The following reagents were used in the present study: Fetal bovine serum (FBS), RPMI-1640 (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), lithium chloride (LiCl; Sigma-Aldrich, St. Louis, MO, USA), CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS assay; Promega Corporation, Madison, WI, USA) and FITC Annexin V (BD Biosciences, San Jose, CA, USA). Rabbit polyclonal anti-β-catenin (cat. no. sc-7199; 1:2,000 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse monoclonal anti-β-actin (cat. no. sc-130065; Santa Cruz Biotechnology, Inc.; 1:1,000 dilution), rabbit monoclonal anti-phosphorylated (p)-β-catenin (cat. no. 4176; 1:1,000 dilution; Cell Signaling Technology, Inc., Boston, MA, USA), rabbit monoclonal anti-Bcl-xl (cat. no. 2764; Cell Signaling Technology, Inc.; 1:1,000 dilution), horseradish peroxidase (HRP)-conjugated polyclonal goat anti-mouse IgG (cat. no. SA0001-1; 1:5,000 dilution; ProteinTech Group, Inc., Chicago, IL, USA) and goat anti-rabbit IgG (cat. no. SA0001-2; 1:5,000 dilution; ProteinTech Group, Inc.) antibodies were used for western blotting. Small interfering RNA (siRNA; Shanghai GenePharma Co., Shanghai, China) and Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were also used.

A549/WT and A549/CDDP cells were obtained from the Chinese Academy of Medical Sciences (Beijing, China) and the Cancer Hospital of Peking Union Medical College, Chinese Academy of Medical Sciences (Beijing, China), respectively. Cells were cultured in RPMI-1640 culture medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (GE Healthcare Life Sciences). A549/CDDP cells were grown in complete culture medium containing 2 mg/l cisplatin. Cell cultures were maintained in a 5% CO₂-humidified incubator at 37°C. To activate β-catenin signaling, cells were incubated with 10 mM LiCl for 24 or 48 h.

An MTS assay was used to determine the proliferative potential of cells. In brief, cells were seeded onto 96-well plates at a density of 5×10³ cells/well. Following different treatments, 20 µl of MTS solution was added to cells. After incubation for 1 h at 37°C, the absorbance was measured at 490 nm using an Infinite M200 microplate spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland). The growth inhibition rate was calculated using the following equation: Growth inhibition rate (%) = (1 − ODSample / ODControl) × 100. The half maximal inhibitory concentration (IC₅₀) value was calculated using the least-squares method. The IC₅₀ values and nonlinear regression graph were calculated using the GraphPad Prism^® (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA) by plotting the log concentration of the cisplatin versus the growth inhibition rate of cells.

Cellular apoptosis was evaluated using FITC Annexin V/propidium iodide (PI; BD Biosciences) double staining followed by flow cytometric analysis. Cells were treated with 0, 5 or 10 mg/l cisplatin for 24 h. Subsequently, cells were collected and resuspended in 100 µl binding buffer containing 5 µl Annexin V-FITC and 5 µl PI. After 15 min incubation in darkness at room temperature (20–25°C), a further 400 µl of binding buffer was added to the cell suspension and the samples were analyzed by a FACScan flow cytometer (BD Biosciences).

Total protein was extracted from cells using radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc.) and the protein concentration was measured using BCA Protein Assay kit, according to manufacturer's instructions (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal quantities of protein extract were separated by 10% SDS-PAGE (Sigma-Aldrich). The gel was run for 20 min at 80 V followed by 100 min at 120 V. Proteins were transferred to a polyvinylidene fluoride or nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% w/v non-fat dry milk (Sigma-Aldrich) dissolved in Tris-buffered saline plus 0.1% Tween-20 (TBS-T; pH 8.3; Sigma-Aldrich) and probed with primary antibodies at 4°C overnight. After washing with TBS-T, membranes were incubated with HRP-labeled secondary antibodies for 1–2 h at room temperature. Immunobands were detected using an enhanced chemiluminescence kit, according to manufacturer's instructions (KPL, Inc., Gaithersburg, MD, USA). The densitometric values of target proteins were analyzed by Quantity One (version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA) or ImageJ (version 1.48; National Institutes of Health, Bethesda, MD, USA) software.

