Dried fruiting bodies of I. obliquus were purchased from a local market and ground in a blender. Milled mushroom (20 g) was extracted with distilled water (600 ml) at 121°C for 2 h. Extracts were centrifuged at 5,000 rpm for 20 min, filtered through 0.45-μm Whatman filter paper (#4; Whatman, UK) to remove insoluble matter and then freeze-dried. Polysaccharides were precipitated from resuspended extracts using 75% ethanol, collected by filtration through 0.45-μm Whatman filter paper, resuspended, and dialyzed against distilled water for 5 days to remove low-molecular-weight compounds (21,22).

Fetal bovine serum (FBS), penicillin G, and streptomycin were obtained from Gibco (Grand Island, NY, USA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Lonza (Walkersville, MD, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and isopropyl alcohol were purchased from Sigma Chemical Co. (St. Louis, MO, USA). β-actin monoclonal antibody (mAb), extracellular signal-regulated kinase (ERK) MAPK Ab, phospho-ERK MAPK Ab, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) Ab, phospho-SAPK/JNK Ab, p38 MAPK Ab, phospho-p38 MAPK Ab, COX-2 Ab, MMP-2 Ab, MMP-9 Ab, and NF-κB p65 mAb were purchased from Cell Signaling Technology (Beverly, MA, USA), Santa Cruz Biotechnology (Santa Cruz, CA, USA), or BD Biosciences (San Jose, CA, USA), respectively. All other chemicals were of analytical grade.

The B16-F10 murine melanoma cell line was obtained from the Korea Cell Line Bank (Seoul, Korea). B16-F10 cells were cultured in DMEM supplemented with 10% heat inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C in a humidified 5% CO₂ incubator.

The effect of PIO on the viability of B16-F10 cells was measured using the MTT assay, which is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenase in viable cells. Cells were pre-incubated in 12-well plates for 24 h at 37°C humidified atmosphere of 5% CO₂ incubator. Then, cells were incubated with PIO (1–1,000 μg/ml) for 24 h. After incubation, cells were washed with 1X PBS to remove dead cells, and then 50 μl of MTT stock solution (2 mg/ml) was added to each well, which was then incubated for 2 h. After incubation, the MTT assay was performed to quantitate cellular viability. Finally, isopropyl alcohol was added to solubilize the formazan salt formed, and the amount of formazan salt was determined by measuring the absorbance at 595 nm using an enzyme-linked immunosorbent assay microplate reader.

The apoptotic death of tumor cells was examined using a fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI) apoptosis detection kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. Briefly, cells were harvested by trypsinization, washed with PBS and centrifuged to collect the cell pellet. The number of cells was adjusted to 1×10⁶ cells/ml. The cells were then resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂, at pH 7.4) and stained with FITC-labeled Annexin V and PI at room temperature for 15 min in the dark. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA, USA) within 1 h after supravital staining. FITC-labeled Annexin V was analyzed using excitation and emission settings of 488 and 535 nm, respectively. PI was analyzed using excitation and emission settings of 488 and 575 nm, respectively. For each flow cytometer run, 10,000 cells were required. The percentages of cells were calculated using the CellQuest software (Becton-Dickinson). The cells in the early stages of apoptosis were Annexin V-positive and PI-negative; however, the cells in the late stages of apoptosis were both Annexin V- and PI-positive. The apoptotic index (%) was calculated as the sum of late apoptotic cells divided for the total number of events.

The wound healing assay was performed as previously described with some modifications (23). Briefly, B16-F10 cells were grown to confluence in 6-well dishes for 24 h in serum-free medium (three dishes per group). The medium was replaced with serum-containing medium followed by the addition of PIO at various concentrations (0, 25, 50 and 100 μg/ml), and the cells in monolayer were disrupted (i.e., wounded) by scraping them with a 100-μl micropipette tip. At the indicated times (0, 24 and 48 h) after scraping, the cells were washed twice with PBS (pH 7.4). Finally, the cells were gently washed three times with PBS and photographed with an optical microscope at ×40.

