Fucoidan was purchased from Sigma-Aldrich (St. Louis, MO). Fucoidan powder was dissolved in phosphate buffer saline (PBS), then sterilized using a 0.22 μm pore filter (Millipore, Billerica, MA) and stored at 4 °C until use.
DU-145, androgen-independent human prostate carcinoma cells, were purchased from American Type Culture Collection (ATCC, Manassas, VA), and were grown in Modified Eagle’s Medium (MEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Grand Island, NY) at 37 °C in a humidified 5% CO2 atmosphere.
Cell viability and proliferation
DU-145 cells were cultured in 96-well plates (2 × 104 cells/well) for 24 h before the serum-free medium was used and cells were treated with 100, 200, 500, 1000 μg/mL of fucoidan for another 24 h. Cell viability and proliferation were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Amresco, Solon, OH) and 5-bromo-20-deoxyuridine (BrdU, Roche Diagnostics, Mannheim, Germany) incorporation assays, respectively, according to the manufacturer’s instructions.
DU-145 cells were seeded into the insert of Transwell (Corning, Tewksbury, MA) at a density of 1 × 105 cells/well, then cultured in serum-free culture media. Fucoidan (500 μg/mL) or vehicle (PBS) was added to the lower reservoirs. Cells were subsequently allowed to migrate across a collagen I-coated polycarbonate filter for 12 h at 37 °C. Non-migrated cells were removed from the top side of the filter by scraping. Migrated cells on the bottom side of the filter were subsequently fixed with 4% paraformaldehyde for 30 min and stained by hematoxylin solution (Beyotime, Shanghai, China) for 5 min. Cells in five random fields of each migration well were counted to determine the average number of migrated cells.
24-well plates were coated with 300 μL Matrigel (BD, San Jose, CA) and incubated at 37 °C for 20 min to allow the Matrigel to solidify. DU-145 cells were plated at a density of 1 × 105 cells/well and incubated with fucoidan (500 μg/mL) or vehicle (PBS) at 37 °C for 6 h. The cells were then photographed using a Zeiss digital camera. Tube formation was quantified by measuring the length of capillary structures using the software ImageJ (NIH, Bethesda, ML). Five randomly selected fields of view were photographed per well. The average value of the five fields was taken as the value for each sample.
Animals and xenograft model
Athymic nude mice (5-week-old) were obtained from Charles River Laboratories (Beijing, China). Animals were housed in a temperature-controlled room (22 °C) with 12 h light/dark cycling under pathogen-free conditions, and had free access to food and water. The experimental procedures were approved by Institutional Animal Care and Use Committee of Ningbo No.2 Hospital. All animals were randomly divided into two groups (n = 6), and treated with vehicle (saline) or fucoidan (20 mg/kg) by oral gavage for 28 days. Subconfluent DU-145 cells were harvested by trypsin/EDTA treatment and washed with cold PBS by centrifugation, then resuspended in PBS and kept on ice before used. Tumor cells (1 × 107 cells in 0.2 mL PBS) were injected subcutaneously into the mice. Tumor size was measured every four days by caliper, and tumor volume was calculated by the formula: 0.5 × (larger diameter) × (smaller diameter)2. At the end of experiment, the animals were sacrificed by CO2 euthanasia and the tumor tissues were harvested and weighted, then stored in −80 °C for further analysis.
Concentration of hemoglobin in tumor tissue was determined using a Hemoglobin Colorimetric Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions.
Trizol reagent (Takara, Dalian, China) was used for isolating total RNA of tumor tissue. 50–100 mg of tissue was directly lysed by adding 1 mL of Trizol reagent and homogenized using a homogenizer. Then 0.2 mL of chloroform was added, and the homogenized sample was incubated for 15 min at room temperature. Subsequently, RNA was precipitated by mixing with isopropyl alcohol. Total RNA yield was quantified by UV spectrophotometry measured at 260 nm. Then mRNA was isolated from total RNA by using Oligo (dT), and reverse transcribed into first-strand complement DNA (cDNA) and amplified using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara). A total volume of 25 μL reaction mixture contained 2 μL of cDNA, 12.5 μL of 2 × SYBR Green 1 Master Mix (Takara, Dalian, China), and 1 μL of each primer. The PCR condition was as follows: pre-incubation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, and annealing/extension at 60 °C for 30 s using iQ5 Real-Time PCR detection System (Bio-Rad, Hercules, CA). The primers used were as follows :
Tumor tissue was lysed with Protein Extraction Reagent (Beyotime), and protein concentration was determined by BCA reagent (Beyotime). About 20 μg of protein was separated in 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinyl difluoride (PVDF, Millipore) membrane. After blocking with TBST containing 5% milk for 1 h, the membrane was incubated with antibodies against JAK, p-JAK, STAT3, p-STAT3 and GAPDH (Cell Signaling, Danvers, MA) overnight at 4 °C. After incubation in horseradish peroxidase-conjugated secondary antibody for 1 h, the membrane was exposed to Immobilon solution (Millipore) for band detection.
Chromatin immunoprecipitation (ChIP)
An Agarose ChIP Kit (Pierce, Rockford, IL) was used to prepare nuclear extracts from tumor tissue homogenate and perform ChIP according to the manufacturer’s instructions. A ChIP-grade primary antibody against STAT3 was purchased from Cell Signaling. Immunoprecipitated DNA was purified with DNA Clean-Up Column (Qiagen, Hilden, Germany) and then quantitated by real-time PCR using PrimeScript RT-PCR Kit (TAKARA). The primers used were as follows :
Data were analyzed and graphed by Prism 6.0 (GraphPad Software, La Jolla, CA), and presented as Mean ± standard deviation (SD). Significance of difference between groups was analyzed by performing two-way RM analysis of variance (ANOVA) for time course study, or one-way ANOVA with Dunnett’s multiple comparison test or unpaired Student’s t test for other studies. P value no more than 0.05 was considered statistically significant.