Study On Antitumor And Antimetastasis Effect Of A Fucosylated Chondroitin Sulfate Isolated From Isostichopus Badionotus

Sea cucumber, an important food and medicine resource, has lots of bioactive substances, such as sea cucumber polysaccharide, saponins, collagen proteins and cerebrosides. The sea cucumber polysaccharide is one of the most important bioactive substance, and weight about 7%-10% of the body wall of the sea cucumber. There are sea cucumber chondroitin sulfate (SC-CHS) and the sulfated fucan of the sea cucumber polysaccharide. In this study, sea cucumber chondroitin sulfate (SC-CHS) was isolated from the Isostichopus badionotus, and its antitumor and antimetastasis effect was studied in tumor cells and animal models. Results are shown as follows:The monosaccharide composition of the SC-CHS was similar to that of chondroitin sulfate, mainly with molar ratio 1:0.7:0.9 of glucuronic acid (GlcUA), galactosamine (GlaN) and fucose (Fuc). IR and NMR analysis of anomer hydrogen signals of sulfate fucose in SC-CHS shows that mainly 2, 4-SO4 disubstituted. It was proved that SC-CHS could significantly inhibit 95D cell proliferation most sensitively by MTT assay; induce 95D cell early apoptosis with flow cytometry; SC-CHS induced expression of the p53 tumor suppressor gene and the Cdk inhibitor p21; The anti-proliferative activity of SC-CHS was accompanied by inhibition of cycling B1and Cdc2 mRNA; Moreover, SC-CHS inhibited the expression of bcl-xl,and induced expression of caspase-3 clearly. The results showed that SC-CHS significantly inhibit 95D cell proliferation through apoptosis and G2/M arrest.The effects of SC-CHS on adhesion, the invasion and metastasis ability of 95D cells were investigated through cell-base adhesion assay, migration assay, invasion assay and RT-PCR assay. The results showed that the SC-CHS could decrease the adhesion rate in dose dependent manner and inhibit the cellular motility(P<0.05); inhibited invsion of 95D cells in dose despondently(P<0.01); inhibited the expression of MMP-2/9, and induce expression of TIMP-1/2. The results suggested that the SC-CHS could inhibit the metastasis of 95D cell with protecting ECM under degradation by MMPs. The chorioallntoic membrane assay and tube formation assay indicate that SC-CHS inhibit the generation of new blood vessels. And RT-PCR and Western Blot assay confirm that its antiangiogenic effect was that SC-CHS can inhibit the expression of VEGF.The Lewis lung carcinoma models of C57BL/6J mice were established and the results showed that the tumor growth was suppressed significantly in SC-CHS groups, the average inhibition rate was 31.5% compared with control group. The metastatic foci occurrence rate of SC-CHS group was lower than control group, and the difference were significant(P<0.05). SC-CHS could significantly down-regulated the expression levels of HIF-1αand VEGF mRNA of the tumor than control. And SC-CHS also could increase amounts of TNF-αandγ-IFN in serum. The results suggested SC-CHS has inhibitory effect on growth and metastasis of Lewis lung carcinoma cell in mice, and may be through inhibiting solid tumor angiogenesis and improve the well-being of the immunity of the mice.To observe the inhibitory effect of SC-CHS on lung metastasis of mouse melanoma cell line B16-F10. The results suggested that SC-CHS clearly suppressed lung metastasis by 65.26% in mice and could reduce the levels of serum saliva acid content andγ-GT energy clearly (P<0.01). Biochemical parameters such as hydroxyproline, hexosamines and uronic acid levels in the lung were also reduced clearly (P<0.01) in the SC-CHS treated animals. Therefore, the results indicate that SC-CHS can inhibit the metastasis of B16-F10 metastatic mouse melanoma cells and its mechanism is maybe that it can inhibit the B16-F10 cells adhering to the ECM and reduce the migration of B16-F10 cells.In this study, we systemically investigated the antitumor and antimetastasis effect of a fucosylated chondroitin sulfate isolated from Isostichopus badionotus, furthermore we do lots of work to do that contribute to the illustration of the mechanism of its effect. Our works provide the scientific approvement for the research of the bioactive function of the sea cucumber polysaccharide and good use in food and medicine.