The human lung adenocarcinoma cell line A549 and its cisplatin-resistant subline A549/DDP were obtained from the Institute of Cell Biology. Cells were cultured in a humidified incubator at 37°C and 5% CO₂ in Roswell Park Memorial Institute (RPMI)-1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated foetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone; GE Healthcare Life Sciences). To maintain drug resistance, A549/DDP cells were maintained in the presence of 0.5 µg/ml cisplatin (Merck KGaA).

The effect of cisplatin on cell growth was determined using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies Inc.) assays. For this, 100 µl A549 and A549/DDP cell suspensions were distributed in quadruplicate in 96-well plates at a density of 5×10⁴ cells/well. Culture medium (100 µl) was added to the wells of 96-well plates as a blank control group, followed by incubation of both sets of plates at 37°C and 5% CO₂ for 24 h. The cells were then treated with or without 15 µM CX4945 (Selleck Chemicals Co., Ltd.) for 24 h, followed by an additional 24 h incubation with 0, 1.25, 2.5, 5, 10, or 20 µg/ml cisplatin. CCK-8 solution (10 µl) was subsequently added to each well of the plates and incubated for 1 h, after which the absorbance at 450 nm was measured. The proliferation of A549 and A549/DDP cells was also determined by the same method after cells were treated with cisplatin (5 µg/ml) for 0, 12, 24, 36, 48 and 72 h. This procedure was performed three times, and the mean value was calculated. The concentration at which cisplatin alone or in combination with CX4945 produced 50% growth inhibition (IC₅₀) was calculated based on the relative survival curve.

A total of 5×10⁵ A549/DDP cells/well were cultured overnight in 6-well plates, followed by the addition of CX4945 at a concentration of 0, 5, 10, 15, 20, or 25 µM. After 24 h, after cells were digested with trypsin with no EDTA, they were centrifuged at 350 × g at 4°C for 5 min, washed twice with phosphate-buffered saline, and resuspended in binding buffer (BD Biosciences) at a concentration of 2×10⁵ cells/ml. Annexin V-conjugated fluorescein isothiocyanate and propidium iodide (BioLegend, Inc.) was added to the binding buffer and incubated for 15 min at room temperature. After incubation, the percentage of apoptotic cells was detected using a flow cytometer (BD FACSCalibur; BD Biosciences) and analysed usig FlowJO version 7.6.1 (Tree Star, Inc.). After dividing the A549/DDP cells into four groups based on different treatments (RPMI-1640, DDP alone, CX4949 alone, or combination treatment), apoptosis was detected using flow cytometry.

After the treatment with RPMI-1640 medium, CX4945, cisplatin or CX4945+cisplatin, and incubation for 24 h, whole-cell lysates were harvested from A549 and A549/DDP cells using cell lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was measured using a bicinchoninic acid assay, and equal quantities (20 µg) of protein were resolved using SDS-PAGE with a 10% gel. After resolving, the proteins were transferring to polyvinylidene difluoride membranes. The membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature, and then incubated with a primary antibody overnight at 4°C, followed by incubation with either horseradish peroxidase (HRP) conjugated goat anti-rabbit (cat. no. 111-035-003; 1:10,000) and goat anti-mouse secondary antibodies (cat. no. 115-035-003; 1:10,000) (both from Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Protein bands were detected using enhanced chemiluminescence reagents (EMD Millipore).

Antibodies against the following targets were used for western blot analysis: CK2α (cat. no. sc-12738; 1:500; Santa Cruz Biotechnology, Inc.), β-catenin (cat. no. sc-7963; 1:500; Santa Cruz Biotechnology, Inc.), DVL-2 (cat. no. 373413; 1:1,000; R&D Systems, Inc.), p-DVL-2^Ser143 (cat. no. ab124933; 1:1,000; Abcam), p-DVL-2^Thr224 (cat. no. ab124941; 1:1,000; Abcam), c-Myc (cat. no. 9402; 1:500; Cell Signaling Technology, Inc.), caspase-3 (cat. no. sc-271759; 1:500; Santa Cruz Biotechnology, Inc.), cleaved caspase-3 (cat. no. 9661; 1:500; Cell Signaling Technology, Inc.); cyclin D1 (cat. no. 2922; 1:500; Cell Signaling Technology, Inc.), MRP1 (cat. no. sc-365635; 1:1,000; Santa Cruz Biotechnology, Inc.) and LRP (cat. no. sc-390134; dilution, 1:500; Santa Cruz Biotechnology, Inc.). Densitometry analysis was performed using ImageJ version 1.52a (National Institutes of Health). The respective total protein was used as the loading control for phosphoproteins. β-actin was used as the loading control for all other proteins.

