The A549 human lung cancer cells and WI-38 normal human fetal lung fibroblast cells were obtained from the Korea Cell Line Bank (Seoul, Korea). RPMI 1640 medium was purchased from GE Healthcare Life Sciences Hyclone Laboratories (Logan, UT, USA). Fetal bovine serum (FBS) and antibiotics (streptomycin/penicillin) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). 5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Materials and chemicals used for electrophoresis were obtained from Bio-Rad Laboratories, Inc, (Hercules, CA, USA). FCP was provided by Animal Bio-Resources Bank (Jinju, Korea). Antibodies targeting CFL1 (cat. no. AB3831), ANXA4 (cat. no. ABC885) and KRT8 (cat. no. MAB1611) and β-actin (cat. no. MABT825) were purchased from EMD Millipore (Billerica, MA, USA). Antibody targeting YWHAE (cat. no. BS6109) was obtained from Bioworld Technology, Inc. (St. Louis Park, MN, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ALX-211-205TS-C100) and anti-rabbit IgG (ADI-SAB-301-J) were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). All other chemicals used in the present study were purchased from Amresco, Inc. (Solon, OH, USA) and Sigma-Aldrich. All chemicals used were of the highest grade commercially available.

The A549 cells and WI-38 cells (1×10⁵) were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin in a humidified incubator with 5% CO₂ in air 37°C. The cells were cultured in 12-well plates and incubated overnight. Subsequently, the cells were treated with 0, 100, 200, 300, 400 and 500 µg/ml of FCP for 24 h. Following incubation, 100 ml of 0.5 mg/ml MTT solution was added to each well and incubated for 3 h at 37°C in the dark. The formazan contained in the cells was solubilized by the addition of 500 ml of DMSO, and the absorbance was measured at 540 nm using an enzyme-linked immunosorbent assay plate reader.

The total proteins were extracted from the A549 cells in the untreated (control) and FCP-treated groups. Following incubation with FCP, the cells were lysed in lysis buffer, containing 7 M urea, 2 M thiourea and 4% (w/v) CHAPS, on ice for 1 h. The lysates were then centrifuged at 13,000 × g for 15 min at 4°C, and the collected supernatant was stored at −80°C until analysis. The total protein was used for 2-DE. The protein concentration was determined using the Non-Interfering™ protein assay kit (G-Biosciences, St. Louis, MO, USA), in accordance with the manufacturer's protocol.

An equal quantity (150 µg) of protein per sample was loaded onto a 18 cm linear IPG strip (pH 4–7; Amersham Biosciences; Uppsala, Sweden) for first-dimensional isoelectrofocusing, which was followed by 12% second dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on an Ettan DALT II system (Amersham Biosciences). The gels were stained with silver nitrate, as described previously with modifications (27), and three independent gels were used in triplicate. Briefly, gels were incubated in fixation solution (50% ethanol and 5% acetic acid) for 15 h, washed once with 30% ethanol for 15 min followed by three times with distilled water for 5 min each. The gels were stained with silver nitrate (0.3%) in the dark for 25 min at room temperature. The gels were subsequently rinsed with water three times and developed with solution containing 3% sodium carbonate, 0.02% sodium thiosulfate and 0.05% formalin. The gels were scanned and image analysis was performed using Progenesis Samespots software (Nonlinear Dynamics, Newcastle, UK). Using this software, the differentially expressed spots were identified by automatic matching of the detected protein spots. Those spots differing significantly (P<0.05) in their intensities (fold-change ≥2), in the FCP-treated A549 cells were used for further analysis.

Selected protein spots were excised manually from the 2-DE gel, and protein digestion was performed (28) with modifications. Briefly, the excised gel pieces were washed with 100 µl 100 mM NH₄HCO₃ for 5 min and then dehydrated in 100 µl acetonitrile for 10 min. Following drying in a lyophilizer (SFDSM06; Samwon Freezing Engineering Co., Busan, South Korea), the gel pieces were rehydrated in 5–10 µl 50 mM NH₄HCO₃ containing 20 ng/µl trypsin (Promega Corporation, Madison, WI, USA) on ice. After 45 min, the trypsin solution was removed and replaced with 10–20 µl 50 mM NH₄HCO₃ without trypsin, and digestion was performed for a minimum of 16 h at 37°C. Subsequently, the peptide mixtures were targeted onto a MALDI-TOF/TOF plate and analyzed using a Voyager-DE STR mass spectrometer (Applied Biosystems; Thermo Fisher Scientific, Inc.), equipped with delay ion extraction.

The proteins were identified using the Mascot program (http://www.matrixscience.com). The Swissprot database and peptide mass fingerprinting (PMF) data were used to identify matching proteins. The following parameters were used for the database searches: Taxonomy, Homo sapiens (human); cleavage specificity, trypsin with one missed cleavage permitted; peptide tolerance of 100 ppm for the fragment ions; permitted modifications, fixed cysteine carbamidomethylation, variable oxidation of methionine. Protein scores >84 were considered statistically significant (P<0.05).

