Background
Colorectal cancer (CRC) is the third most prevalent form of cancer in men and women, with a 5-year survival rate of 63%, decreasing to 10% in patients with metastatic disease [
1]. Thus, the formation of distant metastasis is the decisive and most lethal event during the course of the disease. Although recent advances in chemotherapeutic agents in CRC have been achieved, treatment options are still limited and are associated with significant morbidity and mortality.
Mushroom polysaccharides are widely being used as nonspecific immunostimulants for cancer patients in Asian countries. The Polysaccharide isolated from
Phellinus linteus (PL) is an immunomudulatory agent with a molecular weight of 153 kDa [
2]. PL stimulates the proliferation of T lymphocytes and activates B cells [
3], induces secretory and celluar macrophage response [
4], and inhibits tumor growth and metastasis through the immunopotentiation [
5]. It had been suggested that antitumor effect are not only immunomodulatory, but may result from a direct action on tumor cells.
We previously demonstrated that PL has an antiproliferative effect for SW480 colon cancer cells and the growth inhibition is mediated by induction of apoptosis and G
2/M cell cycle arrest which are associated with decrease in Bcl-2, increase of the release of cytochrome
c, and reduced expression of cyclin B1 [
6]. Since our report, the direct antitumor effect of PL has been demonstrated by others: the inhibition of pulmonary metastasis of melanoma cells through the downregulation of mRNA level of urokinase plasminogen activator (uPA) [
7], suppressed proliferation of lung cancer cells by the inhibition of cyclin-dependent kinases cdk2, 4, and 6, and induced apoptosis through the activation of caspase 3 [
8], apoptosis of prostate cancer cells [
9,
10], and inhibition of tumor growth and invasive behavior of breast cancer cells mediated by cell cycle arrest at S phase and inhibition of serine-threonine kinase AKT signallig [
11].
One important signaling pathway involved in the etiology of colon cancer is Wnt/β-catenin, and more than 90% of colon cancers bear mutations that result in the activation of this pathway [
12]. Activating mutations in genes of the Wnt/β-catenin signaling pathway are observed early in the development of colon cancers. Mutations that activate the Wnt/β-catenin pathway generally affect β-catenin phosphorylation and stability [
13]. Phosphorylated β-catenin is degraded via the ubiquitin pathway. In the absence of efficient degradation such as genetic mutations of adenomatous polyposis coli (APC) or β-catenin, β-catenin accumulates and is transported to the nucleus, where it acts as a transcription factor in concert with T-cell factor/lymphocyte enhancer binding factor (TCF/LEF) [
14,
15]. The resulting β-catein-TCF/LEF complex activates TCF target genes which affect cell proliferation, survival, angiogenesis, invasion and metastasis [
16]. Recent evidences suggested that although mutations of components of the Wnt/β-catenin pathway generally occur early in colon cancer progression, accumulation of β-catenin in the nucleus has been associated with late stages of tumor progression and the development of metastasis [
17‐
19].
In the present study, we have investigated the effects of a PL treatment on multiple steps involved in colon cancer growth, invasion and neoangiogenesis. Herein, we show that PL inhibits proliferation, motility and invasion as well as matrix metalloproteinases (MMPs) and tumor neoangiogenesis of SW480 colon cancer cells in vitro and in vivo. Furthermore, we demonstrated that PL significantly inhibited Wnt/β-catenin signaling pathway.
Our data suggest that the PL-induced down-regulation of Wnt/β-catenin signaling may contribute to the inhibition of tumor growth, invasion and angiogenesis of SW480 colon cancer cells.
Methods
Cell lines and culture conditions
SW480 human colon cancer cells and HT1080 fibro-sarcoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD). The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL). Cultures were maintained at 37°C in a humidified 5% CO
2 atmosphere. PL (Hankook Sin Yak Pharm., Nonsan, Korea) was dissolved in DMEM and adjusted to the indicated final concentrations with culture medium before use. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords obtained by caesarean section by a modification of the technique previously described [
20]. HUVECs were cultured in gelatin-coated plates with Endothelial Basal Medium-2 (EBM-2) supplemented with 10 mL FBS, 0.2 mL hydrocortisone, 2 mL human fibroblast growth factor-basic (hFGF-B), 0.5 mL vascular endothelial growth factor (VEGF), 0.5 mL R
3-IGF-1, 0.5 mL ascorbic acid, 0.5 mL human epidermal growth factor (hEGF), 0.5 mL GA-1000, 0.5 mL heparin (EBM-2 Bullet kit, Clonetics) and incubated at 37°C in a humidified incubator containing 5% CO
2. HUVECs were used at passages 2-5.
Cell proliferation assay
The effect of PL on the growth of colon cancer cells was evaluated using 5 × 10
3 cells seeded onto 96-well plates (Corning, NY), which were treated with PL simultaneously at the time of cell plating. To evaluate the effect of PL at concentrations 125, 250, 500, and 1,000 μg/mL, cells were maintained in media with various concentrations of PL for up to 96 h and cell numbers were determined by a tetrazolium-based colorimetric assay (MTT, Sigma, St. Louis, MO) [
21], and absorbance was read at 570 nm.
