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A number of polysaccharides exhibit pharmacological activities. Polysaccharides derived from
Cancer is an important cause of human mortality worldwide, and numerous cancer treatments, including cancer chemotherapeutic agents, are known to result in adverse side effects (
Our previous studies have demonstrated that polysaccharides from the
Streptomycin, fetal bovine serum (FBS), and penicillin G and were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Dulbecco's modified Eagle's medium (DMEM) was obtained from Lonza Group, Ltd. (Basel, Switzerland). Isopropyl alcohol and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich. (St. Louis, MO, USA). Antibodies against extracellular signal-regulated kinase (ERK; 1:1,000 dilution; rabbit monoclonal antibody; cat. no. 4695), phosphorylated (p)-ERK (1:1,000 dilution; Thr202/Tyr204 rabbit polyclonal antibody; cat. no. 9101S), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK; 1:1,000 dilution; rabbit polyclonal antibody; cat. no. 9252), p-SAPK/JNK (1:1,000 dilution; Thr183/Tyr185 mouse monoclonal antibody; cat. no. 9255S), p38 MAPK (1:1,000 dilution; rabbit polyclonal antibody; cat. no. 9212), p-p38MAPK (1:1,000 dilution; Thr180/Tyr182 rabbit monoclonal antibody; cat. no. 4631S) and NF-κB p65 (1:1,000 dilution; rabbit polyclonal antibody; cat. no. 3034), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:2,000 dilution; cat. no. 7074) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). MMP-2 (1:1,000 dilution; rabbit polyclonal antibody; cat. no. 4022), MMP-7 (1:1,000 dilution; rabbit monoclonal antibody; cat. no. 3801), MMP-9 (1:1,000 dilution; rabbit polyclonal antibody; cat. no. 3852), β-actin (1:1,000 dilution; mouse monoclonal antibody; cat. no. sc-47778), and HRP-conjugated goat anti-mouse IgG (1:2,000 dilution; cat. no. sc-2005) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) or BD Biosciences (Franklin Lakes, NJ, USA). All other chemicals were of analytical grade.
Exopolysaccharide from
The B16-F10 murine melanoma cell line was obtained from the Korean Cell Line Bank (Seoul, South Korea). Cells were grown in complete DMEM medium supplemented with 10% heat-inactivated FBS, 100 µg/ml streptomycin, and 100 U/ml penicillin. Cells were maintained at 37°C in a humidified 5% CO2 incubator.
Cell viability was assessed using the MTT colorimetric assay, as previously described (
A fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) apoptosis detection kit (Molecular Probes; Thermo Fisher Scientific, Inc.) was used to determine the level of apoptosis in tumor cells, according to the manufacturer's protocols. Briefly, cells were harvested using trypsin/EDTA solution, washed with PBS, and centrifuged at 600 × g for 5 min at room temperature to pellet the cells. Cell concentration was adjusted to 1×106 cells/ml and the cells were resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, at pH 7.4) prior to staining with FITC-labeled Annexin V and PI for 15 min at room temperature in light-protected conditions. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (BD Biosciences) within 1 h after staining. The percentage of apoptotic cells was calculated using the CellQuest software program (version 4.0.4; BD Biosciences). The apoptotic cell rate was calculated as the sum of cells in the early and late phase of apoptosis divided by the total number of events recorded by the flow cytometer.
Six-well chambers with polycarbonate filters with a pore size of 8.0 µm were used to perform the migration assays. The filters (Corning Incorporated, Corning, NY, USA) were coated with gelatin (Sigma-Aldrich). The cells were seeded to the upper part of the chamber at a density of 1×106 cells/ml with or without PLIO (50 or 100 µg/ml). In the lower chamber, DMEM containing 10% FBS served as a source of chemoattractants. Following incubtion for 24 h, cells that had migrated through the gelatin were stained with 2% crystal violet. The non-migrated cells in the upper chamber were removed with a cotton swab. Images of the migrated cells were captured and the cells were counted under a light microscope (magnification, ×40). Cell invasion assays were performed using a Matrigel-coated Transwell chamber. The cells (1×106 cells/ml) were seeded to the upper chamber of the Transwell insert with or without PLIO (50 or 100 µg/ml) in serum-free medium. In the lower chamber, DMEM medium containing 10% FBS was used as a source of chemoattractants. Following incubation, cells that had invaded through the Matrigel were fixed with 4% formaldehyde in PBS, stained with 2% crystal violet, images were captured and cells were counted under a light microscope (magnification, ×40) (
Following PLIO treatment (25, 50 or 100 µg/ml), the cells were rinsed with PBS twice and were lysed in lysis buffer [10 mM NaH2PO4/NaHPO4 (pH 7.5), 10 mM Tris-HCl (pH 7.5), 1% Triton X-100, 130 mM NaCl, 10 mM NaPPi, 2 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride] on ice for 30 min. The cell lysates were centrifuged of 12,000 × g for 20 min at 4°C to remove cell debris and the supernatant was collected. Nuclear extracts were prepared using a nuclear extraction kit (Panomics Inc., Fremont, CA, USA) according to the manufacturer's protocol. Protein content was determined using a Bio-Rad Protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Equal quantities of nuclear and cytosolic protein samples (50 µg per lane) were loaded on 10–15% SDS-PAGE for separation, and transferred onto 0.2 mm Immun-Blot nitrocellulose membranes (Bio-Rad Laboratories, Inc.) by electroblotting. The blot was blocked with 1.5% non-fat milk in 1X Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h, followed by incubation with the specific primary antibodies at 4°C overnight. The blot was finally incubated with HRP-conjugated secondary antibodies. The membranes were washed with TBS-T after each antibody binding reaction. Detection of protein-antibody complexes was conducted using an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA) followed by exposure to X-ray film.
