Fucoidan Suppresses Hypoxia-induced Lymphangiogenesis And Metastasize In Mouse Hepatocarcinoma Hca-F Cells

Objective: Hypoxia is an important feature of the tumor microenvironment. In a low oxygen environment, hypoxia-inducible factor-1α(hypoxia inducible factor-1α, HIF-1α) promotes tumorigenesis and evolution. Synthesis and stability of HIF-1α subunit was regulated by oxygen concentration. Under conditions of hypoxia within the tumor presented, HIF-1α prolyl two amine acid residue is an oxygen-dependent prolyl hydroxylase hydroxylated amine acid activity is inhibited, α subunit can be stable in the cytoplasm, ubiquitin degradation pathway is blocked, and the nuclear transcription factor NF-κB family of proteins synergies within the α subunit transported to the nucleus, and the structural subunit HIF-1β to form stable dimers and have the function of transcriptional regulation, bind to HRE(hypoxia responsive element) to express complex can activate downstream genes related to the formation of more than 40 kinds, including cells of blood vessels, lymphatic vessels, energy intake into the biological behavior and survival of tumor cells, proliferation, invasion and metastasis, etc. Therefore, the anti-drug research HIF-1α protein synthesis, translocation change and stability, with the potential for metastasis, treatment recur significant increased clinical theoretical knowledge.Cancer is a serious threat to human health, one of the killer, which, due to the prognosis of liver cancer recur, easy transfer and other features to become the third of cancer mortality, this study mouse hepatoma Hca-F cell, with high lymphatic metastatic ability, function and mechanism studies on environment fucoidan inhibited lymphatic metastasis is the ideal cell lines. Fucoidan, mainly in the brown algae in vivo, is a rich source of soluble sulfate miscellaneous sulfated polysaccharides. Due to abundant marine biological resources, rich active substances have medicinal value, the development of marine drugs have become a hot research, which has a variety of polysaccharides biological activity, and toxic side effects, therefore, the study of the biological activity of fucoidan more with potential. However, in recent year’s fucoidan under hypoxic environment, lymphatic metastasis of liver cancer cells within the tumor cells and lymphatic rare ability neonatal literature.Method: 1. In this paper, chemicals CoCl2(Carbonyl chloride) simulate the interior of a solid tumor hypoxia microenvironment. The experiment was normoxic, hypoxia, hypoxia plus fucoidan group(100-400 g/ mL fucoidan), using the MTT assay of Co Cl2 action to build a low oxygen environment Hca-F cells and the effect of the drug concentration in the optimum time.2. The inquiry under hypoxic conditions, fucoidan on Hca-F cell growth, HIF-1α expression, lymphangiogenesis and invasion and metastasis of fucoidan of intervention. CCK-8 assay using fucoidan under hypoxic environment Hca-F cell viability inhibition. Western bloting, qRT-PCR and IFA detection fucoidan Hca-F cells for expression and translocation of HIF-1α inhibition of its downstream gene regulation, including the promotion of lymphangiogenesis growth factor VEGF-C and liver cells Growth Factors HGF and its receptor VEGFR-3 and C-Met expression. Transwell assay using the influence of hypoxia inducible fucoidan Hca-F cell migration and invasion, and promote lymphatic metastasis ECM degradation, matrix metalloproteinases MMP-2, MMP-9 and the expression of TIMP-1 inhibitors.3. Exploration hypoxic conditions, fucoidan inhibition of mouse hepatoma cell Hca-F expression of HIF-1α expression related signaling pathways critical role factors. Western bloting, qRT-PCR and IFA test has a high expression and effect of lymphatic metastatic potential Hca-F cells HIF-1α synthesis PI3 K / AKT / mTOR signaling pathway fucoidan’s.Results: 1. The results show that: the role of Co Cl2(100 mol/L) in Hca-F cells 24 h, significantly increased the expression of HIF-1α protein and mRNA(P<0.01), thus successfully established in vitro hypoxic microenvironment.2. The role of fucoidan in Hca-F cells under hypoxia 24 h can be reduced NF-κB, the expression of MMP-2, MMP-9, and increase the expression of TIMP-1, while cell migration and invasion also decresased. And fucoidan reduced VEGF-C expression and HGF, while in vivo experiments in mice by immunohistochemistry results showed that: internal lymphatic endothelial cells into the tubular structure is not complete, a reduced ability in vivo mouse tumor significantly reduced the number of lymphatic vessels, the presence of structural defects and so on. MLEC mouse endothelial cell tube formation in vitro experiments, under hypoxic conditions, mLEC ability to enhance cell into a tube, a tube number increased significantly in the intervention of fucoidan, mLEC cells into a tube the ability to reduce the number of relative hypoxia in the control group, significantly reduced.3. High lymphatic metastatic potential of Hca-F cells, HIF-1α synthesis of PI3 K / AKT / mTOR signaling pathway, the functional activity of p-PI3 K / p-AKT / p-mTOR relative expression under hypoxic environment significantly higher than under normal oxygen environment, the effect is significant in the intervention fucoidan, p-PI3 K / p-AKT / p-mTOR expression decreased, the trend is significant.Conclusions: These results indicate that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates PI3K/Akt/mTOR signaling pathways.