured with SGC7901 cells.The western blot results confirmed that protein expression was increased in control group by co-culture and fucoidan decreased the expression of VEGFR3 and phopho-AKT.6.Transwell assays result suggested that fucoidan can decrease the i nvasion of SGC7901 cells cultured with recombinat CXCL1.ELISA assays showed that the vital protein MMP-2 which was from SGC7901 cells cultured with recombinat CXCL1 was up-regulated besides fucoidan inhibited this up-regulation.The results of immunofluorescence assays showed that fucoidan reduced the expression of CXCL1 protein.7.The results that fucoidan inhibited lymphangiogenesis in vivo were showed by animal assay.Conclusion: The results showed that fucoidan inhibited the proliferation,migration and tumor-induced lymphangiogenesis of SGC7901 cells,also affected the microenvironment of the tumor and HLECs.The reasons for these may be that fucoidan inhibited the expression of MMP-2,MMP-9,VEGF-C,VEGFR-3 CXCL1 and PI3K/Akt/NF-?B signaling pathway.