Study On Anti-tumor Effect And The Mechanism Of Sea Cucumber Fucoidan

Sea cucumber fucoidan (SC-FUC), one of the most important bioactive constituentsof the body wall of sea cucumber, has lots of bioactive substances, such as anti-tumor,anti-virus, immune regulation, hypoglycemia and hypolipidemia. The active constituents ofsea cucumber polysaccharide are determined by its structures, the more branch, the morebiological activities. The SC-FUC extracted from Isostichopus badionotus has higherviscosity, and the relative molecular weight is about435kda measured by TSKG4000PWXL chromatographic column. In this study, the hypoxia culture model inducedby CoCl2was established. The anti-tumor activity and mechanism under hypoxia ofSC-FUC isolated from the Isostichopus badionotus in vitro and vivo were studied in tumorcells and animal model. Results are shown as follows:MTT assay was used to determine the anti-proliferation ability of the SC-FUColigosaccharide with different molecular weight isolated from the Acaudina Molpadioidesand the SC-FUC extracted from Isostichopus badionotus. The results suggested thatSC-FUC oligosaccharide from Acaudina Molpadioides rarely inhibited the proliferationactivity of many tumor cells; In contrast, SC-FUC from Isostichopus badionotus showedsignificant inhibitory effects on HGC-27cell proliferation. According to the MTT assay,the investigation of the anti-tumor activities and mechanism of SC-FUC was conductedusing the SC-FUC extracted from Isostichopus badionotus and HGC-27cell.MTT and RT-PCR assay were used to establish the chemical hypoxia. Theconcentration of CoCl2was screened and confirmed at25μM. Under hypoxia, it provedthat SC-FUC could inhibit the HGC-27cell proliferation (At72h and96h, the IC50was169.85and100.04μg/ml); inducing properties of apoptosis in HGC-27cell. Moreover,SC-FUC could significantly arrest the cell cycle in the G2/M phrase. RT-PCR and Westernblotting assay demonstrated that SC-FUC significantly decreased the expression of HIF-1α、Bcl-2and Bcl-xl; increased the expression of Cytochrome c and caspase3; as wellas cleaved the PARP-1. It suggested that SC-FUC exhibited an apoptotic inducing activityin HGC-27cell under hypoxia via blocking cell cycle progression and inducing apoptosisthrough mitochondrial apoptotic pathway.The investigation of the anti-tumor metastasis effects and mechanisms of SC-FUCwere conducted. The results show that SC-FUC treatment significantly inhibited HGC-27cell migration(P<0.01), invasion(P<0.01), and suppressed adhesion of HGC-27cells toboth matrigel and human endothelial cells (HUVEC)(P<0.05, P<0.01) in adose-dependant manner. RT-PCR and Western blotting analysis revealed that SC-FUCsignificantly decreased the expression of HIF-1α、 Hpa and the matrixmetallo-proteinase-2/-9(MMP-2/-9) as well as their specific inhibitors tissue inhibitor ofmetalloproteinase-2/-1(TIMP-2/-1). It is suggested that SC-FUC significantly inhibittumor metastasis under hypoxia by suppressing HIF-1α、Hpa and MMPs expression andenhancing TIMPs expression, protecting ECM under degradation by MMPs.The chicken chorioallantoic membrane assay and tube formation assay indicate thatSC-FUC could also inhibit the tube formation in vitro, decrease the blood vessel growth invivo. RT-PCR and Western blotting assay confirm that SC-FUC can inhibit the expressionof VEGF under hypoxia. It is suggested that SC-FUC could decrease the expression ofangiogenic growth factor and thus prevent the tumor angiogenesis.The activity of anti-tumor and anti-metastasis of SC-FUC were confirmed using themetastatic models of C57BL/6mice transplant with Lewis lung carcinoma (LLC) cells.SC-FUC significantly reduced LLC tumor growth and the pulmonary metastatic nodules inthe surface of lung, the average inhibition rate was45.6%and65.9%compared withcontrol group. RT-PCR and Western blotting assay showed that SC-FUC significantlyup-regulated the expression of caspase3and down-regulated the expression of HIF-1α,HPA, MMPs and VEGF. It is suggested that SC-FUC could prevent the metastasis byinhibiting the expression of ECM degradation related gene and down-regulate the VEGFexpression of tumor cell. In conclusion, the present study provides strong evidence that SC-FUC isolated fromIsostichopus badionotus, in vitro and vivo, possesses marked anti-tumor activity byinducing tumor cell apoptosis, inhibit tumor metastasis and angiogenesis through relatedpathway. Furthermore, we have done lots of work that contribute to the illustration of themechanism of its effect. Researches in this thesis would provide credible theoretical andexperimental foundation for the further studies on sea cucumber polysaccharide andhigh-value use in food and medicine.