Human cervical cancer cell lines HeLa and Siha were obtained from the Institute of Life Sciences Cell Resource Center (Shanghai, China). HeLa and Siha cells were cultured in minimal essential medium (MEM, HyClone; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences), penicillin (100 U/ml)-streptomycin (100 U/ml) liquid (Thermo Fisher Scientific, Inc.) and 0.25 µg/ml amphotericin B (Ameresco, Inc.). Cells were cultured at 37°C in a humidified incubator containing 5% CO₂.

HeLa and Siha cell proliferation was examined using the cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.), according to the manufacturer's protocol. Exponentially growing cells were seeded into 96-well plates at a density of 5×10³ cells/well in a final volume of 100 µl MEM and cultured under normal conditions for 24 h at 37°C in a 5% CO₂-humidified incubator. Subsequently, different concentrations (0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 µmol/l) of gefitinib (cat. no. SML1657; Sigma-Aldrich; Merck KGaA; Darmstadt, Germany) were added to each well, with DMSO used as the vehicle control. After 48 h, 10 µl CCK-8 reagent was added to each well and incubated for 1 h at 37°C. Cell proliferation was calculated by measuring the absorbance at a wavelength of 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.). Growth inhibition was calculated as a percentage of the untreated controls. All experiments were performed in triplicate and the data were expressed as the mean value ± standard deviation of five wells per treatment. For each cell line, the half maximal inhibitory concentration (IC₅₀) was determined using the four-parameter logistic model.

HeLa and Siha cells were seeded into six-well plates at the density of 10⁶ cells/well. Following treatment with 10 µmol/l of gefitinib for 48 h, cells were harvested using trypsin without EDTA, washed three times with ice-cold PBS and fixed with 70% ethanol overnight at 4°C. Cells were subsequently stained 25 µl propidium iodide with 10 µl RNase A at 37°C for 30 min in dark using the Cell cycle and apoptosis analysis kit (cat. no. C1052; Beyotime Institute of Biotechnology, Haimen, China). Cell cycle analysis was performed using a BD FACSCalibur system and CellQuest pro software (version 2.0; BD Biosciences, Franklin Lakes, NJ, USA). All experiments were performed in triplicate.

Cell apoptosis was examined using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection kit (BD Biosciences). Following treatment with 10 µmol/l of gefitinib for 48 h, cells were harvested using trypsin without EDTA and washed three times with ice-cold PBS. Cells were subsequently suspended at 1×10⁴ cells/ml in Annexin V-Binding buffer and incubated with 5 µl Annexin V-FITC for 15 min at 37°C in dark, followed by staining with 5 µl PI. Apoptotic cells were immediately analyzed using a BD FACSCalibur system and CellQuest pro software. All experiments were performed in triplicate.

HeLa and Siha cells were seeded into 24-well plates at a density of 1×10⁵ cells/well and incubated overnight. After 24 h of culture, cells were co-transfected with 200 ng pTOP-Flash (Promega Corporation, Madison, WI, USA) or pFOP-Flash reporter plasmids (Promega Corporation) and 200 ng β-galactosidase (β-gal) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) to monitor for transfection efficiency. After transfection for 48 h, the activity was measured. The TCF-responsive TOP-Flash reporter contains three TCF binding sites, and the corresponding FOP-Flash contains three mutated TCF sites (15). Cells were treated with 10 µmol/l gefitinib for 4 h at 37°C and luciferase activity was analyzed using a Dual-Luciferase Reporter assay system (Promega Corporation), according to the manufacturer's protocol. Luciferase activity was normalized for β-gal activity. The experiment was performed in triplicate.

Total cellular protein was extracted from cells on ice for 15 min using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) supplemented with fresh proteinase inhibitor cocktail and phosphatase inhibitor (Sigma-Aldrich; Merck KGaA). Samples were centrifuged at 11,000 × g for 20 min at 4°C. Total protein was quantified using a bicinchoninic acid assay (Sigma-Aldrich; Merck KGaA) and 20 µg protein/well was separated via SDS-PAGE on a 10% gel. The fractionated proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore) and blocked for 1 h at room temperature with 5% non-fat skimmed milk. The membranes were incubated with primary antibodies, including E-cadherin (1:1,000; cat. no. ab1416), Vimentin (1:1,000; cat. no. ab8978), GSK3β (1:1,000; cat. no. ab93926), p-GSK3β (1:1,000; cat. no. ab131097), β-catenin (1:1,000; cat. no. ab32572) and GAPDH (1:5,000; cat. no. ab181602) overnight at 4°C (all from Abcam, Cambridge, UK). Following primary antibody incubation, membranes were incubated with horseradish peroxidase-conjugated secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ZB-2306; OriGne Technologies, Inc.), for 1 h at room temperature. Protein bands were visualized using a Western Lightning^® Chemiluminescence Reagent Plus according to the manufacturer's protocol (cat. no. NEL105001EA; PerkinElmer, Inc., Waltham, MA, USA). Protein expression was quantified using ImageJ (version 1.8.0; National Institutes of Health, Bethesda, MD, USA).

