Anti-metastasis Effect Of Fucoidan From Undaria Pinnatifida Sporophylls In Mouse Hepatocarcinoma Hca-F Cells And Mechanism Research

Objective: Metastasis is one of the major causes of cancer-related death. It is acomplex biological process involving multiple genes, steps, and phases. It is alsoclosely connected to many biological activities of cancer cells, such as growth, invasion,adhesion, hematogenous metastasis and lymphatic metastasis.Fucoidan is a highly sulfated polysaccharide of brown algae whose main sugar unitis L-fucose. It has been reported to have a wide range of physiological and biologicalactivities. Compared with other fucoidans fucoidan extracted from sporophylls of thebrown seaweed Undaria pinnatifida (Ups-fucoidan) has a higher sulfate and L-fucosecontent and a wider range of physiological and biological activities, such as anti-tumor,anticoagulant, anti-inflammatory, induce-apoptosis, and Immunoregulation activities.The anti-tumor activity of Ups-fucoidan has garnered much attention.In the process of development and transfer, tumor cells secrete serious of relevantsignal factors, which could stimulate the proliferation, the peripheral lymphangiogenesisand the degradation of extracellular matrix, and promote lymph node metastasize. Inthis study, we will measure the expression of relevant key factors to demonstrate themechanism of lymphatic metastasis. Cyclin D1and cyclin-dependent kinase4(CDK4)are key players in the cell cycle and growth. Matrix metalloproteinases (MMPs) andtheir specific inhibitors–tissue inhibitor of metalloproteinases (TIMPs) have beenacknowledged as major critical molecules associated with extracellular matrix (ECM)degradation and cancer cell invasion during metastasis. Integrin family (Integrin α5-β1), CD44and L-Selectin belongs to adhesion signal factors, which are closely related toadhesive between tumor cells to ECM and lymph node. It is also well established thatnuclear transcription factor-κB (NF-κB) is most frequently involved in cell survival andgrowth through the transactivation of anti-apoptotic genes. However, NF-κB activity isconnected with multiple signaling pathways, such as the phosphoinositide3-kinase(PI3K)/Akt and extracellular regulated protein kinases (ERK) signaling pathways.Vascular endothelial growth factor C (VEGF-C) and VEGF receptor3(VEGFR-3) arepotent lymphatic endothelial cell (LEC) molecules involved in growth and are keyregulators of lymphangiogenesis, by the activation of VEGF-C/VEGFR-3signalingpathway. Moreover, some researchers have reported that the proliferative actions ofVEGF require activation of both ERK and Akt signaling cascades. Similarly, hepatocytegrowth factor (HGF) and tyrosine kinase (C-Met) play important roles in the control oftumor growth and invasion. These pathways play a pivotal role in cancer formation andprogression by modulating cell growth, growth, apoptosis, and metastasis.In the present study, we evaluated the effects of Ups-fucoidan on tumor metastasisof Hca-F cells. Our findings will aid understanding of the anti-tumor, anti-invasion andanti-adhesion effects of Ups-fucoidan and the mechanism of anti-metastasis.Method:1. The inhibitory effects of different concentration of Ups-fucoidan onHca-F cell growth were assessed via measurement of cell viability by MTT assay invitro. The Annexin V/PI double staining was used to determine the apoptosis rate. Theprotein expression levels of cyclin D1and CDK4were measured by western blot assay.2. Cell death rate was identified by trypan blue staining assay, after the treatment ofdifferent concentrations of Ups-fucoidan for24h to exclude the cyto-toxicity to Hca-Fcells.3. In the migration and invasion analysis, the inhibitory effects of Ups-fucoidan oninvasive and migratory capabilities of Hca-F cells were evaluated by Transwell assay.ELISA and western blot assays were used to evaluate the relative expression quantity ofMMP-2, MMP-9, TIMP-1and TIMP-3.4. The adhesion assay was used to measure the binding of Hca-F cells to peripheral lymphatic endothelium. The adhesion factors protein levels of Integrin α5-β1, CD44andL-Selectin were measured by western blot assay.5. The expression of key factors like VEGFR-3, C-Met, PI3K/Akt and ERK wasmeasured by western blot assay. The secretion levels of HGF and VEGF-C wereassessed by ELISA assay. The mRNA expression levels of timp-1, vegf-c, vegfr-3andgapdh were measured by Semi-q RT-PCR.Results:1. Ups-fucoidan inhibits Hca-F cell proliferation. The results show aconcentration-and time-dependent manner of Ups-fucoidan on Hca-F cell growth.Apoptosis was measured by Annexin V/FITC double staining using flow cytometry. Theresults showed that the percentage of apoptotic cells was increased significantly byUps-fucoidan treatment. Cyclin D1and CDK4expression were significantly decreasedfollowing Ups-fucoidan treatment.2. After Ups-fucoidan withdrawal, counting the death cell by trypan blue stainingand calculating the relative cell death rate (RD) compared with control, and all the RD <5.2%in each concentrations group of Ups-fucoidan. These findings indicate thatUps-fucoidan could inhibit Hca-F cells growth, but not cyto-toxicity.3. In the Transwell assay, Ups-fucoidan suppressed Hca-F cell migration andinvasion in vitro. ELISA results show that both MMP-2and MMP-9levels in cellmedium were decreased following Ups-fucoidan treatment, but were not significantlydifferent from the control. Western blot results show that Ups-fucoidan increasedTIMP-1and TIMP-3expression in a concentration-dependent manner.4. The adhesion assay result show that the adhesive capability of Hca-F cellstreated with1000μg/ml Ups-fucoidan was significantly lower than that of the control.The expression of Integrin α5-β1, CD44and L-Selectin were significantly decreasedfollowing Ups-fucoidan treatment in western blot results.5. Western blots result show that the expression of VEGFR-3, C-Met, p-PI3K,p-Akt, p-ERK1/2, and NF-κB was downregulated following Ups-fucoidan treatment ina concentration-dependent manner. ELISA result demonstrate significantly lower of therelative expression quantity (RE%) of VEGF-C and HGF following Ups-fucoidan treatment compared to the control. The Semi-q RT-PCR results indicate thatUps-fucoidan could down-regulate the expression of vegf-c and vegfr-3, and up-regulatethe expression of timp-1.Conclusions: Our findings demonstrate the inhibitory effect of Ups-fucoidan onHca-F cell growth, adhesion, and invasion capabilities and the mechanism involvingsuppress the activity of NF-κB-dependent PI3K/Akt and ERK signaling pathwaysthough the inactivation of HGF/C-Met and VEGF-C/VEGFR-3dual signalingpathways.