After the indicated treatments, total RNA was extracted from cells from the four groups (blank control, cisplatin alone, CX4945 alone and CX4945+cisplatin) using Trizol^® (Thermo Fisher Scientific, Inc.) according to manufacturer instructions. Total RNA was reverse transcribed to cDNA at 42°C for 60 min followed by 72°C for 10 min, using a PrimeScript RT reagent kit (Bio-Rad Laboratories, Inc.). qPCR was conducted using the UltraSYBR reagent (CWBIO, Technology, Co. Ltd.) on a thermocycler with the following cycling conditions: Initial denaturation for 3 min at 94°C, followed by 40 cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at 60°C, extension for 30 sec at 72°C, and a final extension step for 2 min at 72°C. Primer sequences are listed in Table I. The RNA levels of target genes were standardized against that of β-actin using the 2^−ΔΔCq method (30). All assays were performed in triplicate to ensure minimum deviation.

Statistical analysis was performed using GraphPad Prism software (v5.01; GraphPad Software, inc.). Data are expressed as the mean ± standard deviation. Independent samples t-tests and paired t-tests were used to compare data between two groups. One-way analysis of variance with Bonferroni's correction was used to compare data among three or more groups. A P<0.05 was considered to indicate a statistically significant difference.

A549 and A549/DDP cells were treated with cisplatin at increasing concentrations (0, 1.25, 2.5, 5, 10 or 20 µg/ml), and the viability of treated cells was detected using CCK-8 assays. The viability of the cells was also determined by the same method after treatment with cisplatin (5 µg/ml) for 0, 12, 24, 36, 48 and 72 h. Cisplatin significantly inhibited A549 and A549/DDP cell growth in a dose- and time-dependent manner (Fig. 1A and C), with IC₅₀ values for cisplatin of 3.74±0.22 and 19.28±2.17 µg/ml in A549 and A549/DDP cells, respectively (P=0.0002; Fig. 1B). These results indicated that A549/DDP cells were 5.16-fold more resistant to cisplatin compared with that of A549 cells. Moreover, A549/DDP cell viability decreased until the concentration increased to 5 µg/ml; therefore, we chose this as the optimal concentration for treatment.

Activation of Wnt/β-catenin signaling not only leads to a variety of human diseases (31) but it is also associated with cisplatin resistance (13). CK2 and DVL-2 participate in and activate Wnt/β-catenin signaling (26,32). Moreover, S143 and T224 of human DVL-2 are phosphorylated by CK1δ/ε, and the S143A mutation inhibits polo-box domain (PB)-DVL-2 interaction, while the T224A mutation partially inhibits this interaction (33). The phosphorylation of DVL-2 and DVL-2-PBD binding are critical for subsequent interaction with polo-like kinase 1, which is involved in non-canonical Wnt signaling (33). However, whether these two phosphorylation sites are associated with the canonical Wnt signaling pathway remains unclear. Therefore, to determine whether differences in responses to cisplatin between A549 and A549/DDP cells were related to disparities in CK2 and DVL-2 levels, western blot analysis was performed to evaluate CK2, total-DVL-2, p-DVL-2^Ser143 and p-DVL-2^Thr224 levels in both cell lines. As shown in Fig. 2, CK2 and p-DVL-2^Ser143 expression levels were significantly higher in A549/DDP cells compared with the A549 cells (P<0.001). Additionally, β-catenin levels were significantly higher in A549/DDP cells (P<0.001), as were c-Myc and cyclin D1 (P<0.001), which are direct targets of lymphoid-enhancer-binding factor/T cell factor (LEF/TCF) transcription factors and function downstream of Wnt signaling (34). However, the levels of total DVL-2 and p-DVL-2^Thr224 in A549 and A549/DDP cells were not significantly different (Fig. 2A and B). To further investigate whether the resistance of A549/DDP was associated with resistance-related proteins, levels of MRP1, LRP, caspase-3 and cleaved-caspase-3 were analyzed using western blot, revealing significantly higher MRP1 and LRP levels (P<0.001) and lower caspase-3 (P<0.001) and cleaved caspase-3 (P<0.001) levels in A549/DDP cells compared with that in A549 cells (Fig. 2C and D). These results suggested that differences in cisplatin resistance between A549 cells and A549/DDP cells might be associated with upregulated levels of MRP1, LRP, CK2 and p-DVL-2^Ser143, which are important proteins associated with the Wnt/β-catenin pathway.

Previous studies have not addressed DVL-mediated regulation of cancer cell resistance to chemotherapy. To investigate whether cisplatin resistance in lung adenocarcinoma cells is associated with DVL-2 activity, total DVL-2, p-DVL-2^Ser143 and p-DVL-2^Thr224 levels were detected after cisplatin treatment of A549 and A549/DDP cells. No changes in total DVL-2 or p-DVL-2^Thr224 levels were found upon cisplatin treatment in either cell line; however, cisplatin treatment increased levels of CK2 and p-DVL-2^Ser143 in A549/DDP cells but decreased these levels in A549 cells. Additionally, β-catenin, c-Myc and cyclin D1 levels were associated with CK2 levels in A549 and A549/DDP cells (Fig. 3). A previous study showed that the upregulated expression of β-catenin, cyclin D1 and c-Myc resulted in the activation of the Wnt/β-catenin pathway (35); therefore, it was hypothesized that cisplatin activates Wnt/β-catenin signaling in A549/DDP cells, which might be related to upregulated CK2 and p-DVL-2^Ser143 levels. Moreover, previous studies suggest that the mechanisms associated with cancer drug resistance are closely related to resistance proteins and apoptosis (36–38). Therefore, MRP1 and LRP levels were analyzed after cisplatin treatment of A549 and A549/DDP cells, which revealed significantly higher levels of these markers in A549/DDP cells (P<0.001), whereas they were unchanged in A549 cells (P<0.001). Furthermore, the levels of caspase-3 decreased and the levels of cleaved caspase-3 increased in both cell lines following cisplatin treatment compared with no cisplatin treatment (P<0.001), but this upregulation was more significant in A549 cells compared with that the A549/DDP cells (Fig. 4).