The A549 cells (2×10⁵) were cultured in 6-well plates and incubated with FCP (363 µg/ml) for 24 h. The cell lysates was prepared, and 30 µg of proteins were separated by 12% SDS-PAGE and transferred onto a PVDF membrane. The blots were blocked with 5% nonfat dry milk for 1 h at room temperature and then incubated with primary antibodies overnight (dilution, 1:1,000), followed by incubation with HRP-conjugated goat anti-mouse IgG (dilution, 1:1,000) for 2 h at room temperature. The signal was visualized using ECL detection reagent (GE Healthcare Life Sciences) and quantified by densitometry using the Image J (http://rsb.info.nih.gov) program. The densitometry readings of the bands were normalized to the expression of β-actin. The experiment was repeated three times.

GO analysis was performed using the Agbase database (http://www.agbase.msstate.edu/), as previously described (29). GO annotations were obtained from GORetriever by submitting the spot identities. The annotation results were summarized based on the GOA and whole proteome GOSlim set using GOSlimViewer (agbase.msstate.edu/cgi-bin/tools/goslimviewer_select.pl).

All statistical analyses were performed using SPSS software (SPSS for Windows, ver. 10.0; SPSS, Inc. Chicago, IL, USA). The data are presented as the mean ± standard deviation of at least three independent experiments. The statistical significance between the control and test groups was determined using one-way analysis of variance followed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the anticancer activity of FCP, A549 human lung cancer cells and WI-38 human fetal lung fibroblasts were treated with the indicated concentrations (0–500 µg/ml) of FCP for 24 h. FCP inhibited the growth of the A549 cells in a dose-dependent manner, however, no definite cytotoxic effects were found in the WI-38 cells (Fig. 1A) and cytotoxicity was negligible at concentrations of ≥200 µg/ml. These results showed that FCP exhibited a level of specific cytotoxicity towards cancer cells. The 50% inhibitory concentration for FCP treatment was found to be ~363 µg/ml. In addition, morphological changes, including cell shrinkage and density, and decreased cell numbers were observed in the FCP-treated A549 cells (Fig. 1B). These results suggested that the FCP specifically inhibited the A549 lung cancer cells.

Subsequently, to investigate the mechanism underlying the inhibitory effects of FCP on A549 cell proliferation, 2-DE gel analysis was performed. The representative 2-DE patterns of the untreated (control) and FCP-treated A549 cells were obtained by resolving 150 µg of total proteins on IPG strips (18 cm; pH 4–7) in the first dimension and 12% SDS-PAGE in the second dimension (Fig. 2). In total, 15 differentially expressed protein spots were identified (≥2-fold; P<0.05) using Progenesis Samespots image analysis software (version 4.0). Finally, eight differentially expressed proteins, one upregulated and seven downregulated, were detected in the FCP-treated A549 cells using MALDI-TOF/TOF-MS analysis and database searching (Fig. 2 and Table I). Specifically, YWHAE was upregulated, and ANXA4, CFL1, tsf, HSP90B1, ANXA1, KRT8 and KRT79 were downregulated in the FCP-treated A549 cells, compared with the control cells (enlarged 2-DE images in Fig. 3). The annotation of all identified proteins with their corresponding Swissprot accession number, experimental and theoretical molecular weight, experimental and theoretical isoelectric point, sequence coverage and number of peptide matches, Mascot score, expression and statistical values are shown in Table I. Based on protein functions, the identified proteins were divided into the following categories: Cytoskeletal proteins (CFL1, KRT8 and KRT79), signal transduction (YWHAE, ANXA1 and ANXA4), molecular chaperons/heat shock proteins (HSP90B1) and protein metabolism (tsf). The identified proteins were predominantly involved in tumor growth, cell cycle, apoptosis, migration and signal transduction.

The immunoblotting was performed to confirm the expression of proteins, which were identified in the FCP-treated A549 cells using proteome analysis. The results showed that YWHAE was significantly upregulated, whereas CFL1, ANXA4 and KRT8 were significantly downregulated in the FCP-treated A549 cells, compared with the control (P<0.05; Fig. 4A). These findings suggested that the results of the immunoblotting were consistent with those of the comparative proteomic analysis.

The GO terms for biological processes were examined for all eight identified proteins. The most notable functional categories in terms of the protein expression pattern are shown in Fig. 4B. The highest associations were with biological processes (14%; GO:0008150). Another 8% of the associations were with cell differentiation (GO:0030154) and anatomical structure development (GO:0048856), whereas 6% were associated with signal transduction (GO:0,007165) and cell death (GO:0008219). Of the remainder, 4% were associated with response to stress (GO:0006950), cellular protein modification process (GO:0006464), cytoskeleton organization (GO:0007010), membrane organization (GO:0061024) and transport (GO:0006810).