Dry sea cucumber C. frondosa was purchased from a local market (Changchun, Jilin Province, China). Antibodies against focal adhesion kinase (FAK) (product code ab131435), phospho-FAK (product code ab81298), paxillin (product code ab32115) and phospho-paxillin (product code ab4833) were purchased from Abcam. Antibodies against p21-activated kinase 1 (PAK1) (cat. no. sc-166887) and phospho-PAK1 (sc-135755) were purchased from Santa Cruz Biotechnology, Inc. LIM domain kinase 1 (LIMK1) (product no. 3842), phospho-LIMK1 (product no. 3841), cofilin (product no. 5175) and phospho-cofilin (product no. 3311) were obtained from Cell Signaling Technology, Inc. ECL chemiluminescent detection reagent and Glutathione Sepharose 4B affinity chromatography resin were obtained from GE Healthcare Life Sciences. Fibronectin, DMSO, EDTA, cysteine and TIRTC-conjugated phalloidin were purchased from Sigma-Aldrich; Merck KGaA. Fetal bovine serum, penicillin, streptomycin and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco Life Technologies; Thermo Fisher Scientific, Inc. All other chemical reagents used in this study were analytical grade.

The preparation of sea cucumber (C. frondosa) fucoidan was performed according to a previous method (22) with some modifications. Briefly, the dry body wall of the sea cucumber (200 g) was ground and degreased by reflux extraction with anhydrous acetone at 60°C for 4 h. Then, the sample was digested with 0.5% papain solution containing 5 mM EDTA and 5 mM cysteine at 60°C for 10 h. Subsequently, the digested sample was centrifuged at 8,000 × g for 15 min at 4°C. Polysaccharides were precipitated from the supernatant with 200 ml of 10% cetylpyridinium chloride solution at 4°C overnight. The precipitate obtained by centrifugation was re-dissolved with 1.5 l of 3 M NaCl:ethanol (100:15, v/v), and then 1 l of 95% ethanol was added into the mixture. After centrifugation at 8,000 × g for 15 min at 4°C and removal of the precipitate chondroitin sulfate, another 1.5 l of ethanol was added to the supernatant. The precipitate collected by centrifugation was re-dissolved and dialyzed (MWCO 1000 Da) against deionized water for 48 h. The sample was lyophilized to obtain sea cucumber C. frondosa fucoidan named Cf-Fuc. Cf-Fuc was composed of L-fucose and a sulfate ester group. The purity of Cf-Fuc was determined as 98.6% (total carbohydrate content 72.5% quantified by phenol-sulfuric acid method (23); the sulfate content 26.1% determined by BaCl₂-gelatin method (24).

U2OS cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology. U2OS cells were routinely cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin, incubated in a 5% CO₂ incubator at 37°C.

Cells (1×10⁴) were seeded into each well of 96-well plates. After incubation at 37°C for 24 h, Cf-Fuc was administered to the cells at 50 and 100 µg/ml. After 24 h of incubation at 37°C, the medium was replaced with 100 µl fresh medium containing 0.5 mg/ml of MTT. Subsequently, the cells were incubated at 37°C for another 4 h, and 150 µl of DMSO was added after removal of the supernatant. The absorbance at 570 nm was determined using a microplate reader.

U2OS cells were detached by trypsin digestion and suspended in serum-free medium with or without Cf-Fuc (50 and 100 µg/ml) for 30 min. Then, the cells were seeded into a 96-well plate coated with fibronectin (10 µg/ml), and incubated for 0.5, 1, 2 or 4 h. After washing with PBS, the adherent cells were fixed with 4% paraformaldehyde at room temperature (RT) for 30 min, and stained with 0.5% crystal violet at RT for 15 min. The dye, extracted with 33% acetic acid after washing with PBS, was quantified using a microplate reader.

U2OS cells incubated with or without Cf-Fuc (50 and 100 µg/ml) at 37°C for 4 h were then placed in 24-well Transwell™ (Corning Costar, Corning, NY) filter inserts (8 µm pore diameter). DMEM containing 10% FBS as the chemoattractant was added in the lower well. After incubation for 12 h, the non-migrated U2OS cells inside the inserts were gently removed using cotton swab. The migrated cells were then fixed and stained with 0.1% crystal violet at RT for 15 min. Migrated cells were counted in five random fields under a light microscope at ×400 magnification.

U2OS cells were seeded into a CELLview cell culture dish (Greiner Bio-One) pre-coated with fibronectin (10 µg/ml), then incubated with or without Cf-Fuc (50 and 100 µg/ml) at 37°C for 4 h. U2OS cells were then placed in a 37°C heating chamber with CO₂ supply. Images were captured at 5 min intervals with a CCD camera for 4 h. Image stacks were quantitatively analyzed and wind-rose plots of tracked migration paths of U2OS cells were plotted by NIH ImageJ software (version 1.30; National Institutes of Health).

Six-well plates were pre-coated with fibronectin (10 µg/ml) at 37°C overnight, and then U2OS cells were seeded into each well and incubated at 37°C for 2 h. Cells were further treated with or without Cf-Fuc (50 and 100 µg/ml) for 0.5, 1, 2 or 4 h. After being washed with PBS the cells were fixed and stained with rhodamine phalloidin (0.1% Triton X-100, 3.7% formaldehyde, and 2 µM rhodamine phalloidin in PBS) for 1 h. Cells were incubated with methanol to extract the dye and rhodamine fluorescence (excitation wavelength: 530 nm, emission wavelength: 590 nm) was further assessed with a fluorescence microplate reader (TECAN GENios; Tecan Austria GmbH). F-actin content at time 0 was considered as 1; F-actin content at 0.5, 1, 2 and 4 h are expressed as a relative fold change of mean fluorescence intensity at time 0.

