Study On Anti-metastasis Activity Of Exopolysaccharide From Rhizopus Nigricans

Metastasis is the major cause of death for cancer patients.However,there is less research on the drugs of anti-metastasis which was used in clinic.It has reported that most chemotherapeutics used for anti-metastasis are primarily to inhibit the growth of cancer cells,rather than the invasion and metastasis.Therefore,it is an urgent issue to find an effective medicine for anti-tumor invasion and metastasis without side effect.Invasion and metastasis is the committed steps which can be as the targets for finding new drugs to inhibit the tumor progression.It is reported that the drugs with anti-angiogenesis could increase the survival rate and prolong life of terminal cancer patients in clinic.However,because of the limitation of chemotherapy drug and serious side effects,chemotherapy drug is not appropriate for all cancer.Increasing studies have revealed that bioactive polysaccharides possess various biological activities without side effects,including immunoenhancement and antitumor activities,which can effectively inhibit the tumor growth without damage for human organism.In this article,we researched the anti-metastasis activity of polysaccharides on CT26 which would be of great significance in model pharmacological research.Exopolysaccharides 1-1(EPS 1-1),separated from the fermentation liquor of Rhizopus nigriccans,is a natural polysaccharide which possesses antitumor and immnomodulating activities.Previous studies illustrated that EPS 1-1 could inhibit the tumor growth and improve the immunocompetence of CT26 bearing mice.Moreover,EPS 1-1 could significantly boost the immunity of normal and immunosuppressed mice through cellular immunity and humoral immunity.In this research,we analyzed the potential anti-metastasis activity and mechanism of EPS 1-1.In-vitro study:As an initial step of metastasis,invasion was evaluated by Transwell assay.The result suggested that that EPS1-1 could inhibit the invasion of CT26 cells with the inhibition ratio of 25.00%,45.37%and 50.93%at the dose of 0.1,0.2 and 0.4 mg/ml,respectively.It is well known that metastasis of malignant cell is closely associated with migration.The detection of migration illustrated that the migration ability of CT26 cells was inhibited by EPS1-1 with inhibition ratio of 36.23%,48.30%and 65.70%at the dose of 0.1,0.2 and 0.4 mg/ml,respectively.Research has reported that the adhesion of tumor cell and ECM were involved in migration and invasion.Results showed that EPS1-1 could decrease the adhesion of CT26 cells with the inhibition ratio of 36.1%and 50.4%at the dose of 0.2 and 0.4 mg/ml.Furthermore,the tube formation in tumor plays an important role in the metastasis and can be a target for the anti-metastasis drugs.Our study proved that EPS1-1 could significantly inhibit the tube formation of endothelial cells.It is generally known that MMP-2 and MMP-9 play an important role to degrade proteins of extracellular matrix.And the levels of Vimentin and VEGF represent the invasive ability and the tube formation in cancer.What's more,E-cadherin plays a crucial role in adhesion and metastasis.Gelatin zymography assay indicated that the activityies of MMP-2 and MMP-9 in CT26 cells after treated with EPS1-1 were significantly declined.qPCR showed that the mRNA levels of MMP-2,MMP-9,VEGF and Vimentin in CT26 cells after treated with EPS1-1 were significantly down regulated with and the up-regulated expression of E-cadherin.Besides,Western-blot revealed that the protein expression of MMP-9,VEGF and Vimentin were also down-regulated in EPS1-1 treated group.In-vivo study:The lung metastasis model with CT26 and B16F10 cells was established to research the anti-metastasis.Pulmonary index showed that EPS1-1 could inhibit the metastasis of CT26 and B16F10 to lung with the inhibition ratio of 48.50%and 38.37%,respectively.Furthermore,we studied the expression levels of related gene and protein in lung tissue from CT26 bearing mice.qRT-PCR analysis suggested that the mRNA levels of MMP-9,MMP-2 and Vimentin were remarkably down-regulated in EPS1-1 treated group.Western-blot showed that protein levels of MMP-9,MMP-2 and Vimentin were also reduced by EPS1-1.Immunohistochemistry staining suggested that EPS1-1 attenuated the positive expression of CD31 in lung tissue.Besides,thymus index and the content of IL-2 in serum were both elevated in EPS1-1 treated group,which revealed that EPS1-1 might enhance the immune activity of mice.Result:In conclusion,our research indicated that EPS 1-1 could inhibit the adhesion,migration and invasion of CT26 cell to inhibit metastasis in vitro.And e tube formation of ndothelial cell could be damaged by EPS 1-1.In vivo,the metastasis of CT26 and B16F10 cells to lung was inhibited after administrated with EPS 1-1.What's more,EPS1-1 could enhance the immunologic function of CT26 bearing mice and might reduce the micro vessel density of lung.The anti-metastasis of EPS 1-1 might be correlated with the regulation of immunity and expression of related gene and protein in cancer.