SW579, a human thyroid cancer cell line, was purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were cultured at 37°C with 5% (v/v) CO₂ in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Gaithersburg, MD, USA), which was supplemented with 10% (v/v) fetal calf serum (FCS; Life Technologies) and antibiotics (100 µM penicillin and 100 µM streptomycin; Sigma-Aldrich, Carlsbad, CA, USA).

Cells were seeded at 200 cells per well in 24-well tissue culture plates for 24 h under 5% CO₂ at 37°C. Subsequently, the cells were treated with various concentrations (0, 50, 100, 150, 200 or 250 ng/ml) of recombinant ING4 protein (Sino Biological, Inc., Beijing, China), and the plates were incubated for 1 week in a humidified incubator at 37°C. Colonies were stained with 0.05% crystal violet (Beyotime Institute of Biotechnology, Shanghai, China) containing 50% methanol, and counted. The colonies were counted in four or five random fields of vision for each of the duplicate samples using a CX71 microscope (Olympus Corporation, Tokyo, Japan) at a magnification of ×100. The IC₅₀ value of the recombinant ING4 protein was determined and for use in later experiments.

To detect the rate of apoptosis, an annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (KeyGen, Nanjing, China) was used in accordance with the manufacturer's instructions. The samples were immediately analyzed on a FACSCalibur flow cytometer (Becton-Dickinson Medical Devices, Shanghai, China).

A Transwell migration assay was performed using the Boyden chamber (8 µM pore size; polycarbonate membrane; Cell Biolabs, San Diego, CA, USA). The cells were resuspended in FCS-free DMEM to a concentration of 3×10⁵ cells/ml. The upper chamber was loaded with 100 µl cell suspension, while the lower chamber was loaded with 600 µl DMEM containing 10% FCS. Following incubation for 12 h in normal culture conditions, the filter was fixed in 4% paraformaldehyde (Sigma-Aldrich) and stained with crystal violet. The cells on the upper side of the filter were removed using a cotton swab, and the cells that had migrated to the undersurface of the membrane were counted using a light microscope (6XC; Shanghai Guangmai Instruments Ltd., Shanghai, China). In total, 10 microscopic fields (magnification, ×400) were randomly selected for the counting of the cells.

A gelatin zymography assay was performed according to the methods outlined by Song and Zhao (17). Briefly, 50 mg protein was applied to 10% polyacrylamide gels, with 1% gelatin incorporated as a substrate for the gelatinolytic proteases. Subsequent to running the gel, the sodium dodecyl sulfate (SDS) was removed by washing twice in 2.5% Triton X-100 for 30 min. The gels were incubated overnight in a zymography development buffer, which contained 50 mM Tris-HCl (pH 7.4), 2 mM NaN₃ and 5 mM CaCl₂. Following development, the gels were stained for 3 h in 45% methanol/10% glacial acetic acid containing 1% (w/v) Coomassie Brilliant Blue R-250 (Beyotime Institute of Biotechnology), and subsequently partially destained with the same solution without dye. The gelatinolytic activity of each matrix metalloproteinase (MMP) was qualitatively evaluated as a clear band against the blue-stained gelatin background.

A CAM assay was conducted according to the methods outlined by Lokman et al (18). Chick eggs (Dezhou Food Imp & Exp Co. Ltd., Dezhou, China) were incubated in a MultiQuip incubator (MultiQuip, Austral, NSW, Australia) at 37°C with 60% humidity. Under aseptic conditions, a small window was made in the shell on day 3 of chick embryo development to observe the underlying vasculature. The window was resealed with adhesive tape and the eggs were returned to the incubator until day 11 of chick embryo development. On day 11, all three groups of SW579 cells were labeled with CellTracker™ Green 5-chloromethylfluorescein diacetate (Invitrogen Life Technologies, Carlsbad, CA, USA) in suspensions (1×10⁶ cells/well) and mixed with growth factor reduced Matrigel (8.9 mg/ml; BD Biosciences, Franklin Lakes, NJ, USA) to a total volume of 30 µl. Subsequently, the Matrigel grafts were placed on top of the CAM, and the eggs were resealed and returned to the incubator for 72 h until day 14 (n=6 chicken embryos per cell line; SW579, PBS and ING4). Matrigel grafts with the surrounding CAM were harvested from each embryo, fixed with 4% paraformaldehyde for 24 h and embedded in Optimum Cutting Temperature compound (Tissue-Tek; Sakura Finetek USA, Inc., Torrance, CA, USA). The samples were stored at −80°C, and frozen samples were cut into 5-µm sections using a Cryomicrotome CM 1850 (Leica Microsystems, Bannockburn, IL, USA). Images were acquired using an Olympus fluorescence microscope (Olympus Corporation, Osaka, Japan).

