Inhibitory Effect Of Fucoidan In Mouse Hepatocellular Carcinoma Hca-F Cells Against Lymphatic Metastasis

Objective:Metastasis is an important symbol of the basic characteristics of malignant tumors and tumor metastasis, it is a multi-factor, multi-step, multi-gene co-regulation of the complex process, involving a large number of related changes in gene structure and expression regulation of tumor cell proliferation and tumor metastasis, the degradation of the extracellular matrix, invasion and migration are closely related.Polysaccharides are a class of natural polymer products, widely found in animal cell membranes, cell walls of plants and microorganisms, which had multiple benefit effects, and less side effects. Fucoidan is a class of rich L-fucose and sulfate groups acidic heteropolysaccharide. Extracts only found in marine brown algae and echinoderms water-soluble polysaccharide,which directed immune regulation, anti-cancer, lowering blood, anti-aging and other biologically active, had a high research value.In the view of fucoidan’s anti-tumor activity in recent years, it became one of the most popular researches.Highly metastatic mouse hepatoma cells line Hca-F and low metastatic cells lines Hca-P were separated from mice ascites hepatoma cell line H22 by professor Ling Maoying, who is in the Department of Pathology.Hca-F cell transfer rate higher than 70% and Hca-P cell transfer rate was less than 30%, which is the classic model of tumor lymphatic metastasis, but the mechanism was unknown.Vascular endothelial growth factor-C (VEGF-C) closely related with tumor cell growth, proliferation, and metastasis. VEGFR-3 is a VEGF-C specific receptor, has only expressed in tumor cells, early experiments showed that VEGFR-3 in Hca-F cells would be expressed. Binding of VEGF-C to VEGFR-3, can activate hepatocyte growth factor (HGF) expression, and activation of downstream signaling pathways, regulation of proliferation, tumor metastasis and other biological activity.Metastasis of tumor can be blood metastasis and lymphatic metastasis, lymphatic metastasis is the key to early steps of tumor development. Tumor metastasis especially need proliferation, with thus adhesion occurs, and then degraded extracellular matrix. Family of matrix metalloproteinases (MMPs) are key members of the degradation of the extracellular matrix, tissue inhibitor of matrix metalloproteinases (TIMPs) can inhibit the activity of MMPs, altered tumor cell metastasis.Method:1. The use of ethanol fractionation method purified Fucoidan, Hydrogen peroxide and ascorbic acid combined degradation for N-Fucoidan to get LMW-Fucoidan.2. The use of phenol-sulfuric acid method, gelatin nephelometry barium sulfate-carbazole method automatically indicate polarimeter, NMR were measured fucoidan’s total sugar content, uronic acid content, polarimetry and functional groups.3. MTT method was used to detecte the difference between Hca-F cell which had highly metastatic ablity and Hca-P cell which had low metastatic talent in hepatocellular carcinoma, which was shown the ability to grow.In the Transwell assays, the migrative ablity of Hca-F cells and Hca-P were different. In the Elisa assay Hca-F cell and Hca-P cell supernatants secreted VEGF-C, HGF, MMP-2, MMP-9 growth factor differences.4. By trypan blue assay Fucoidan treated Hca-F cells, calculated the survival rate for cytotoxicity.5. After Fucoidan treatment for Hca-F cell by using the MTT assay at different time, which explored the influence for growth in Hca-F cell.In the Transwell assay was using to impact on Hca-F cell invasion ability.6. Western blot assay was measured Hca-F cell’s protein expression of VEGFR-3, C-MET, CXCR-4, TIMP-1 in total protein, and detected the changes of cell growth factor’s secretion level which collected from cell’s supernatant through the ELISA assay,such as MMP-2, MMP-9, VEGF-C and HGF.In the RT-PCR experiment shown the detection of vegf-c, c-met, timp-1 mRNA expression levels after fucoidan treatment.7. In MTT assay to compare the influence between N-Fucoidan and LMW-Fucoidan on Hca-F cell growth, ELISA method shown after the treatment by N-Fucoidan and LMW-Fucoidan for VEGF-C, HGF, MMP-2, MMP-9 different expression levels, Western blot assay compared Fucoidan and LMW-Fucoidan effected for Hca-F cell’s VEGFR-3, C-MET, CXCR-4, TIMP-1 protein expression in total protein.8. In vivo experiments, after injected Hca-F cells on footpad of 615 mice, fucoidan inhibited tumor cell to metastasising. By ELISA assay to text the expression changes for VEGF-C and HGF growth factor, HE staining morphology, immunohistochemistry assay expounded lymphatic vessel density.9. Incomplete Freund’s adjuvant-induced mLEC, MTT assay Fucoidan impacted on mLEC cells proliferation, immunofluorescence identified mLEC cells LYVE-1 protein expression, were observed fucoidan impact on mLEC into the tube.Results:1. N-Fucoidan obtained was 38.91%, LMW-Fucoidan obtained rate was 60%2. The total sugar content of N-Fucoidan was 44.7%, group content of sulfuric acid, uronic acid contents were,18.5%,12.5%. Compared with the total sugar content of LMW-Fucoidan increased 4% than N-Fucoidan, uronic acid content increased by 4%, the sulfate content decreased 12%.3. MTT results showed that, Hca-F cell proliferation was 1.79 times than Hca-P cells, Transwell assay showed that Hca-F cell invasion capacity was 1.67 times Hca-P cells, Hca-F cells expressed VEGF-C, HGF, MMP-2, MMP-9 capacity higher than that of Hca-P cells.4. MTT assay showed that after fucoidan treatment decreased cell growth in a dose-and time-dependent manner.5. After fucoidan treatment, Hca-F cell death is less than 1%, indicated that fucoidan inhibited Hca-F cell growth was not by cytotoxic effects. Transwell experiments shown fucoidan which decreased ability of Hca-F cell invasion.6. Western blot results indicted that, fucoidan influenced Hca-F cell’s VEGFR-3, C-MET, CXCR-4 protein expression were inhibited in total protein, and TIMP-1 expression was activated. ELISA results shown that, after fucoidan treatment, expression of MMP-2, MMP-9, VEGF-C, HGF were decreased, vegf-c, c-met, vegfr-3 mRNA levels lower, increased timp-1 expression.7. MTT experimental results show that LMW-Fucoidan owned stronger inhibitory effect than Fucoidan in Hca-F cells proliferation. ELISA inhibition results were shown in Hca-F cells expression of MMP-2, MMP-9, VEGF-C, HGF levels and mRNA expression levels, N-Fucoidan effect was stronger than that of LMW-Fucoidan.8. In animals experiments proved that fucoidan could inhibit tumor cell growth, increased spleen index and thymus index, VEGF-C and HGF levels were decreased tumor blood vessel density.9. mLEC induction successed rate was 100%, immunofluorescence LYVE-1 positive cells were greater than 85%, fucoidan inhibited mLEC growth in a dose-dependent manner.