| Objective: Angiogenesis, the formation of new blood capillaries by vascularendothelial cells from existing vessels, is the result of s complex effect on cell-cell andcell-matrix interactions. It mainly involves the degradation of extracellular matrix(ECM), endothelial cells proliferation, migration and tube formation, as well assprouting of new capillary branches. The complex process is regulated by stimulatoryand inhibitory factors. Angiogenesis is not only necessary for the growth of cancer butalso for tumor transplantation and metastasis. There are two stages in tumor growth, thefirst stage involves the absence of blood vessel formation, whereas the second stagerequires the formation of new capillaries. Under the second stage, tumor growthrequires angiogenesis to supply nutrients and oxygen. In addition, the tumor alsoutilizes the newly formed blood vessels as conduits to disseminate invasive tumor cells.Angiogenesis is an essential component for the growth and progression of neoplasms,and no doubt blocking it is an effective strategy for inhibiting tumor growth. Hence,anti-angiogenic therapy has become a promising approach in the development of novelanticancer therapy as well as therapies for other pro-angiogenic diseases.Fucoidan, a kind of intercellular polysaccharide of brown algae, is a water-solublepolysaccharide mixture including L-fucose and sulfate as main composition, galactose,xylose, mannose, alduronicacid et al. Recently，it has been to reported to have variousbiological activities such as antiviral, antioxidative, anti-inflammatory, antiangiogenic,anticoagulant, and antitumor properties. In this study, we evaluated and confirmed theanti-angiogenic effect of fucoidan in vitro, ex vivo and angiogenesis abilities. Westernblotting and RT-PCR were used to investigated the mechanism on angiogenesis offucoidan.Method:1.The fucoidan extract from U. pinnatifida sporophyll was prepared byenzymatic hydrolysis and alcohol precipitation and the contents of total carbohydrate, sulfate radical and uronic acid were measured by the phenol-sulfuric acid reaction,BaCl2-gelation and sulfuric acid-carbazole colorimetric method, respectively.Furthermore, the molecular weight of the sample was evaluated by size exclusionchromatography using a TSK-gel G3000PWXL(TOSOH, Tokyo, Japan). The opticalrotation was measured at589nm by the WZZ-1polarimeter at20oC.2.MTT assay was used to measured the effect of fucoidan on growth inhibition ofHUVECs.3.Using Transwell to assay the chemotactic motility of HUVECs treated withdifferent concentrations of fucoidan.4.Formation of capillary tube-like structures by HUVECs with differentconcentrations of fucoidan was assessed in a matrigel-based assay.5.New vascular network damaging assay based on the formation of capillary tubewas used to evaluate the effect of fucoidan on angiogenesis.6.Using rat aortic ring model to test the effect of fucoidan ex vivo.7.Western blot and RT-PCR were used to evaluate the level of VEGF-Aexpression in HUVECs to investigate the mechanism of fucoidan on angiogenesis.Results:1.The fucoidan purified from U. pinnatifida was a beige fibrous powder(purity﹥90%). This fucoidan sample consisted mainly of carbohydrates (59.2%)，sulfates (21%) and uronic acid (9.13%) with fucose and galactose constituting themonosaccharide composition, and the percentage of protein content was detected to be0.13%. The molecular weight of the purified fucoidan was about9.52×10~4Da. Theoptical rotation of the fucoidan (0.6mg/ml) showed a value of0.99°at20°C.2.MTT results indicate that HUVECs were inhibited significantly by fucoidan,which depended on the drug dosage and treat-time closely. It also shows growthinhibition was not remarkable when HUVECs were treated with fucoidan for24h. Byincreasing the treatment time to48h, growth inhibition exerted by the sameconcentrations of fucoidan became stonger and statistically significant. As much as40%of growth was inhibited by the maximum concentration (400 g/ml) of fucoidan tested.3.Transwell assay shows fucoidan induced the in vitro migration of HUVECs in adose-dependent manner. In contrast, treatment of HUVECs with fucoidan at100,200or400μg/ml led to significant reduction (P <0.05, P <0.01) in the number of migratingcells compared to control.4.Fucoidan exhibited inhibitory effect on tube formation. And treatment withvarious concentrations of fucoidan clearly inhibited the tube formation of HUVECs in a dose-dependent manner.5.Tubes developed from cells grown on fucoidan treated (400μg/ml) membranefor24h and48h showed no significant change whereas tubes from cells grown onuntreated membrane showed strong promoting effect on HUVEC new vascularstructures at48h. These results suggested that fucoidan could prevent the growth ofexisting capillary tube.6.Rat aortic ring model is the complex result of the proliferation, migration andtube formation.Inhibition of microvessel formation by fucoidan was observed even atlow concentration (100μg/ml). At400μg/ml, fucoidan inhibited about61%ofmicrovessel formation compared to control. These results suggested that fucodiansuppressed ex vivo microvessel formation in a dose-dependent manner.7.Western blot and RT-PCR results show that VEGF-A decreased in a dose-dependent manner after being treated with fucoidan which suggested that inhibition ofVEGF-A by fucoidan may contribute to a reduced level of angiogenesis by HUVECs.Conclusions: In our experiment, fisrtly, the results showed that fucoidan is rich insulfates and high molecular weight.Secondly, it showed that fucoidan significantly inhibit the migration, tubeformation, new vascular network formation and microvessel outgrowth in a dosedependent manner.Further, the effects of the fucoidan on angiogenesis of HUVECs was measured bywestern blotting and RT-PCR, and suggested that fucoidan suppressed expression of anangiogenesis factor vascular endothelial growth factor A (VEGF-A), resulting insuppressed vascular tubules formation. Hence, fucoidan has a great value as a tumorangiogenesis inhibtor. |
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