Fucus vesiculosus

Polymeric, oligomeric, and monomeric sulfated
carbohydrates were used as substrates to determine the specificity
of AMOR_S1_16A. κ-carrageenan (Tokyo Chemical Industry), ι-carrageenan
(Tokyo Chemical Industry), λ-carrageenan (Tokyo Chemical Industry),
and agar (Sigma-Aldrich) were tested in their native form and as hydrolysates.
To generate a variety of substrates, partial hydrolysis of these polysaccharides
was performed with 1 M TFA at 60 °C for 1 h, followed by neutralization
with 5% NH₄OH. Additionally, κ-carrageenan oligomers
were enzymatically produced using the κ-carrageenase ZgCgk16A
from Z. galactanivorans(36 (link)) (EC number 3.2.1.83) (NZYTech, Portugal) using
10 mg/mL substrate and 1 μL enzyme (0.25 mg/mL) in 50 mM Tris-HCl,
pH 7.2, 150 mM NaCl at 25 °C overnight. In addition, AMOR_S1_16A
was tested on a wide range of commercial fucoidan substrates: Macrocystis pyrifera, Undaria pinnatifida, Fucus vesiculosus, Laminaria japonica from Sigma-Aldrich, and Alaria sp. Fucus serratus, Laminaria digitata, Ascophyllum nodosum, Lessonia nigrescens, Ecklonia sp., Durvillaea sp., Cladosiphon sp. from Biosynth. Fucoidan substrates were
used in their native form and as hydrolysates, as described above
for agar and carrageenans. The monosaccharides galactose-4-sulfate
(G4S) and galactose-6-sulfate (G6S) were obtained from Biosynth Ltd.,
UK, and N-acetylgalactosamine-4-sulfate (4S-GalNAc)
from Dextra, UK (Kindly provided by Dr. Alan Cartmell, Newcastle University,
UK), respectively.

Fucoidan (Fucus vesiculosus, Sigma Aldrich, Arklow, Ireland) was prepared as a 10 mg/mL stock solution in PBS, filtered using 0.2 μm filter and sterilised via autoclaving. The effect of autoclaving on fucoidan was evaluated by comparing the fast-performance liquid chromatography (FPLC) spectral profile with the profile of a non-autoclaved fucoidan sample (results not shown) which confirmed that autoclaving did not have any detrimental effect. For cell culture studies, 200, 100, 50, 25, 12.5, 6.25 and 3.125 μg/mL solutions were prepared from the stock solution using a complete growth medium.

The coating compositions used in this investigation consisted of two components: room temperature vulcanizing, medical grade, platinum silicone elastomer (MED-4210, Factor II, Incorporated, Lakeside, OK, USA); 10 wt.% siloxane oil (1000 cP, DMS-T31, Gelest, Morrisville, PA, USA); and 0–8 wt.% fucoidan, extracted from brown seaweed Fucus vesiculosus (Sigma Aldrich, St. Louis, MO, USA). The coating compositions were prepared by mixing [7 ].

