Background
Prostate cancer is the most common cancer in men and the second leading cause of death from cancer in men in the United States [
1,
2]. Many risk factors, such as genetic, dietary, medication exposure, infectious disease and sexual factors, can lead to the development of prostate cancer [
3]. The therapy for prostate cancer usually involves a combination of surgery, chemotherapy and radiotherapy, however, the adverse effect is obvious [
4,
5]. On the contrary, bioactive ingredients extracted from food resources can provide complementary and alternative strategies to treat prostate cancer [
6].
Angiogenesis is the physiological or pathological process through which new blood vessels form from pre-existing vessels [
7]. Angiogenesis does not initiate malignancy but promotes tumor progression and metastasis, therefore, intensive efforts have been undertaken to develop therapeutic strategies to inhibit angiogenesis in cancer over the past decades [
7]. Signal transducers and activators of transcription 3 (STAT3) is a member of the STAT protein family. In response to cytokines and growth factors, STAT3 is phosphorylated by receptor-associated Janus kinases (JAK), then form homo or heterodimers, and translocate to the cell nucleus where they act as transcription activators [
8,
9]. The abnormal activation of STAT3 can cause unrestricted cell proliferation, malignant transformation and tumor angiogenesis [
8,
10]. Activation of STAT3 signaling is essential in the metastatic progression of prostate cancer, and targeting STAT3 pathway can yield a potential therapeutic intervention for prostate cancer [
11‐
13].
Fucoidan is a sulfated polysaccharide obtained mainly in various species of brown algae and brown seaweed such as
Undaria pinnatifida,
Laminaria angustata,
Fucus vesiculosus, and
Fucus evanescens [
14,
15]. It is reported that fucoidan has anti-tumor activity on lung, breast, liver, colon, prostate and bladder cancer cells [
16]. Compared to medications, fucoidan is food-grade ingredient which can provide complementary and alternative strategies without intolerable side effects [
17,
18]. In previous study, fucoidan induced the apoptosis of PC-3 human prostate cancer cells in vitro, but the possible role in vivo was still unknown [
19]. Therefore, here we investigated anti-tumor and anti-angiogenic effects of fucoidan in both cell-based assays and mouse xenograft model, as well as tried to clarify a role of JAK-STAT3 pathway in the protection.
Methods
Reagents
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.
Cell culture
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.
Cell migration
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.
Hemoglobin assay
Concentration of hemoglobin in tumor tissue was determined using a Hemoglobin Colorimetric Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions.
Real-time PCR
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 [
20]:
Western blot
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 [
21]:
Statistical analysis
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.
Discussion
In the past decades, many therapies, such as androgen-ablation therapy, prostatectomy, radiation therapy and cytotoxic chemotherapy, were developed to treat prostate cancer, but the subsequent adverse effects are also obvious [
22,
23]. As a food-grade ingredient, fucoidan is extracted from marine plant. Previous clinical studies showed that long-term intake of fucoidan was safe in both healthy people and cancer patients [
17,
18,
24]. In our study, we proved anti-tumor activity of fucoidan in both cell-based assays and mouse xenograft model, shedding new light for complementary and alternative therapy of prostate cancer.
Targeting angiogenesis is a new direction of cancer therapy [
25]. Angiogenesis involves several sequential phases, in which sprout formation is initiated with the release of proteolytic enzymes from endothelial cells to degrade surrounding basement membrane, followed by cell proliferation and migration, finally the migrating cells form tube-like structures [
26,
27]. In previous study, fucoidan was reported to Inhibit migration and invasion of A549 human lung cancer cell and tube formation of Hela cells in vitro [
28,
29]. Furthermore, fucoidan reduced microvessel density and expression of VEGF in mice xenograft of 4 T1 mammary carcinoma cells [
30]. Although Boo reported inhibitory effect of fucoidan on viability of PC-3 human prostate cancer cells, whether anti-angiogenic mechanism was involved was still unknown [
19]. Here, we first reported inhibitory effects of fucoidan on proliferation, migration and tube formation of DU-145 prostate cancer cells, more importantly, we disclosed anti-angiogenic effects of fucoidan using a mouse xenograft model, in which hemoglobin assay and CD31 analysis directly proved fucoidan reduced vascular density in the tumor.
STAT3 is a candidate molecular target in angiogenesis-mediated therapy [
31]. VEGF expression correlates positively with STAT3 activity in diverse human cancer cell lines [
10]. An activated STAT3 mutant could up-regulate VEGF expression and stimulates tumor angiogenesis [
10]. On the contrary, targeting STAT3 could block expression of VEGF induced by multiple oncogenic growth signaling pathways, and then inhibit tumor angiogenesis [
32]. In this study, we also found reduction of STAT3 phosphorylation in tumor tissue, in which angiogenesis was inhibited by fucoidan.
As a transcription factor, STAT3 is phosphorylated to form dimers and then translocate to nucleus, where the dimers directly regulate the expression of genes responsible for survival (
Bcl-xL,
Survivin,
p53), proliferation (
Myc,
Cyclin D1/2) and angiogenesis (
VEGF,
HIF) [
31]. In this study, using ChIP, we disclosed reduced activation of
VEGF,
Cyclin D1,
Bcl-xL promoters after fucoidan treatment, suggesting expression inhibition of these genes. VEGF is a vital regulator in angiogenesis and it is mainly secreted by tumor cells and targets VEGF receptor on endothelial cells to promote angiogenesis [
33]. VEGF-mediated autocrine loop in endothelial cells is also an essential component of solid tumor angiogenesis [
34]. Cyclin D1 is a protein required for progression through the G1 phase of the cell cycle [
35]. Overexpression of cyclin D1 contributes to malignant properties of tumor cells by increasing VEGF production and decreasing Fas expression [
36]. Bcl-xL, one member of Bcl-2 family, acts as an anti-apoptotic protein by preventing the release of mitochondrial cytochrome c to cytoplasm, which leads to caspase activation and programmed cell death [
37]. Therefore, expression inhibition of
VEGF,
Cyclin D1 and
Bcl-xL could prevent angiogenesis and promote apoptosis to hinder tumor growth.
Conclusions
Taken together, we first disclosed anti-tumor and anti-angiogenic effects of fucoidan, a food-grade ingredient, on prostate cancer in both cell-based assays and mouse xenograft model, as well as clarified a role of JAK-STAT3 pathway in the protection. All these findings provided novel complementary and alternative strategies to treat prostate cancer.