The migration of B16-F10 cells was also measured by chemotactic directional migration using a 6-well Transwell insert. The 8-μm pore filters (Corning Incorporated, Corning, NY, USA) were coated with gelatin (Sigma) and B16-F10 cells (1×10⁶ cells/ml) were placed in the upper chamber with or without PIO (50 or 100 μg/ml) and allowed to undergo migration for 24 h. The non-migrated cells in the upper chamber were removed with a cotton swab. The filters were stained with 2% crystal violet. Migrated cells adherent to the underside of the filter were counted and photographed using an optical microscope at ×40. The invasion of B16-F10 cells was measured using Matrigel-coated Transwell cell culture chambers (8-μm pore size) as previously described. After the cells were cultured for 24 h in serum-free DMEM, they were collected, resuspended in serum-free medium, placed in the upper chamber of the Transwell insert (1×10⁶ cells/ml), and incubated with or without PIO (50 or 100 μg/ml). DMEM containing 10% FBS was placed in the lower chamber. All the cells in each treatment group were incubated for 24 h at 37°C in a humidified atmosphere with 95% air and 5% CO₂. The non-invasive cells that remained in the upper chamber were removed by wiping with a cotton swab, and the invasive cells were fixed with 4% formaldehyde in PBS and stained with 2% crystal violet in 2% ethanol. The invasive cells in the lower surface of the filter that penetrated through the Matrigel were counted and photographed using an optical microscope at ×40 (24).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) substrate-embedded enzymography (zymography) was used to identify collagenase and gelatinase activities (25,26). Briefly, the supernatant collected from the cell culture was resolved in 10% SDS-PAGE gels, which were prepared by the incorporation of gelatin (1 mg/ml) before casting. After electrophoresis, the gels were washed twice for 30 min in 2.5% Triton X-100 with shaking. The gels were then incubated at 37°C for 24–48 h in reaction buffer containing 50 mM Tris-HCl (pH 7.6), 10 mM CaCl₂, 150 mM NaCl, and 20% sodium azide, followed by staining with 0.25% Coomassie brilliant blue G-250 in 50% methanol and 10% acetic acid for 1–2 h. The completely stained gels were appropriately destained with 40% methanol and 10% acetic acid. The enzyme activities were evident as clear (unstained) regions against the dark background.

After treatment, the cells were washed in 1X PBS and lysed in lysis buffer [10 mM Tris-HCl (pH 7.5), 10 mM NaH₂PO₄/NaHPO₄ (pH 7.5), 130 mM NaCl, 1% Triton X-100, 10 mM NaPPi, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml pepstatin A] for 30 min on ice. Lysates were centrifuged at 12,000 × g for 20 min at 4°C. The supernatant was collected, and its protein content was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) before analysis. The cytosolic or nuclear protein samples were loaded at 10 μg of protein/lane, separated by SDS-PAGE in 10–15% gel, and transferred to NC membranes (Immun-Blot NC membrane, 0.2 μm; Bio-Rad Laboratories). Membranes were blocked with 1.5% skim milk in 1X Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h, and they were incubated with primary antibodies at 4°C overnight. Finally, the membranes were treated with horseradish peroxidase-coupled secondary antibodies for 1 h at 4°C. The membranes were washed with TBS after each antibody binding reaction. The detection of each protein was performed using an enhanced chemiluminescence kit (Millipore Co., Billerica, MA, USA).

Nuclear extracts were prepared by lysing nuclei in high-salt buffer supplemented with protease and phosphatase inhibitors using a nuclear extraction kit (Panomics Inc., Fremont, CA, USA) according to the manufacturer’s protocol. Protein concentrations were quantified using the Bio-Rad protein assay.

Data are expressed as mean ± standard error (SE) values, and the results were obtained from at least three independent experiments performed in triplicate. The data were analyzed using Student’s t-test to evaluate significant differences. A P<0.05 was regarded as statistically significant.

To investigate the cytotoxicity of PIO in B16-F10 melanoma cells, cells were treated with PIO at various concentrations ranging from 0 to 1,000 μg/ml for 24 h and cell viability was determined by the MTT assay. We found that PIO did not significantly affect the growth of B16-F10 cells (Fig. 1A). To evaluate whether the growth-inhibitory of PIO was associated with apoptosis, a double-staining method using FITC-labeled Annexin V and PI was performed. During the early stage of apoptosis, cells display phosphatidylserine on their outer cell membranes, which is readily detectable using Annexin V. During the later stages of apoptosis, as the plasma membrane becomes increasingly permeable, PI can move across the cell membrane to bind to cellular DNA. Double staining the cells with Annexin V and PI allowed us to detect apoptotic cells by flow cytometry. Low concentrations of PIO (25, 50 or 100 μg/ml) did not have an apoptotic effect on the cells (Fig. 1B and C). Therefore, these results demonstrated that PIO at concentrations ranging from 0 to 100 μg/ml did not induce cell death and apoptosis in the highly metastatic B16-F10 melanoma cell line. This concentration range was then applied in all subsequent experiments.

