F. vesiculosus was collected from the coast of the Baltic Sea and the identity of the specimens was confirmed as previously described (21,22). The fucoidan used in the present study was isolated from the extract of F. vesiculosus in 20:80 ethyl acetate:hexane using column chromatography and was characterized using ¹H nuclear magnetic resonance (NMR), ¹³C NMR and mass spectrometry.

Human FTC133 and TPC1 ATC cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin. The cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

A Cell Counting Kit-8 (CCK-8) cell proliferation kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to analyze the effect of fucoidan on cell proliferation. The cells were seeded into 96-well plates at a density of 2.5×10⁵ cells in each well. After 24 h, different concentrations of fucoidan (0, 1, 2, 4, 8, 10, 15 µM) were added to each well containing the cells. Following incubation for 48 h at 37°C, 20 µl of CCK-8 solution (5 mg/ml) was added to each well and incubation was continued for another 4 h at 37°C in an incubator with 5% CO₂. The proliferation of the cells was measured using a Multiskan Go spectrophotometer (Thermo Fisher Scientific, Inc.) at 450 nm. The proliferation index was calculated from the resulting optical density values.

Following treatment of the cells with the aforementioned concentrations of fucoidan for 72 h, the cells were washed three times in ice-cold phosphate-buffered saline (PBS) and then lysed in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 137 mM NaCl, 10% glycerol, 100 mM sodium vanadate, 1 mM PMSF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1% NP-40 and 5 mM cocktail. The concentration of proteins in the lysate was determined using a bicinchoninic acid assay. The proteins (2 µg) were resolved on a 10% polyacrylamide gel by electrophoresis. For the transfer of proteins onto a polyvinylidene difluoride membrane, a semi-dry method was used. The membrane was blocked in 5% non-fat dry milk overnight and then washed in Tris-buffered saline with Tween-20 (TBST). The membrane was then incubated overnight at 4°C with anti-hypoxia inducible factor (HIF)-1α (1:50; cat. no. 100–449), anti-B cell lymphoma-2 (Bcl-2)-associated X protein (Bax; 1:50; cat. no. 100–56097), anti-Bcl-2 (1:50; cat. no. 100–2087), anti-cleaved caspase-3 (1:50; cat. no. 100–56113).) and anti-cleaved poly ADP-ribose polymerase (PARP; 1:50; cat. no. 100-56599; Cell Signaling Technology, Inc., Danvers, MA, USA). The incubation with antibodies was followed by washing with TBST. Subsequently, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. no. 610-103-121; GE Healthcare Life Sciences, Chalfont, UK) for 1 h at room temperature. Enhanced chemiluminescence (Pierce Biotechnology; Thermo Fisher Scientific, Inc.) was used for the antigen detection. β-actin was used as the control.

Onto 18 mm cover slips, the cells in minimal essential medium (Gibco; Thermo Fisher Scientific, Inc.) were plated at a density of 3.0 10⁶ and ~80% confluence for 24 h at 4°C. For angiogenesis analysis, cells were treated with 100 µM CoCl₂ for 18 h to induce hypoxic conditions Treatment of the cells with 100 µg/ml fucoidan for 24 h was followed by fixing in 10% paraformaldehyde. The cells were then washed twice with PBS and stained with 2 µg/ml DAPI for 20 min at 37°C. A fluorescent microscope was then used to analyze the nuclear fragmentation in the stained cells. A TUNEL kit (Chemicon, Temecula, CA, USA) was used for the TUNEL assay, which was performed according to the manufacturer's protocol.

At 90% confluence, HUVECs were plated onto the 60 mm diameter culture dishes and then scratched with pipette tip. The cells were then subjected to PBS washing followed by incubation in endothelial cell growth medium MV2 (ECGM2) supplemented with 2% FBS along with recombinant human VEGF (50 ng/ml), thymidine (1 mM) and fucoidin (10 µM) obtained from PromoCell GmbH (Heidelberg, Germany). The cell cultures used as negative control were incubated with ECGM2 medium containing 2% FBS alone. After 16 h, HUVECs were rinsed in PBS followed by methanol fixation. The experiments were performed in triplicates and the data are presented as the mean of three experiments.

To each Matrigel-coated well (BD Biosciences, Franklin Lakes, NJ, USA), 0.2 ml of cell suspension comprising cells suspended in growth medium at a density of 2.5×10⁵ cells/ml, was added. The cells were incubated at 37°C in an incubator with 5% CO₂ in the presence or absence of fucoidan for 12 h. The cells were also incubated at 4°C in media containing 50 ng/ml VEGF and 1 mM thymidine along with fucoidan (10 µM) for 16 h. Subsequently, a phase contrast microscope was used to examine changes in the morphology of the cells.

To determine the migration potential of the fucoidan-treated cells, Transwell cell culture inserts were used. The cells (3×10⁵) in 200 ml of growth medium were seeded into the upper chambers of the Transwell inserts. To the lower chamber, 750 ml of medium was added, which contained 20% FBS as a chemoattractant. In the control wells, medium was added to the upper and the lower chambers. The non-migrated cells in the upper chamber were removed by swabbing following 24 h of incubation at 4°C. The inserts were observed under an inverted fluorescent microscope subsequent to fixing and staining. The number of cells in five randomly selected fields of view were counted in triplicate.

One-way analysis of variance and Student's t-test were used for statistical analysis. The data are presented as the mean ± standard deviation. P≤0.05 was considered to indicate a statistically significant difference. SPSS software (version 10.0; SPSS, Inc., Chicago, IL, USA) was used for statistical calculations.

Treatment of the FTC133 and TPC1 ATC cell lines with different doses of fucoidan (1–15 µM) led to inhibition of cell growth 36 h following treatment. The inhibition of cell growth by fucoidan was observed to be concentration-dependent (Fig. 1). For the FTC133 and TPC1 cell lines, the half maximal inhibitory concentrations for growth inhibition were 8 and 10 µM, respectively.

The results of the western blot analysis revealed that the expression levels of Bax, cleaved PARP and caspase-3 were increased, and the expression of Bcl-2 was decreased in the FTC133 cells treated with fucoidan for 36 h, compared with the control cells (Fig. 2A). DAPI staining showed the appearance of DNA fragmentation and perinuclear apoptotic bodies in the FTC133 cells treated with 8 µM of fucoidan (Fig. 2B). Thus, fucoidan led to apoptosis of the FTC133 cells. The fucoidan-induced apoptosis was indicated by DNA strand breakage, which were also observed in the results of the TUNEL analysis.

The cells were treated with CoCl₂ (100 µM) for 18 h to induce hypoxia-like conditions, and were then treated with different doses of fucoidan. Under the hypoxic conditions, the expression of HIF-1α was promoted, however, fucoidan treatment (10 µM) reduced the hypoxia-induced expression of HIF-1α. The expression of VEGF was also enhanced under the hypoxic conditions and this hypoxia-induced expression of VEGF was decreased by treatment with fucoidan. The results from the tube formation assay clearly revealed that the formation of vessel-like structures was suppressed following treatment with fucoidan (Fig. 3A). Incubation of the wounded cells in media containing 50 ng/ml VEGF and 1 mM thymidine in the presence of fucoidan (10 µM) for 16 h showed failure of wound healing capacity (Fig. 3B). Investigation of the effect of fucoidan on the expression of VEGF revealed a reduction in the expression of VEGF by fucoidan in hypoxia (Fig. 3C). Thus, tube formation and the migration of cells were inhibited by fucoidan, suggesting that fucoidan has a potent anti-angiogenic effect.