HeLa and Siha cells were cultured in a six-well plate with glass coverslips and following treatment with 10 µM gefitinib at 37°C for 48 h, cells were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were washed three times with PBS for 5 min and blocked for 2 h at room temperature with 8% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA). Subsequently, cells were incubated with primary antibodies against E-cadherin (1:50; cat. no. ab1416) and vimentin (1:50; cat. no. ab92547; both Abcam) in a humidified chamber overnight at 4°C. Cells were washed three times with PBS. Following primary incubation, cells were incubated with tetramethylrhodamine-conjugated anti-rabbit IgG (1:500; cat. no. ZDR5209; OriGene Technologies, Inc.) at room temperature for 30 min. Cell nuclei were counterstained with DAPI (1:1,000; cat. no. C0060; Beijing Solarbio Science & Technology Co., Ltd.) for 20 min at room temperature. Cells were subsequently washed three times with PBS in the dark and the coverslips were mounted with mounting medium at room temperature for ~1 h in the dark. Fluorescence intensity was observed under a fluorescence microscope (magnification, ×40; XDS-500D; Shanghai Caikon Optical Instrument Co., Ltd.).

Data were presented as the mean ± standard error of the mean of at least three experiments. All statistical analyses were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). All experimental data were analyzed using the unpaired Student's t-test or one-way analysis of variance followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of gefitinib on CC cell growth, cell proliferation was examined in HeLa and Siha cells following treatment with gefitinib. Treatment with gefitinib exhibited strong cytotoxicity in HeLa cells [IC₅₀, 16.19±0.26 µmol/l; 95% confidence interval (CI): 1.077–1.341; Fig. 1A] and Siha cells (IC₅₀, 11.87±0.21 µmol/l, 95% CI: 1.003–1.146; Fig. 1B) compared with the control. Furthermore, the effect of gefitinib on cell cycle distribution was examined in HeLa and Siha cells. The number of cells in the G₀/G₁ phase was significantly increased in Hela and Siha cells following treatment with gefitinib compared with the control (Fig. 1C and D), which suggests that gefitinib may induce CC cell cycle arrest in the G₀/G₁ phase.

To examine the effect of gefitinib on CC cell apoptosis, apoptosis was examined in HeLa and Siha cells following treatment with gefitinib. Treatment with gefitinib significantly induced apoptosis in CC cells in a dose-dependent manner compared with the control (Fig. 2A and B). Furthermore, the relative protein expression levels of apoptosis-related proteins, Bax and Bcl-2, were examined. Following treatment with gefitinib, the relative protein expression level of Bcl-2 was significantly reduced, whereas the protein expression level of Bax was significantly increased in HeLa and Siha cells compared with the control (Fig. 2C and D).

EMT is a key regulator of CC progression (16). Therefore, the expression levels of E-cadherin, an epithelial cell marker, and vimentin, a mesenchymal cell marker were examined in HeLa and Siha cells following treatment with gefitinib. Following treatment with gefitinib, the protein expression level of E-cadherin was significantly increased, whereas the protein expression level of vimentin was significantly reduced in HeLa and Siha cells compared with the control (Fig. 3A and B). Immunofluorescence demonstrated increased E-cadherin expression and reduced vimentin expression in HeLa and Siha cells following treatment with gefitinib compared with the control (Fig. 3C and D. These results suggest that gefitinib may suppress the EMT process in CC cells.

The Wnt/β-catenin signaling pathway is a major contributor to CC tumorigenesis (17). GSK3β is a known negative regulator of β-catenin. Therefore, the phosphorylation of GSK3β at Ser9, an indicator of the activation state of GSK3β, was examined by western blot analysis in HeLa and Siha cells following treatment with gefitinib. The protein expression levels of p-GSK3β and β-catenin and were significantly reduced in HeLa and Siha cells following treatment with gefitinib compared with the control (Fig. 4A and B). Furthermore, luciferase reporter assays demonstrated that activation of the Wnt/β-catenin pathway was significantly suppressed following treatment with gefitinib in HeLa and Siha cells compared with the control (Fig. 4C and D). Taken together, these results suggest that gefitinib may inactivate the Wnt/β-catenin signaling pathway to inhibit EMT in CC cells.