Anti-metastasis Mechanism Study Of Exopolysaccharides EPS11 From Marine Bacterium

Cancer,as one of the major public health problems in the world,has seriously threatened human life and health,and its morbidity and mortality rates continue to rise.Metastasis,as the main factor of poor prognosis in cancer patients,is still one of the main causes of death in patients with malignant tumors.Nowadays,the clinical treatment for tumor metastasis is mainly through chemotherapy.But most of the chemotherapy drugs used in the market have certain toxic side effects.Long term chemotherapy can induce cancer cells to produce drug resistance,which will further reduce the effectiveness of chemotherapy drugs in the clinical treatment of tumors.Marine microbial polysaccharides have become one of the key points in marine scientific research because of their good biocompatibility,low toxicity and easy to get,which can provide candidate compounds for the development of high efficiency and low toxicity new drugs for anti-tumor metastasis.EPS11 is a marine bacterial exopolysaccharide found by our laboratory in the early stage,which has an anti-tumor effect.It has potential anti-tumor metastasis effect,but the corresponding molecular mechanism is not clear.In this paper,the molecular mechanism of EPS11 anti-tumor metastasis was studied in detail from in vitro and in vivo two levels by proteomics and cell biology,which provided a reliable theoretical basis and drug precursor for the development of polysaccharide anti-tumor metastatic drugs.In this study,MTT assay was used to detect the cytotoxic effects of EPS11 on hepatocellular cancer cells Huh7.5,Bel-7402 and HepG2,and the results indicate that EPS11 could inhibit the growth and proliferation of hepatocellular carcinoma cells in a dose-and time-dependent manner.At the same time,optical microscope observation showed that EPS11 could cause the aggregation and detachment in hepatocellular carcinoma cells.Furthermore,the effects of EPS11 on the adhesion rate and cell surface structure of hepatocellular carcinoma cells Huh7.5,Bel-7402 and HepG2 were detected by crystalline purple in quantity,scanning electron microscope(SEM),respectively.The results showed that EPS11 could both reduce the adhesion ability and destroy the cell filiform structure on the surface of hepatocellular carcinoma cells Huh7.5,Bel-7402 and HepG2 in a dose-and time-independent manner.We found that the decrease of cell adhesion rate was consistent with the decrease of cell filiform structure,which indicated that the cell filiform structure of cell surface plays an important role in the adhesion of hepatocellular carcinoma cells.In order to further explore the function of EPS11 anti-tumor metastasis,we used wound healing assay and Transwell Boyden chamber assay to prove the fact that EPS11 could inhibit the migration of hepatocellular carcinoma cells Huh7.5 by reducing its adhesion ability and destroying their surface structure.In this study,we used proteomics technology to reveal the mechanism of EPS11 on the biological process of hepatocellular carcinoma cells Huh7.5,such as inhibiting the growth,proliferation and metastasis of hepatocellular carcinoma cells.According to the data,we found that the expression levels of mainly cell adhesion molecules were significantly decreased in hepatocellular carcinoma cells Huh7.5 after EPS11 treatment.Among them,the cell adhesion molecule CD99 was most obviously decreased.CD99 plays an important role in cell proliferation,adhesion,migration and differentiation,so we select it as a key effector molecule to deeply study the role of EPS11.After verifying it with quantitative real-time PCR(qRT-PCR)and Western blotting,we found that EPS11 could significantly reduce the expression of CD99 at mRNA and protein levels,especially in protein level.In turn,we overexpress CD99 molecules in hepatocellular carcinoma cells Huh7.5 to detect the effects of EPS11 on the proliferation,adhesion and mobility,and which were aimed to verify the importance of CD99 in EPS11 inhibiting the proliferation and migration ability of hepatocellular carcinoma cells Huh7.5.The results showed that CD99 could significantly rescued the proliferative rate,adhesion ability and migration ability decrease of hepatocellular carcinoma cells Huh7.5 after EPS11 treatment,which further confirmed that CD99 played an important role in the mechanism of anti-hepatocellular carcinoma metastasis for EPS11.It also provides evidence that CD99 can be an important marker for the target treatment of hepatocellular carcinoma.In order to further explore the effect of EPS11 inhibition on tumor cell metastasis in vivo,the model of pulmonary metastasis of melanoma cells was used to verify the result.Compared with the control group,the pulmonary metastasis of melanoma tumor lung colonization in mice could be significantly reduced after injection of EPS11,but it has no effect on the growth indicators in mice such as weight.It is proved that EPS11 has a good anti-tumor metastasis effect in animals and can be a potential anti-tumor metastasis candidate drug.