Preparation of cell lysates and western blot analysis
Proteins were extracted with RIPA buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 5 mM ehylenediaminetetraacetic acid [EDTA], 1% Nonidet P-40, 0.1% sodium dodecyl sulphate [SDS], and 1% sodium deoxycholate) containing protease inhibitor cocktail tablets (Roche Diagnostics Indianapolis, IN). Samples were resolved through a 10% SDS-polyacrylamide gel and transferred to Hybond ECL membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were blocked in 1× TBS (1 L of 10× TBS was prepared by mixing 24.2 g of Trizma base and 80 g NaCl, and the pH was adjusted to 7.6), containing 0.1% Tween 20 with 5% non-fat skim milk, for 1 h at room temperature and incubated with a primary antibody for 1 h at room temperature. After 3 washes (5 min each) in TBST (TBS containing 0.1% Tween 20), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After 3 washes (5 min each) in TBST, proteins were visualized using the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Immuno-cytochemical analysis
Cells were grown to 60% confluence on 12-well chamber slides. The cells were treated with PL for 24 h before simultaneous paraformaldehyde fixation and permeabilisation with Triton X-100. After blocking with 1% bovine serum albumin (BSA), the cells were incubated with anti-β-catenin antibody (Santa Cruz Biotechnolgy, CA) overnight at 4°C. The cells were labeled with Alexa Fluor 488-conjugated anti-mouse secondary antibody (Molecular Probes, Eugene, Oregon, USA). The slides were covered with a mounting solution (Dako, Carpinteria, CA) and photos were obtained using an LSM5 confocal microscope (Carl Zeiss, Inc., Jena, Germany).
Luciferase reporter activity
Cells were seeded and allowed to achieve 80% confluence in 6-well plates. The cells were transiently transfected with 1 μg per well of TCF/LEF-Luc by using Lipofectamine Plus transfection reagents (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions. After transfection, the cells were treated with various concentrations of PL for 24 h. Cell lysates were prepared using 1× reporter lyses buffer (Promega, Madison, WI). Luciferase activity was measured as previously described, by using an AutoLumat LB953 Luminometer (Berthold, Stevenage, UK) and using the luciferase assay system from Promega [
22,
23]. The relative luciferase activity was calculated after normalization of cellular proteins. All values are expressed as the percentage of activity relative to basal activity.
In vitroinvasion and motility assay
Transwell culture chambers containing polycarbonate filters of diameter 6.5 mm and pore size 8 μm (Costar, Cambridge, MA) were used for the assay according to a previously described method [
24]. For the invasion assay, filters coated with Matrigel (160 μg per filter) were used. To investigate the effect of PL on invasion, we added various concentrations of PL to the media. After 72 h of incubation, cells on the top of the filter, which was generated by non-invasive cells, were removed using cotton swabs. The filters were removed, and the invasive cells beneath the filters were stained with Gill's hematoxylin and counted under a microscope. For the motility assay, the same system was used, but the Matrigel coating was omitted.
Protease analysis by substrate-embedded gel
For zymography analyses [
25], cells grown to 80% confluence were washed 3 times with calcium-magnesium-free phosphate-buffered saline (PBS) and cultured in DMEM without FBS. Conditioned media (CM) were obtained after 24 h of culture and centrifuged at 3,000 ×
g for 10 min to remove cells and debris. Cell-free CM was concentrated approximately 10-fold by using a Centricon-10 device (Amicon, Beverly, MA), and aliquots of the concentrated CM were normalized for cell number. Proteins in the normalized CM were then separated by electrophoresis on 10% polyacrylamide gels impregnated with 1 mg/mL gelatin (Fisher Chemical Co., Fair Lawn, NJ) or 1 mg/mL casein (Sigma Chemical Co.) containing 13 μg/mL plasminogen (Sigma Chemical Co.) under non-reducing conditions. After electrophoresis, the gels were washed twice in 2.5% Triton X-100 for 30 min, proteolysed with a reaction buffer (50 mM Tris-HCl, 5 mM CaCl
2, and 0.02% NaN
3 [pH 8.0]) for 72 h at 37°C, and stained with Coomassie Brilliant Blue G-250. To investigate the effect of PL on proteases, we added various concentrations of PL to the incubation buffer.