Cells were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) for 15 min, permeabilized in 0.1% Triton-X100 (Sigma-Aldrich) for 15 min, and stained with DAPI (1 µg/ml; Sigma-Aldrich) for 5 min in the dark. After washing with PBS, cell samples were imaged using an IX51 fluorescent microscope (Olympus Corporation, Tokyo, Japan) at ×100 magnification. Three fields were randomly captured from each sample and the mean number of DAPI-positive cells was calculated.

Cells were fixed with 4% PFA for 20 min. After washing with PBS, cells were treated with PBS containing 0.5% Triton-20 (Sigma-Aldrich) for 10 min. Cell samples were blocked with PBS supplemented with 4% bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature and probed with primary antibodies at 4°C overnight or at 37°C for 3 h. After PBS washing, samples were incubated with FITC-labeled secondary antibody for 1 h at 37°C. The nuclei were counterstained with 5 µg/ml DAPI and the samples were visualized using confocal laser scanning microscopy (Leica TCS SP5II; Leica Microsystems GmbH, Wetzlar, Germany).

To knock down β-catenin expression, A549 cells were seeded onto six-well plates at a density of 1×10⁵ cells/well and maintained in 500 µl antibiotic-free culture medium. After 24 h, Lipofectamine 2000 and 33 nM siRNA diluted in 250 µl serum- and antibiotic-free culture medium were added to the cell culture. The culture medium was replaced with fresh medium 4–6 h after transfection. The sequences of siRNAs targeting β-catenin were as follows: Sense, 5′-GGACACAGCAGCAAUUUGU-3′ and anti-sense, 5′-ACAAAUUGCUGCUGUCCTT-3′. Control cells were transfected with negative control siRNA with the following sequences: Sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and anti-sense, 5′-ACGUGACACGUUCGGAGAATT-3′.

Total RNA was extracted using an RNeasy Mini kit, according to the manufacturer's instructions (TianGen Biotech Co., Ltd.). Total RNA (2 µl) was reverse transcribed in a 20-µl reaction system using a Quant Reverse Transcriptase kit (TianGen Biotech Co., Ltd.). qPCR was performed using a 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.) and a SuperReal PreMix (SYBR Green) kit (TianGen Biotech Co., Ltd.). Primers used for qPCR amplification were as follows: Bcl-xl, forward, 5′-CCTGAATGACCACCTAGAGCCTT-3′ and reverse, 5′-TCATGCCCGTCAGGAACCAG-3′; 18S rRNA, forward, 5′-GTAACCCGTTGAACCCCATT-3′ and reverse, 5′-CCATCC AATCGGTAGTAGCG-3′. The cycling conditions used were as follows: Pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 sec; annealing at 55°C for 20 sec; extension at 68°C for 35 sec. A total of 40 cycles were performed. The relative expression of Bcl-xl was normalized to 18S rRNA and was calculated using the 2^−ΔΔCq method (12).

Data were calculated from three independent experiments and are presented as means ± standard deviation. Data were analyzed using SPSS software (version 19.0; IBM SPSS, Armonk, NY, USA). Statistical significance was assessed using Student's t-test, Wilcoxon rank-sum test and one-way analysis of variance (ANOVA). Bonferroni correction and Fisher's Least Significant Difference test were performed following one-way ANOVA. P<0.05 indicated a statistically significant difference. Figures were constructed using GraphPad Prism (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA).

The current study initially determined the sensitivity of human lung adenocarcinoma A549/WT and A549/CDDP cells to different concentrations of cisplatin. An MTS assay demonstrated that cisplatin dose-dependently inhibited the proliferation of A549/WT and A549/CDDP cells; however A549/WT cells were more sensitive to cisplatin exposure. The IC₅₀ value of A549/CDDP cells was two-fold higher than that of A549/WT cells (14.67±0.66 vs. 7.87±0.57 mg/l; n=3; Fig. 1A). In addition, 5 and 10 mg/l cisplatin treatment induced apparent apoptosis in A549/WT and A549/CDDP cells, with an increased number of apoptotic cells among A549/WT cells as compared with A549/CDDP cells (Fig. 1B). To investigate the potential role of Wnt/β-catenin signaling in cisplatin resistance, the protein expression of β-catenin in A549/WT and A549/CDDP cells was determined by immunoblotting. As demonstrated in Fig. 2, a significant upregulation of β-catenin was detected in A549/CDDP cells compared with A549/WT cells (P<0.05), suggesting that β-catenin may participate in the cisplatin resistance of lung adenocarcinoma cells.