Rac1 activation was determined according to a previous method (25). Briefly, U2OS cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) and the lysate supernatant was then incubated with GST-PAK1 PBD fusion proteins immobilized on Glutathione Sepharose 4B affinity chromatography resin. After washing with lysis buffer, the resin was boiled in Laemmli sample buffer. Then, Rac1 protein bound to GST-PAK1 PBD as well as the endogenous total Rac1 were analyzed by SDS-PAGE and immunoblotting.

Cells were harvested and lysed with RIPA lysis buffer. Cell lysates were collected and the concentration of proteins was quantified by BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg of total protein per lane) were subjected to 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 3% BSA for 2 h at room temperature and then incubated with indicated primary antibodies (1:1,000 dilution) at 4°C overnight. HRP-conjugated secondary antibody (Beyotime Institute of Biotechnology) was incubated at RT for another 1 h after washing with TBST three times. Protein bands were subsequently visualized using ECL Western blotting chemiluminescent detection reagent. The band intensities for quantitative analysis were measured by densitometric analysis using NIH ImageJ software (version 1.30; National Institutes of Health).

Data were expressed as the mean ± SD. Significant differences were determined by one-way ANOVA, followed by Bonferroni post hoc test using GraphPad Prism 6 (GraphPad Software, Inc.). P-values <0.05 were considered to indicate a statistically significant difference.

As revealed in Fig. 1, U2OS cell viability was not impacted by Cf-Fuc treatment at concentrations of 50, 100, and 200 µg/ml for 24 h. When the concentration of Cf-Fuc increased to ≥400 µg/ml, the U2OS cell viability was significantly decreased compared to the control group. Therefore, the non-cytotoxic concentrations of 50 and 100 µg/ml were selected for the following experiment to evaluate the inhibitory effect of Cf-Fuc on U2OS cell adhesion and migration, in case the phenotype presented in the present study was caused by cytotoxicity.

As revealed in Fig. 2, the adherent percentage of U2OS cells without treatment gradually increased, and eventually reached 83.2% at 4 h. While, the adherent percentage of Cf-Fuc-treated cells increased slowly, and only reached 59.0 and 43.8% at 4 h, respectively, with the Cf-Fuc concentration of 50 and 100 µg/ml, indicating that Cf-Fuc treatment could inhibit U2OS cell adhesion.

First, the inhibitory effect of Cf-Fuc on U2OS cell migration was evaluated by Transwell assay. Cf-Fuc caused strong inhibition on U2OS cell migration, leading to a significant decrease in the number of migrated cells compared with the control group (Fig. 3). Then, the migratory capacity of Cf-Fuc-treated U2OS cells was quantitatively analyzed by NIH ImageJ. As revealed in Figs. 4 and 5, the control cells exhibited a strong migration ability, and the velocity reached 64.8 µm/h. Notably, in comparison to the cells in the control group, Cf-Fuc impaired the migratory capacity of U2OS cells. The migratory distance of Cf-Fuc-treated cells significantly decreased and the velocity was reduced to 28.5 and 18.1 µm/h, respectively, at concentrations of 50 and 100 µg/ml.

Cytoskeleton remodeling is a critical event involved in cancer metastasis (26). During the adhesion and migration of cancer cells, G-actin polymerizes into F-actin filaments, which acts as stress fiber of the cytoskeleton and participates in the formation of lamellar pseudopods required for osteosarcoma metastasis (27,28). Therefore, the effect of Cf-Fuc on actin polymerization was further investigated. As revealed in Fig. 6, the actin polymerization of U2OS cells increased in a time-dependent manner. However, F-actin content of Cf-Fuc-treated cells was reduced compared to the control cells.

The effect of Cf-Fuc on cell adhesion signaling of U2OS cells was further examined. As revealed in Fig. 7A, Cf-Fuc treatment significantly suppressed the phosphorylation of FAK (Fig. 7B) and paxillin (Fig. 7C) compared to the control group, indicating that Cf-Fuc inhibited the U2OS cell migration possibly by modulating the FAK/paxillin cell adhesion signaling axis.

Small GTPase Rac1 plays a crucial role in the remodeling of the actin cytoskeleton. Whether Cf-Fuc could inhibit Rac1 activation in U2OS cells was subsequently examined. As revealed in Fig. 8A, compared to the control group, once treated with Cf-Fuc, the level of GTP-Rac1 (activated form of Rac1) was reduced by 72 and 91%, respectively, at concentrations of 50 and 100 µg/ml (Fig. 8B).

The Rac1/PAK1/LIMK1/cofilin signaling axis could regulate the assembly of the actin cytoskeleton and has been revealed to be involved in cancer metastasis (29,30). Therefore, to determine whether Cf-Fuc inhibited U2OS cell adhesion and migration via the PAK1/LIMK1/cofilin signaling axis, the phosphorylation levels of PAK1, LIMK1 and cofilin were further investigated. As revealed in Fig. 9, the phosphorylated forms of PAK1, LIMK1 and cofilin were significantly inhibited by Cf-Fuc treatment at concentrations of 50 and 100 µg/ml compared to the control group. The results indicated the potential molecular mechanism of Cf-Fuc in inhibiting U2OS cell adhesion and migration by blocking the activation of the Rac1/PAK1/LIMK1/cofilin signaling axis.