HUVEC proliferation and capillary tube formation on matrigel
Growth assays of HUVECs were carried out according to the procedure described by Bae et al. [
24], with some modifications. Briefly, HUVECs were seeded in 0.2% gelatin-coated wells in a 96-well culture plate (Corning, NY) at an initial density of 5 × 10
3 cells/well in 200 μL of EBM-2 and then grown under standard conditions at 37°C in 5% CO
2. On the next day, 125, 250, 500, and 1,000 μg/mL of PL were added to each well for 3 d, and the number of viable cells was measured using the MTT assay. To further assess the anti-angiogenic effect of PL, we performed vascular tube formation experiments. HUVECs were seeded at a density of 10
4 cells/well in Matrigel-coated 24-well plates and incubated at final PL concentrations of 250, 500, and 1,000 μg/mL. During these incubations, HUVEC morphological changes were monitored using an inverted phase-contrast microscope (Model IX 70; Olympus, Tokyo, Japan) and photographs were obtained.
Nude mice tumourigenicity
All animal-related procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chungnam National University. Confluent colon cancer cell cultures were harvested by brief trypsinization, washed 3 times with calcium and magnesium-free PBS, and re-suspended at a final concentration of 5 × 10
7 cells/mL in serum-free DMEM. Single-cell suspensions were confirmed by phase-contrast microscopy, and cell viability was determined using trypan blue exclusion; only single-cell suspensions with a viability > 90% were used. Pathogen-free female BALB/cAnNCrj-nu athymic nude mice (4 weeks old; Charles River Laboratories, Kanazawa, Japan) were anesthetized with diethyl ether by inhalation, and 5 × 10
6 SW480 cells in serum-free DMEM were inoculated subcutaneously (s.c.) into the right flank. From the day of tumor cell inoculation, the mice received a daily intratumoral injection (i.t.) of PL (100 μg/100 μL of saline) or intravenous injection (i.v.) of PL (25, 50, 100 μg/100 μL of saline) as well as the same amount of physiologic saline as a control for 14 d. The mice were regularly examined, tumor sizes were measured with a caliper, and tumor volumes were determined using the following formula: volume = 0.5 × (width)
2 × length [
26]. Each experimental group consisted of 8 animals.
Immuno-histochemical analysis
The mice were euthanized and tumors were removed and bisected. One part of the tumor was placed in neutral buffered formalin for paraffin block preparation, and the other part was frozen for preparation of cryo-cut sections. The degree of apoptosis was evaluated using an ApopTag apoptosis detection kit (S7101; Intergen, Norcross, GA), according to the manufacturer's recommendations. The apoptotic index was calculated as the percentage of nuclei stained by peroxidase, showing nuclear halo or apoptotic bodies. Positive cells were quantified by counting the number of brown-stained nuclei/total number of cells in 5 randomly selected 400× magnified fields.
To evaluate the proliferation index, the paraffin sections were incubated with a monoclonal mouse Ki-67 antibody (MIB-1; Dako, Carpinteria, CA) at a dilution of 1:100. Staining was carried out with a universal labeled streptavidin-biotin kit (Dako, Carpinteria, CA), according to the standard protocol. The proliferation index was determined by counting stained cells at 400×. To immuno-localize tumor blood vessels, the cryo-sections were stained with a monoclonal rat anti-mouse CD31 antibody (PECAM-1; BD PharMingen, San Diego, CA) at a dilution of 1:50. The antigen-antibody reaction was visualized using an anti-mouse immunoglobulin horseradish peroxidase detection kit (BD PharMingen, San Diego, CA), according to the manufacturer's recommendations. Vessel density was determined by counting the Positive cells were quantified by counting the standard vessels in 5 randomly selected 200× magnified fields.
To confirm tissue β-catenin levels, paraffin sections were de-parafinised in xylene and dehydrated in serially diluted ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0). The sections were blocked with a protein blocker (Dako, Carpinteria, CA) and stained with an anti-β-catenin antibody (Santa Cruz Biotechnology, CA). The sections were then stained with hematoxylin and photographed under a light microscope. Positive cells were quantified by counting the number of brown-stained cells/total number of cells in 5 randomly selected 400× magnified fields
Statistical analysis
Data are expressed as mean and standard deviation (SD), and significance was established by unpaired Student's t test. For all analyses, the level of statistical significance was more than the 95% confidence level (P < 0.05). *, **, or *** indicates P < 0.05, P < 0.01, or P < 0.001, respectively.
Conclusions
In conclusion, the present results show that PL isolated from Phellinus linteus causes a significant reduction in β-catenin protein levels and the down-regulation of certain downstream genes in the Wnt/β-catenin pathway in SW480 colon cancer cells in vitro and in vivo. In addition, we showed that PL significantly reduces invasiveness of SW480 cells through a direct effect on the activity of cellular MMPs, motility, and angiogenesis, which are strongly associated with Wnt/β-catenin signaling. The present data suggest that PL can be developed as an effective therapeutic agent for patients with colon cancer through its effect on the inhibition of multiple steps involved in colon cancer growth, invasion, and neo-angiogenesis by suppression of Wnt/β-catenin signaling.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KSS, GL and JSK carried out the molecular studies and drafted the manuscript. KPJ helped to prepare the manuscript. TDK, JPK, SBS and JKY participated in the immunoassays and performed the statistical analysis. HDP, BDH, KL and WHY conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.