Fucoidan Reverse EMT Phenotype In Human Liver Cancer Cell HepG2 And Its Mechanism Research

Objective: Epithelial mesenchymal transformation(EMT)is that the epithelial cells lose its polarity and then convert to mesenchymal phenotypic cells in certain situations.The characteristic of this process is that the adhesion of cell decline or even disappear,the polarity of epithelial cell loses and the ability of cell movement and transfer enhance.In the study of nearly decade years,epithelial mesenchymal transformation phenotype transformation often has abnormal activation in tumors process and development,the tissue fibrosis and other pathologic changes.Snail as a member of the Snail family is the major nuclear transcription factor in EMT,can promote the occurrence and development of EMT in tumor cells.During the process of EMT,Snail can take part in the abduction of Vimentin directly and inhibit the production of E-cadherin.For all about these reasons,the compound synthesis and drug that target Snail factor or focus its nuclear transfer possessed of the potentials of development and exploitation.It has utmost significance influence for anti-tumor cells EMT process phenomena and tumor recurrence.Polysaccharide which is widely exists in animals,plants and microorganisms,has positive impact about a variety of physiological processes in our body.Fucoidan is a kind of miscellaneous sulfated polysaccharides which is rich in L-fucose and sulfate groups.Among all the functional group the sulfate groups is its most major function group.Fucoidan is one of the important bioactive substances in the marine brown algae,many pharmacological activities such as immunomodulatory,antitumor,anticoagulant,antiviral and radiation protection are all its biological activity.During the previous stage work and reseach in our laboratory,we extract the Fucoidan from sporophyll of undaria pinnatifida suringar.In this kind of marine brown algae,we get a kind of Fuocidan which is richer in sulfate groups,so it has more formidable biological activities.In this experiment,we want to confirm that if Fucoidan could reverse EMT phenotype in Hep G2 cell and explore the possible molecular mechanisms.This experiment is based on human liver cancer cell line Hep G2 cell line and used the method and means of cell biology,biochemistry and molecular biology.Methods: 1.MTT assay to determine the effect of Fucoidan on cell survival in Hep G2 cell.2.The experiment of wound healing in vitro and transwell to ascertain the influence of Fucoidan on migration about cancer cell.3.Immunofluorescence method assay and evaluate the effect of Fucoidan about cell membrane and cytoskeleton system of cancer cell.4.Western blot and Real-Time PCR method to detect the influence of Fucoidan of the expression of Vimentin,E-cadherin protein which is the biomaker of EMT in tumor.5.Immunofluorescence method,Western blot and Real-Time PCR assay Fucoidan influence the expression and nucleus translocation of Snail protein.6.Western-blot assay the expression of HMGA2、HIF-1α and NF-κB protein during the effect of Fucoidan.7.Western-blot testify the expression of TGFRⅡ,Smad2/3 and Smad4,which are the dominating protein of TGF-β/Smads cell signaling pathway which is the chief signaling pathway of the creation about Snail during the dispose of Fucoidan.8.The experiment of Western-blot assay the expression of p PI3 K,p AKT and pm TOR which is the main protein of PI3K/AKT/m TOR signaling pathway.Results: 1.Fucoidan restrains the vitality of Hep G2 cells,Fucoidan reduces the survival ability of cancer cell.2.Fucoidan influences the migration of cancer cell obviously.3.Fucoidan changes the cell membrane and cytoskeleton of cancer cell.Fucoidan transformed the cell membrane from rhombus to cobblestone-likely and altered cytoskeleton from rhombus to polygon.4.Fucoidan reduces Vimentin and boosts E-cadherin on m RNA and protein level.5.Fucoidan the expression of Snail at m RNA and protein levels,also Fucoidan could influence the nuclear translocation of Snail.6.Fucoidan reduces the protein level of NF-κB,HMGA2 and HIF-1α,which is the main nuclear transcription factor of Snail.7.Fucoidan could influence TGF-β/Smads signaling pathway protein,the expression of TGFRⅡ and Smad2/3 decline,but the expression of Smad4 is ascent.8.Fucoidan could reduce the expression of p PI3 K,p AKT and pm TOR which is the dominating protein of PI3K/AKT/m TOR signaling pathway,but Fucoidan had no significant influence about GSK-3β.Conclusions: Fucoidan has obvious inhibitory effect about EMT during tumor cells.The function of Fucoidan affects EMT in cancer cell maybe connected with that Fucoidan influences the expression of EMT biomarker E-cadherin and Vimentin,restrains the expression and nuclear translocation of Snail and its nuclear transcription factor,adjusts TGF-β/Smads signaling pathway and decreases the expression of PI3K/AKT/m TOR signaling pathway.