All measurements were from at least three independent experiments and all results are presented as the mean ± standard error of the mean. The data were analyzed using Student's t-test or nonparametric analysis of variance Duncan's multiple range tests were performed to compare multiple groups when appropriate. P<0.05 was considered to indicate a statistically significant difference.
The effect of PLIO on the viability of murine melanoma cell B16-F10 was determined using the MTT assay. Various concentrations (0–1,000 µg/ml) of PLIO were added to the cells followed by incubation for 24 h. Cell viability was determined to be 89% at 200 µg/ml PLIO compared with the control (
To determine whether PLIO induces cellular apoptosis in melanoma cells, FITC-labeled Annexin V and PI nucleic acid binding dye were used. Following staining of the cell population with the double staining method, apoptotic cells exhibit green fluorescence, dead cells exhibit red and green fluorescence, and live cells exhbit little or no fluorescence (
To investigate whether PLIO had
ECM degradation, which is key in cellular invasion, involves matrix-degrading proteinases, including MMPs. To determine whether PLIO suppressed MMP protein expression levels, western blotting was used. PLIO treatment decreased the expression of MMP-2 and MMP-9 in B16-F10 cells. Particularly, PLIO was observed to reduce the expression levels of MMP-7 (
To investigate whether PLIO inhibits the activation of the NF-κB signaling pathway in melanoma cells, the current investigated the effects of PLIO on translocation of the NF-κB protein from the cytoplasm to the nucleus. Western blotting was used to determine the levels of NF-κB translocation. Cytosolic protein levels of NF-κB in B16-F10 cells were higher in PLIO-treated cells than those in untreated cells (
A previous study demonstrated that the activation of the PI3K/AKT and MAPK signaling pathways promotes cancer cell invasion and migration (
Research has recently focused on the anti-tumor properties of natural components for their potential chemotherapeutic applications. Polysaccharides are often associated with notable pharmacological activities. For example, polysaccharides extracted from mushrooms, including
In conclusion, inhibition of metastasis is a key issue in cancer research. PLIO may inhibit the invasion of highly invasive melanoma cells by inhibiting MMPs expression via downregulation of the NF-κB, AKT, and/or MAPK signaling pathways. Based on these findings, the exact underlying anti-metastatic mechanism of PLIO is remains unclear, however, it is concluded that PLIO exhibits potent anti-metastatic effects.
The present study was supported by a grant from the National Institute of Biological Resources, funded by the Ministry of Environment of the Republic of Korea (grant no. NIBR201528101).
Effect of PLIO on viability of B16-F10 cells. Cells were treated with the indicated concentrations of PLIO for 24 h. Cell viability was measured by the MTT assay. The results are presented as the mean ± standard error of the mean. PLIO, polysaccharides derived from
Effects of PLIO on apoptosis of B16-F10 cells. Cells were treated with the indicated concentrations of PLIO for 24 h. (A) Percentage of apoptotic cells after the treatment of PLIO in B16-F10 cells were measured by the Annexin V/PI flow cytometric analysis. (B) Apoptotic index (%) was calculated as the sum of early and late apoptotic cells divided by the total number of events. Con, control; PI, propidium iodide; FITC, fluorescein isothiocyanate; PLIO, polysaccharides derived from
Effects of PLIO on the migration of B16-F10 cells. Cells were treated with the indicated concentrations of PLIO for 24 h. (A) Cell migration assays were performed using gelatin-coated Transwell chambers. (B) Cells that migrated through the gelatin were stained with 2% crystal violet, images were captured and the cells were counted under a light microscope (magnification, ×40). PLIO, polysaccharides derived from
Effect of PLIO on the invasiveness of B16-F10 cells. Cells were treated with the indicated concentrations of PLIO for 24 h. (A) Cell invasion assays were performed using Matrigel-coated Transwell chambers. (B) Cells that invaded through the Matrigel were stained with 2% crystal violet, images were captuted and cells were counted under a light microscope (magnification, ×40). *P<0.05 vs. the control group. PLIO, polysaccharides derived from
Effect of PLIO on the expression of MMPs in B16-F10 cells. Cells were treated with the indicated concentrations of PLIO in serum-free medium for 24 h. Western blot analysis was used to determine the expression levels of MMP-2, MMP-7, and MMP-9. PLIO, polysaccharides derived from
Effect of PLIO on NF-κB translocation in B16-F10 cells. Cells were treated with the indicated concentrations of PLIO for 24 h. The nuclear (N) and cytosolic (C) fractions from the PLIO-treated cells were isolated and analyzed for the expression of NF-κB using western blot analysis. PLIO, polysaccharides derived from
Effect of PLIO on AKT and MAPK pathways in B16-F10 cells. Cells were treated with the indicated concentrations of PLIO for 24 h. The expression and phosphorylation levels of ERK, JNK, p38 MAPK, and AKT were assessed by western blot analysis. PLIO, polysaccharides derived from