To assess the effects of CX4945 on apoptosis, A549/DDP cells were exposed to increasing concentrations of CX4945 (0, 5, 10, 15, 20, or 25 µM) for 24 h, followed by flow cytometric analysis. Initially, the proportions of apoptotic cells increased from 4.78 to 5.69%, 6.08 and 6.87% after treatment with 5, 10 and 15 µM CX4945, respectively; however, this percentage increased markedly to 13.59% at a CX4945 concentration of 20 µM (Fig. 5A). Western blot analysis of similarly treated A549/DDP cells for proteins associated with drug resistance revealed decreasing levels of MRP1 and LRP with increasing CX4945 concentrations (Fig. 5B); however, levels of caspase-3 decreased and cleaved-caspase-3 increased notably up until a CX4945 concentration of 20 µM, consistent with the flow cytometry data. Therefore, according to a previous finding (39) and these results, 15 µM (as apoptosis only increased at concentrations <20 µM) and 24 h was chosen as the optimal concentration and time for pretreatment.

To investigate whether CX4945 affects the proliferation of A549/DDP cells, they were incubated with different concentrations of cisplatin (0, 1.25, 2.5, 5, 10, or 20 µg/ml) in the presence or absence of CX4945 (15 µM) for 24 h, followed by assessment of cell viability using CCK-8 assay. The IC₅₀ value for cisplatin in CX4945 treated cells was lower compared with that for parental A549/DDP cells (6.43±0.32 vs. 18.43±1.56 µg/ml, respectively) (Fig. 6A), indicating that CX4945 treatment increased the cisplatin sensitivity of A549/DDP cells. To confirm that CX4945 enhances cisplatin-mediated apoptosis, flow cytometry was performed to detect apoptosis in A549/DDP cells treated with RPMI-1640 (as a blank control group), CX4945, cisplatin, or CX4945+cisplatin. The results revealed that total apoptotic rates in groups treated with CX4945, cisplatin, or CX4945+cisplatin were 8.62±0.43% (compared with the control group; P<0.05), 9.64±0.38% (compared with the control group; P<0.05) or 18.54±0.74% (compared with the control group; P<0.05), which were higher compared with that of the control group (8.05±0.36%). Furthermore, the apoptotic rate in the combination treatment group (18.54±0.74%) was significantly higher compared with that in the groups treated with CX4945 alone (8.65±0.43%) compared with CX4945+cisplatin group (P<0.001), or cisplatin alone (9.64±0.38%) compared with CX4945+cisplatin group (P<0.001; Fig. 6B). These data indicated that inhibition of CK2 activity by CX4945 enhanced cisplatin-induced apoptosis in A549/DDP cells.

The present study revealed that cisplatin can lead to the activation of Wnt/β-catenin signaling, which might be associated with CK2 and p-DVL-2^Ser143 upregulation. To supporting this finding, A549/DDP cells were treated with RPMI-1640, CX4945, cisplatin, or CX4945+cisplatin, followed by detection of mRNA and protein levels of Wnt signaling-related molecules using RT-qPCR and western blot analysis, respectively. p-DVL-2^Ser143, β-catenin, c-Myc and cyclin D1 mRNA and protein levels decreased significantly in the combined treatment group compared with cisplatin treatment alone, whereas total DVL-2 and p-DVL-2^Thr224 levels did not differ significantly among the groups (P>0.05). Similar results were obtained upon comparison of CX4945 only group with the control group (Fig. 7A-D). These results revealed that inhibition of CK2 activity decreased p-DVL-2^Ser143 levels and implied that DVL-2 phosphorylation at Ser143 by CK2 promotes activation of Wnt/β-catenin signaling.

Additionally, MRP1, LRP, caspase-3 and cleaved-caspase-3 protein and mRNA levels were analyzed, and revealed that MRP1, LRP and caspase-3 levels in the combined-treatment group were significantly lower compared with that in the cisplatin-only group (P<0.01), whereas cleaved-caspase-3 levels were significantly upregulated in the combined-treatment group (P<0.001). Moreover, compared with that in the control group, MRP1 and LRP mRNA and protein levels decreased in the CX4945-only group, whereas caspase-3 mRNA and protein levels and cleaved-caspase-3 protein levels remained virtually unchanged between both groups. Levels of MRP1, LRP and caspase-3 mRNA are consistent with their protein levels (Fig. 8A-C). These results indicated that CX4945 reversed cisplatin resistance by blocking Wnt/β-catenin signaling, downregulating levels of resistance-associated proteins, and inducing apoptosis.