The effect of PIO on B16-F10 cell migration was determined using the wound-healing assay in which cells were stimulated to migrate by physical wounding cells. As shown in Fig. 2A, when cells were treated with PBS for 24 and 48 h, an apparent and gradual increase of cells in the denuded zone was observed under light microscopy. B16-F10 cells treated with 25, 50 and 100 μg/ml of PIO displayed a reduced ability to migrate and fill the wounded area compared with untreated cells. The quantitative data in Fig. 2B revealed that PIO significantly inhibited the migration of B16-F10 cells.

To further evaluate the anti-metastatic activity of PIO, we assessed the inhibition of B16-F10 melanoma migration and invasion by PIO using Transwell assay with polycarbonate filters (pore size, 8 μm) precoated with gelatin or Matrigel. The results indicated that B16-F10 cells moved from the upper chamber to the lower chamber in the absence of PIO (control group), suggesting that B16-F10 melanoma cells can migrate across a Transwell insert precoated with gelatin. PIO at 50 and 100 μg/ml significantly inhibited melanoma cell migration by 56 and 72%, respectively (Fig. 3). As shown in Fig. 4, the results of the invasion assay illustrated that untreated B16-F10 cells moved from the upper to the lower chamber, suggesting that B16-F10 cells can invade through Matrigel-coated Trans-well cell culture chambers. However, the addition of PIO to the B16-F10 cells resulted in inhibitory effects on cellular invasion in a concentration-dependent manner. Data in Fig. 4B indicate that 50 and 100 μg/ml PIO significantly inhibited invasion by 31 and 61%, respectively. Thus, these results suggested that PIO effectively reduced melanoma cell migration and invasion.

ECM degradation is crucial to cellular invasion, indicating the inevitable involvement of matrix-degrading proteinases (27). Therefore, the effect of PIO on MMP activities was investigated by gelatin-zymography under a condition of serum starvation to clarify the contribution of MMPs to the inhibitory effect of PIO on the invasive ability of cells. As shown in Fig. 5A, gelatin-zymography was used to analyze the effect of PIO on MMP-2 and MMP-9 activities in B16-F10 melanoma cells. MMP-2 and MMP-9 activities were markedly reduced by exposure to PIO at concentrations of 25, 50, and 100 μg/ml for 24 h. To further understand the effects of PIO on MMP-2 and MMP-9, western blot analysis was performed. As shown in Fig. 5B, PIO inhibited the expression of MMP-2 and MMP-9 in B16-F10 melanoma cells. Therefore, these results indicated that PIO regulated the expression and activities of MMP-2 and MMP-9.

Activation of NF-κB in metastatic cancer cells is involved in the expression of MMP-2 and MMP-9 (28). COX-2 also affects the expression of MMP-2 and MMP-9 in highly metastatic cancer cells (13). To investigate whether PIO could regulate the NF-κB signaling pathway in B16-F10 melanoma cells, cells were treated with the indicated concentrations of PIO. The translocation of NF-κB and COX-2 expression levels were determined by western blot analysis. As shown in Fig. 6, the total cytosolic NF-κB protein levels in B16-F10 cells was increased by PIO treatment compared with that in untreated cells. By contrast, the protein levels of NF-κB in the nucleus of B16-F10 cells markedly decreased after PIO treatment compared with the control levels. Moreover, we found that the expression levels of COX-2 in PIO-treated B16-F10 cells were markedly lower than those of the untreated control. These data indicated that PIO inhibited NF-κB activation and COX-2 expression in melanoma cancer cells.

Recent studies reported that MAPK and PI3K/Akt signaling pathways are involved in cancer cell migration and invasion (29). MAPKs and Akt have been demonstrated to be involved in MMP induction in various tumor types (30). To examine whether PIO could regulate MAPK and Akt signaling pathways in B16-F10 melanoma cells, we analyzed the phosphorylation levels of all three MAPKs (ERK, JNK and p38 MAPK) and Akt by western blot analysis using respective anti-phospho MAPK and Akt mAbs in B16-F10 cells after treatment with PIO (25, 50 and 100 μg/ml) for 24 h. As shown in Fig. 7, PIO did not affect the expression levels of all three MAPKs and Akt, but PIO suppressed the phosphorylation levels of ERK, JNK, p38 MAPK and Akt in comparison with the findings in the untreated control. In particular, the phosphorylation levels of JNK, p38 MAPK and Akt were highly inhibited by the addition of PIO at concentrations of 50 and 100 μg/ml. These data indicated that PIO inhibited the phosphorylation of MAPKs and Akt in melanoma cells.