Fucoidan and cell culture95% purified fucoidan from Fucus vesiculosus was obtained from Sigma–Aldrich, St. Louis, MO. The 200 mg/mL of fucoidan stock solution was prepared by dissolving in phosphate-buffered saline (PBS) and stored at −20 °C. HSC-3 oral squamous carcinoma cell line was obtained from Japanese Collection of Research Bioresources Cell Bank. Cells were cultured in DMEM medium supplemented with 1% penicillin-streptomycin (10,000 U/mL) and 10% heat-inactivated fetal bovine serum (Gibco BRL, Gaithersburg, Md., U.S.A.) and maintained at 37°C in an incubator containing 5% CO2.
Cytotoxicity analysisTo determine the half-maximal inhibitory concentration (IC₅₀) of fucoidan, HSC-3 cells were seeded into 96-well plate for 24 h and then treated with fucoidan diluted in PBS in 0, 10, 50, 100, 150, 200, 250, 300, 350, and 400 µg/ml for 24 h. Thereafter, the MTT working solution (Sigma-Aldrich, USA) was added into each well at a final concentration of 30μg/ml and further incubated for 3 h. OD values were measured by using a spectrophotometer with wavelength at 562 and 630 nm and the results were used to calculate IC₅₀ value. Thereafter, HSC-3 cells were treated with three concentrations of fucoidan including the concentration below IC₅₀ (100 μg/ml), approximate IC₅₀ (200 μg/ml), and above IC₅₀ (400 μg/ml) for 24, 48 and 72 h and analyzed cytotoxicity effect by MTT assay. These three concentrations of fucoidan were used for further studies.
Annexin V/PI apoptosis assayCells were treated with fucoidan for 48 h. After that, treated cells were analyzed the distribution of apoptotic stages by using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen™). Briefly, 1 × 10⁵ harvested cells were incubated with FITC Annexin V and PI in the binding buffer and incubate for 15 min at room temperature in the dark. The distribution of apoptotic stages was evaluated by the FACSCalibur flow cytometer (Becton Dickinson, USA).
Hoechst 33342 stainingThe nuclear condensation and fragmentation were determined by Hoechst 33342 staining (Sigma-Aldrich, USA). After treatment with fucoidan, the culture media were removed and cells were washed in PBS. After that, treated cells were stained with 10 mg/mL Hoechst stock solution diluted at 1:2,000 in PBS and incubated for 15-20 minutes at room temperature in a dark condition. Finally, cells were washed 3 times in PBS and visualized by a fluorescence microscope at 480 nm.
Determination of mitochondrial membrane potentialDetermination of mitochondrial membrane potential was performed using the JC-1 mitochondria staining Kit (Sigma-Aldrich, USA). Treated HSC-3 cells were incubated with a JC-1 staining solution for 20 min at 37°C with 5% CO₂. After that, cells were washed twice and overlaid with a growth medium. JC-1 fluorescence intensities of red aggregates and green fluorescence monomers were read with Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific Inc., USA). The ratio of the JC-1 aggregates (red fluorescence) versus JC-1 monomer (green fluorescence) for each treatment was evaluated.
Furthermore, HSC-3 cells (1 × 10⁵) were seeded in 6 well plates for 24 h and were subsequently treated with various concentrations of fucoidan for 48 h at 37°C. Cells were incubated with JC-1 staining liquid for 20 min at 37°C, washed 3 times with growth medium, and examined under the fluorescence microscope.
Flow cytometry analysis of DNA contentTreated cells were evaluated cell cycle distribution by using propidium iodide as previously described (Castro et al., 2011). Briefly, after incubated with various concentrations of fucoidan for 48 h, 2 x 10⁶ treated cells were fixed with 70% ethanol and overnight incubated in a -20°C. Cells were washed twice in PBS to remove the ethanol. Cells were then re-suspended in PBS containing 40mg/ml propidium iodide with 100 mg/ml DNAse-free RNAseA (Sigma-Aldrich, USA) and incubated in the dark at room temperature for 15 min. The DNA content was analyzed using the FACSCalibur flow cytometer (Becton Dickinson, USA).
Western blot analysisAfter treatment with fucoidan, total cellular proteins were obtained using RIPA cell lysis buffer (Cell Signaling Technology, USA) and determined the concentrations by using Lowry protein assays (Bio-rad, USA). From each sample, 30 μg of total proteins were separated by using 15% SDS-PAGE and further transferred onto nitrocellulose membranes. The membranes were blocked in blocking buffer (5% BSA in Tris-buffered saline with 0.1% Tween-20 (TBST)) for 1 h then incubated with primary antibodies (Cell Signaling Technology, USA) including rabbit anti-Bax, rabbit anti-Bcl-2, rabbit anti-cleaved caspase-3, rabbit anti-cleaved PARP, rabbit anti-LC3, rabbit anti-Beclin-1 and rabbit anti-β-actin diluted in 5% BSA in 0.01M TBST at 4°C overnight. The membranes were washed in TBST and further incubated with horseradish peroxidase (HRP) conjugated secondary antibody (Cell Signaling Technology, USA) diluted in 0.01M TBST at 1:5,000 for 1 h at room temperature. The corresponding targeted proteins were visualized by using enhanced chemiluminescent (ECL) detection (Thermo Fisher Scientific Inc., USA). Protei n bands’ intensity was quantified by using ImageJ software.
Statistical analysisData were expressed as the mean ± SD obtained from triplicate experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) test provided in SPSS 22. The values obtained in the assays were considered to be statistically significant when P < 0.05.

