Dried fruiting bodies of Inonotus 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 #4 filter paper to remove insoluble matter and freeze-dried. Polysaccharides were precipitated from resuspended extracts by ethanol precipitation via the addition of 75% (v/v) aqueous 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 (18,19).

Fetal bovine serum (FBS), penicillin G, streptomycin and RPMI-1640 media were obtained from Gibco (Grand Island, NY, 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) Ab, phospho-ERK Ab, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) Ab, phospho-SAPK/JNK Ab, p38 MAPK Ab, phospho-p38 MAPK Ab, AKT Ab, phospho-AKT Ab, COX-2 Ab, MMP-2 Ab, MMP-9 Ab, TIMP-2 Ab, and NF-κB p50 Ab were purchased from Cell Signaling Technology (Boston, MA, USA), Santa Cruz Biotechnology (Santa Cruz, CA, USA), or BD Bioscience (San Diego, CA, USA). All other chemicals used were of analytical grade.

Human A549 NSCLC cell line was obtained from the Korea Cell Line Bank (Seoul, Korea). A549 cells were cultured in RPMI-1640 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.

A549 cells were seeded into 12-well cell culture plates and incubated for 24 h. After various treatments, cell viability was evaluated using the MTT assay, which is based on the reduction of a tetrazolium salt by mitochondrial dehydrogenase in viable cells. After treatments, 500 μg/ml MTT solution was added to each well and incubated for 3 h at 37°C. The formazan crystals in each well were dissolved in isopropyl alcohol and absorbance was measured at 595 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Apoptotic index was determined 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, washed with phosphate-buffered saline (PBS) and centrifuged to collect the cell pellet. The number of cells was adjusted to 1×10⁶ cells/ml. Then, the cells were resuspended in binding buffer [10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂ (pH 7.4)] and stained with FITC-labeled Annexin V/PI at room temperature for 15 min in light-protected conditions. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton-Dickinson, Mountain View, CA, USA). The percentage of apoptotic cells was calculated using CellQuest software (Becton-Dickinson). Cells in the early phase of apoptosis were Annexin V-positive and PI-negative, whereas those in the late phase of apoptosis were positive for both Annexin V and PI. The apoptotic index (%) was calculated as the sum of cells in the early and late phases of apoptosis divided by the total number of events.

Wound healing assay was performed as previously described with some modifications (21). Briefly, A549 cells were cultured to confluence in 6-well cell culture plates for 24 h in serum-free medium. The medium was replaced with serum-containing medium followed by the addition of PFIO at various concentrations (25, 50 and 100 μg/ml) and the cell monolayers were disrupted by scraping them with a 100-μl micropipette tip. At the indicated time points (0, 24 and 48 h) after scraping, the cells were washed twice with PBS (pH 7.4) and photographed using an optical microscope at ×40 magnification.

The migration of A549 cells was also measured by chemotactic directional migration using a 6-well transwell insert. The 8-μm pore filters (Corning, NY, USA) were coated with gelatin (Sigma). A549 cells (1×10⁶ cells/ml) were seeded in the upper chambers with or without PFIO (50 and 100 μg/ml) and allowed to undergo migration for 24 h. Non-migrated cells in the upper chambers were then removed with a cotton swab. The filters were stained with 2% crystal violet. Migrated cells adhered to the underside of the filters were counted and photographed using an optical microscope at ×40 magnification. The invasion of A549 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, seeded in the upper chambers of the transwell inserts (1×10⁶ cells/ml), and incubated with or without PFIO (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 chambers 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 that penetrated through the Matrigel coating and present on the lower surface of the filters were counted and photographed using an optical microscope at ×40 magnification.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) substrate-embedded enzymography (zymography) analysis was used to identify enzyme activities with collagenase and gelatinase (22). Briefly, the supernatant collected from 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. They were then incubated at 37°C for 24–72 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 activity was evident as clear (unstained) regions against the dark background.

Treated 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 phenylmethylsulphonyl fluoride and 2 μg/ml pepstatin A] for 30 min on ice. The lysates were centrifuged at 15,600 rpm for 30 min at 4°C. The supernatant was collected and its protein content was measured using a Bio-Rad protein assay kit before analysis. The total or fractionated protein samples were loaded and separated using SDS-PAGE and transferred to nitrocellulose membranes (Immun-Blot NC membrane, 0.2 μm; Bio-Rad). Membranes were blocked with 1.5% skim milk in 1X Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h and incubated with primary antibodies at 4°C overnight. Finally, the membranes were treated with horseradish peroxidase -linked secondary antibodies for 1 h at room temperature. TBS washing was performed after each antibody binding reaction. The expression of each protein was detected 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 values, and results were obtained from at least three independent experiments performed in triplicates. All data were analyzed using Student’s t-test to evaluate significant differences. A p-value of <0.05 was considered statistically significant.