Nuclear and cytoplasmic protein fractions were isolated using a CelLytic™ NuCLEAR™ Extraction kit (Sigma-Aldrich), according to the manufacturer's instructions. Protein concentrations were determined using a bicinchoninic acid protein assay, with bovine serum albumin used as a standard (Pierce Biotechnology, Inc., Rockford, IL, USA).

Protein extracts were resolved by SDS-PAGE, which was followed by electrotransfer to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Philadelphia, PA, USA). Following a blocking step using 5% milk in Tris-buffered saline with Tween® 20, the membranes were incubated with primary antibodies at room temperature overnight (Table I). Subsequently, the membranes were developed and visualized with enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA). Secondary monoclonal antibodies were purchased from the Beyotime Institute of Biotechnology. The secondary monoclonal antibodies included anti-mouse IgG (#A0216), anti-rabbit IgG (#A0239) and anti-goat IgG (#A0181). The membranes were incubated with secondary antibodies for 2 h at room temperature.

Numerical data are expressed as the mean ± standard deviation, and differences among the mean values were evaluated using the Student's t-test. Statistical analyses were conducted using SPSS software (version 11.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Cell viability was assessed using the colony formation assay. As shown in Fig. 1A, the proliferative rate of the cells in the recombinant ING4-treated group was inhibited significantly in a dose-dependent manner (P<0.05). The IC₅₀ value of the recombinant ING4 protein was determined to be 192.5 ng/ml. When compared with the untreated SW579 cells or the phosphate-buffered saline-treated cells, the apoptotic ratio of the cells following treatment with recombinant ING4 protein was observed to increase significantly using annexin V-FITC and propidium iodide double staining (P<0.05; Fig. 1B). In addition, the Transwell assay revealed that cell motility was significantly decreased in the recombinant ING4 protein-treated group, as compared with the untreated cells (P<0.05; Fig. 1C). The activity levels of MMP-2 and −9 were shown to be inhibited by ING4 protein in SW579 cells (Fig. 1D). Additionally, using the CAM model, ING4 protein was demonstrated to inhibit SW579 cells from escaping primary tumor sites and scattering among blood vessels (Fig. 1E).

To identify the mechanisms underlying the effects of recombinant ING4 protein in SW579 cells, the expression levels of various proteins were detected by western blot analysis. When compared with the untreated cells, the ING4-treated SW579 cells exhibited a higher expression level of the epithelial marker, E-cadherin, while lower expression levels of the mesenchymal markers, vimentin and N-cadherin, were detected (Fig. 2). Notably, changes in Wnt5b expression were observed in the SW579 cells following treatment with ING4. Furthermore, the western blot analysis assays detected a significant inhibition of low-density lipoprotein receptor-related protein 6 expression and phosphorylation following treatment with ING4 (Fig. 2). A concomitant increase in Axin2 and glycogen synthase kinase-3β expression was detected in the ING-treated SW579 cells, as compared with the control groups, while decreased expression levels of nuclear and cytosolic β-catenin were observed (Fig. 2). Pathway enrichment analysis was performed in the Kyoto Encyclopedia of Genes and Genomes database (Institute for Chemical Research, Kyoto University, Kyoto, Japan; Institute of Medical Science, University of Tokyo, Tokyo, Japan), and the analysis results revealed that the Wnt signaling pathway was involved in the antitumor effects of ING4 (Fig. 3).