LiCl has previously been demonstrated to confer chemotherapy resistance in several tumor cell types, such as hepatoblastoma (13), ovarian carcinoma (14) and A549 lung cancer cells (8). In the current study, treatment of A549/WT cells with 5 or 10 mg/l cisplatin resulted in a marked loss of cell viability, which was abolished by treatment with 10 mM LiCl (P<0.01 compared with cisplatin treatment alone; Fig. 3A and B). Furthermore, 10 mM LiCl reduced the sensitivity of A549/WT cells to different concentrations of cisplatin. The IC₅₀ value of the LiCl plus cisplatin treatment group was elevated compared with that of cisplatin treatment alone (9.79±0.76 vs. 7.87±0.57 mg/l; n=3; Fig. 3C). In addition, LiCl markedly reduced cellular apoptosis induced by cisplatin (Fig. 3D). These results indicate that LiCl reduced the sensitivity of A549/WT cells to cisplatin, suggesting that LiCl-conferred drug resistance may be a general phenomenon in cancer cells.

To assess the molecular mechanism by which LiCl promoted cisplatin resistance in A549/WT cells, the protein expression level of total and p-β-catenin in LiCl-treated A549/WT cells was determined. As demonstrated in Fig. 4A–C, LiCl significantly increased β-catenin and p-β-catenin compared with the non-treated controls (P<0.05). In addition, LiCl treatment led to nuclear translocation of β-catenin in A549/WT cells, indicating the activation of β-catenin signaling (Fig. 4D). These results suggest that activation of Wnt/β-catenin signaling may be one of the mechanisms by which LiCl promotes cisplatin resistance in A549/WT cells.

Bcl-xl is a member of the Bcl-2 protein family and functions as a pro-survival factor, inhibiting cellular apoptosis (15,16). As evasion of apoptosis is the key to drug resistance in cancer treatment, the current study examined the potential involvement of Bcl-xl in cisplatin resistance of lung adenocarcinoma cells in the presence and absence of LiCl. The results demonstrated that the protein expression level of Bcl-xl was significantly elevated in A549/CDDP cells compared with A549/WT cells (P<0.05; Fig. 5). Furthermore, RT-qPCR analysis revealed that LiCl treatment significantly increased the mRNA expression levels of Bcl-xl in A549/WT cells compared with the non-treated control cells (P<0.05; Fig. 6A). Consistent with the upregulation of Bcl-xl mRNA, western blot analysis demonstrated that treatment with 10 mM LiCl significantly elevated the protein expression level of Bcl-xl in A549/WT cells (P<0.05; Fig. 6B and C). Thus, LiCl treatment of A549/WT cells resulted in increased expression of Bcl-xl at the mRNA and protein level.

In order to explore the potential association between Bcl-xl and β-catenin signaling pathways in cisplatin resistance of lung adenocarcinoma cells, β-catenin expression was silenced using siRNA in A549/WT cells. Transfection of siRNA targeting β-catenin efficiently down-regulated the mRNA (Fig. 7A) and protein (Fig. 7B) levels of β-catenin in A549/WT cells compared with negative control siRNA (P<0.01). Additionally, β-catenin siRNA significantly inhibited the growth of A549/WT cells (P<0.05) and caused a further reduction of cisplatin-induced growth inhibition (P<0.01; Fig. 8A and B). Furthermore, silencing of β-catenin with siRNA decreased the inhibition rate of cisplatin, as demonstrated by MTS assay. The IC₅₀ value of cisplatin was reduced in cells transfected with β-catenin siRNA (β-catenin siRNA, 4.98±1.37 mg/l; control, 7.87±0.57 mg/l; negative control, 7.45±0.49 mg/l; Fig. 8C). Furthermore, β-catenin siRNA increased cisplatin-induced apoptosis in A549/WT cells (Fig. 8D). In accordance with a previous observation that β-catenin promoted transcription of Bcl-xl (17), the present study observed that silencing of β-catenin in A549/WT cells significantly decreased the mRNA (P<0.001; Fig. 9A) and protein (P<0.01; Fig. 9B and C) expression of Bcl-xl compared with negative control cells. In addition, knockdown of β-catenin reduced β-catenin phosphorylation in A549/WT cells (Fig. 9B). These results indicate that silencing of β-catenin sensitized A549/WT cells to cisplatin and this effect may be mediated by downregulation of Bcl-xl expression.