To investigate the cytotoxicity of PFIO, A549 cells were treated with PFIO 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 0–200 μg/ml of PFIO did not significantly affect A549 cell growth (Fig. 1A). In these experiments, cell viability was 82% at 200 μg/ml. Results of the FITC-labeled Annexin V and PI double-staining experiments revealed that PFIO at 100 μg/ml induced apoptosis in 7.1% of the cells (Fig. 1B and C), whereas concentrations ranging from 0 to 100 μg/ml of PFIO did not induce cell death and apoptosis in the highly metastatic A549 cell line. This concentration range was then applied in all subsequent experiments.

The effects of PFIO on A549 cell migration was determined using the wound-healing assay in which cells were stimulated to migrate by physical wounding. 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. A549 cells treated with 25, 50 or 100 μg/ml of PFIO displayed a reduced ability to migrate and fill the wounded area compared with untreated cells. The quantitative data in Fig. 2B revealed that PFIO could significantly inhibit the migration of A549 cells.

To further evaluate the anti-metastatic activity of PFIO, we assessed the inhibition of A549 migration and invasion by PFIO using transwell assay with polycarbonate filters (pore size, 8 μm) pre-coated with gelatin or Matrigel. Our results indicated that A549 cells were observed moving from the upper to the lower chamber in the absence of PFIO (control group), suggesting that A549 cells could migrate across a transwell insert precoated with gelatin. PFIO at 50 and 100 μg/ml significantly inhibited cell migration by 25 and 37%, respectively (Fig. 3). As shown in Fig. 4, results of the invasion assay illustrated that untreated A549 cells moved from the upper to the lower chamber, indicating the ability of A549 cells to invade through Matrigel-coated transwell cell culture chambers. However, the addition of PFIO to A549 cells resulted in an inhibitory effect on cellular invasion in a concentration-dependent manner. Data in Fig. 4B indicated that 50 and 100 μg/ml of PFIO significantly inhibited A549 cell invasion by 27 and 46%, respectively. Thus, these results suggested that PFIO effectively reduced cell migration and invasion.

ECM degradation is crucial to cellular invasion, indicating the inevitable involvement of matrix-degrading proteinases (23). Matrix degradation could, however, be suppressed by altering the balance between MMP-2 and TIMP-2 (24). Therefore, the effects of PFIO on protein expression and gelatinolytic activity of MMPs were investigated by gelatin-zymography and western blot analysis under serum starvation condition. As shown in Fig. 5A, MMP-2 and MMP-9 activity was remarkably decreased in A549 cells after PFIO treatment at 25, 50 and 100 μg/ml for 24 h compared to control. When MMP-2, MMP-9, and TIMP-2 protein expression was determined using western blot analysis, PFIO was found to reduce the expression levels of MMP-2 and MMP-9 but increase that of TIMP-2 in A549 cells (Fig. 5B). Therefore, our results indicated that PFIO could regulate the expression and activity of MMP-2 and MMP-9.

Previous reports have demonstrated that the MMP promoter contains several transcription factor-binding elements, including binding sites for AP-1 and NF-κB. Additionally, nuclear translocation of AP-1 and NF-κB in metastatic cancer cells involves the expression of MMPs (25,26). Therefore, NF-κB signal transduction pathway may play an important role in the regulation of MMP-2 and MMP-9 expression. Moreover, COX-2, which is regulated by NF-κB activation, also affects the expression of MMP-9 in highly metastatic cancer cells (27). To investigate whether PFIO could regulate the NF-κB signaling pathway, A549 cells were treated with the indicated concentrations of PFIO. The translocation of NF-κB and COX-2 expression levels were determined by western blot analysis. As shown in Fig. 6, PFIO treatment increased the total cytosolic NF-κB protein levels in A549 cells was compared to untreated control. In contrast, nuclear levels of NF-κB protein in A549 cells remarkably decreased after PFIO treatment compared to control. Moreover, PFIO suppressed the expression levels of COX-2. These results indicated that PFIO could regulate NF-κB nuclear translocation and COX-2 expression in A549 cancer cells.

Recent studies reported that MAPKs and PI3K/AKT signaling pathways are involved in cancer cell migration and invasion (25,28,29). MAPKs and AKT have been demonstrated to be involved in MMP induction in various tumor types (30–33). To examine whether PFIO could regulate MAPKs and AKT signaling pathways in A549 cells, we analyzed the phosphorylation levels of all three MAPKs (ERK, JNK and p38 MAPK) and AKT protein in A549 cells by western blot analysis after PFIO treatment (25, 50 and 100 μg/ml) for 24 h. As shown in Fig. 7, PFIO did not affect the expression levels of the three MAPKs and AKT, but it suppressed the phosphorylation of ERK, JNK, p38 MAPK, and AKT compared to controls. In particular, the phosphorylation levels of JNK, p38 MAPK, and AKT were inhibited by the addition of PFIO at 50 and 100 μg/ml. These data indicated that PFIO could inhibit the phosphorylation of MAPKs and AKT in A549 cells.