VEGF was detected in the supernatants of ARPE19 and primary RPE cells using commercially available ELISA kits (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. To establish the parameters of VEGF ELISA, we investigated time-dependent VEGF secretion in ARPE19 and primary RPE cells in the presence and absence of commercially available fucoidan from Fucus vesiculosus (Sigma-Aldrich, F8190) for 1 day, 3 days, and 7 days. A cell viability assay (MTT) was conducted after 7 days. According to these results, an incubation time of 3 days was chosen for experiments with the five different fucoidans. The medium was changed 24 h prior in ARPE19 cells and 4 h prior in primary RPE cells and the supernatant collected. Measured VEGF content was normalized for cell survival and is depicted in relation to that of untreated control cells.

A-549 cells (lung carcinoma cell line, ATCC CCL-185™), WiDr cells (colon adenocarcinoma cell line, ATCC CCL-218™), MCF-7 cells (breast adenocarcinoma cell line, ATCC HTB-22™), Malme-3M cells (lung melanoma cell line, ATCC HTB-64™), HEK-293 cells (human embryonic kidney cell line, ATCC CRL-1573™), HUVEC cells (human umbilical vein endothelial cell line, ATCC PCS-100-010™), LoVo cells (human colon adenocarcinoma cell line, ATCC CCL-229™), and HDFb cells (human derma fibroblast cell line, Invitrogen NZ Limited) were each cultured in 5 mL of Roswell Park Memorial Institute 1640 (RPMI 1640) complete medium (GIBCO, USA) supplemented with 1% Penicillin–Streptomycin (Invitrogen, USA), 1% l-glutamine (Invitrogen, USA) and 10% fetal bovine serum (Medica Pacifica, USA) at 37°C in a 5% CO₂ atmosphere.
The cytotoxicity/cell reducing capacity of crude fucoidan (F0) and its fractions (F1 and F3) was determined using the MTT cell proliferation assay (29 ). Each cell line (A-549, WiDr and MCF-7) was sub-cultured (100 μL) in 96-well plates at a density of 1 × 10⁵ cells per well and allowed to attach to the well bottom for 24 h in 5% CO₂ at 37°C. Different concentrations (0.2–1.0 mg mL⁻¹) of crude fucoidan (F0), its fractions (F1 and F3), and commercial fucoidan (Fucus vesiculosus, Sigma, USA) were added into the wells (100 μL) and were incubated for a further 24 h. The supernatant was removed and washed with 150 μL of PBS (pH 7.2) and removed again. The plates were re-incubated for 4 h after the addition of 30 μL of MTT (5 mM, in phosphate buffered saline solution at pH 7.2) reagent and a further 30 min after adding 150 μL of DMSO. Absorbance of each well was measured at 540 nm using a FLUOstar Omega microplate reader (Alphatech, New Zealand). Assays were performed after treatment of cells for 24, 48, and 72 h. The data were used to calculate the cell viability percentage using the equation below. The IC₅₀ value was calculated using Prism 5 (GraphPad Software Inc., San Diego, CA, USA).
$\begin{array}{llll}\hfill & \text{Cell Viability\hspace{0.17em}}\left(\%\right)& \hfill & \\ \hfill & =\left(1-\frac{\begin{array}{c}\left(\text{Abs of control}-\text{Abs of blank}\right)\\ -\left(\text{Abs of sample}-\text{Abs of blank}\right)\end{array}}{\left(\text{Abs of control}-\text{Abs of blank}\right)}\right)\times 100& \hfill & \end{array}$