Human alveolar basal epithelial adenocarcinoma cells (A549 cells) and human lung fibroblasts (MRC-5 cells) were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (cat. nos. SCSP-503 and SCSP-5040, respectively). A549 cells were cultured in Ham's F-12K (Thermo Fisher Scientific Inc.) medium (cat. no. 21127-022; Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). MRC-5 cells were cultured in MEM (cat. no. 11090081; Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1X GlutaMAX (cat. no. 35050061; Invitrogen; Thermo Fisher Scientific, Inc.), 1X non-essential amino acids (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 11140050) and 1 mM sodium pyruvate solution (cat. no. 11360070; Invitrogen; Thermo Fisher Scientific, Inc.). A549/DDP cells were obtained from Procell Life Science & Technology Co., Ltd. (cat. no. CM-0519) and were cultured in Ham's F-12K supplemented with 10% FBS, 2 µg/ml DDP and 1% penicillin/streptomycin. The medium was changed every two days and cells were passaged when the cell density reached 80–90%.

All cells were seeded into 96-well plates at the density of 5×10³ cells/well (A549 and A549/DDP cells) or 1×10⁴ cells/well (MRC-5 cells). A549 cells were treated with DDP at various concentrations (1.25, 2.5, 5, 10, 20 and 40 µM). A549/DDP cells were treated with higher concentrations of DDP (5, 10, 20, 40, 60, 80 and 100 µM). A549, A549/DDP and MRC-5 cells were treated with DMC at doses of 5, 10, 15, 20, 25, 30 and 35 µM. After culturing the cells for a specified period of time (24, 48, 72 or 96 h), 20 µl MTT (5 µg/ml; Sigma-Aldrich; Merck KGaA) was added and the cells were cultured in the incubator for 1 h. Next, the culture medium was discarded carefully, 100 µl DMSO was added and the plate was agitated for 10 min on a shaker. The absorbance was read at 550 nm on a microplate reader (Bio-Rad Laboratories Inc.).

Coverslips were inserted into a 24-well plate and then A549/DDP cells were seeded on the coverslips at the density of 5×10⁴ cells per well. After 24 h, the medium was replaced with medium containing drugs (10 µM DDP, 5 µM DMC or both), and after 48 h incubation, TUNEL staining was performed. Briefly, cells were washed gently with PBS to remove non-adherent cells and were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were then incubated with PBS containing 0.1% Triton at 4°C for 10 min for permeabilization. The solution was discarded and 50 µl of TUNEL detection solution (cat. no. C1090; Beyotime Institute of Biotechnology) (2 µl of TdT enzyme with 48 µl of fluorescent labeling solution) was added at 37°C for 60 min in the dark. Finally, slides were mounted with an anti-fluorescence quencher (cat. no. G1401; Wuhan Servicebio Technology Co., Ltd.) and were kept at 4°C. Slides were observed under a fluorescence microscope (magnification, ×200) within one week.

A549 cells were lysed in RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) after 48 h of drug treatment. Total proteins were extracted and were denatured at 95°C for 10 min. The protein concentration was determined by the BCA method, and protein concentration was subsequently normalized using loading buffer. Proteins (30 µg) were separated by 10% SDS-PAGE and were then transferred onto PVDF membranes. Membranes were blocked with 5% skimmed milk in PBST for 2 h at room temperature and were incubated with primary antibodies overnight at 4°C. Next, membranes were washed three times with PBS for 15 min and were incubated with secondary antibodies at room temperature for 2 h. Membranes were washed three times with PBS for 15 min and were exposed to 200 µl enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.). Bands were detected on a Bio-Rad instrument and relative expression levels were normalized to endogenous control using Image-Pro Plus software v.6.0 (National Institutes of Health). The primary antibody against ERCC1 was obtained from Abcam (1:2,000; cat. no. ab129267). The antibodies against Bcl-2 (cat. no. 4223), Bax (cat. no. 5023), caspase 3 (cat. no. 9662), PARP (cat. no. 9532), cleaved caspase 3 (cat. no. 9664), cleaved PARP (cat. no. 5625) and β-actin (cat. no. 4970) were purchased from Cell Signaling Technology, Inc. and were used at a dilution of 1:1,000. The secondary goat anti-rabbit antibodies (cat. nos. 7074 and 7076; Cell Signaling Technology, Inc.) were used at a dilution of 1:5,000.

A total of 16 male BALB/c nude mice (4–5 weeks, 20–24 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. The mice were housed under specific pathogen free (SPF) conditions and fed with SPF standards of care, The mice were housed with a 12 h light/dark cycle at 25±2°C and 50±10% humidity, and were provided with sterile food and water. A549/DDP (5×10⁵) cells were subcutaneously injected into the left axilla of 16 nude mice. When the maximum tumor volume reached 100 mm³, 16 mice were randomly divided into four groups of four mice as follows: Control group, monotherapy groups (DDP or DMC) and a combination treatment group. DDP was injected intraperitoneally at a dose of 6.0 mg/kg every three days and DMC was injected intraperitoneally at a dose of 30 mg/kg every day for 20 days. DDP and DMC were dissolved in DMSO (50 µl in DMSO per mouse for injection). The control group was treated with DMSO (50 µl per mouse). The tumor volume was measured every 4 days until day 20 using the following formula: Tumor volume (units, mm³)=width² × length/2. The mice were sacrificed on day 20. Mice were injected intraperitoneally with 300 mg/kg ketamine and 20 mg/kg xylazine for euthanasia, and the death of mice was confirmed when the mice stopped breathing.

Mouse subcutaneous tumors were collected following euthanasia. Subsequently, tissue sections (4-µm) were dewaxed with xylene (twice) for 10 min and hydrated using a descending alcohol series (100% twice, 90% twice, 75% twice) for 3 min at room temperature. Antigens were retrieved in a 95°C water bath for 20 min. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 20 min at room temperature. After blocking with 5% BSA (Sigma-Aldrich; Merck KGaA), the sections were incubated with primary antibody (Ki-67, 1:200; cat. no. 9027S; Cell Signaling Technology, Inc.) at 4°C overnight. The next day, sections were incubated with secondary antibody (1:200; cat. no. ZDR-5306; OriGene Technologies Inc.) for 2 h at 37°C, followed treating with DAB chromogen (1 drop of the DAB Chromogen per ml of substrate buffer; Dako; Agilent Technologies, Inc.) and hematoxylin (cat. no. C0107; Beyotime Institute of Biotechnology) for 2 min at room temperature. Finally, the slides were dehydrated and fixed by resin and observed with an Olympus inverted microscope (Olympus Corporation; magnification, ×200).

Tumor size data were presented as mean ± SEM, all other data were represented mean ± SD. Student's unpaired t-test was used to analyze the statistical significance among two groups. One-way ANOVA followed by the post hoc Bonferroni's test was used to compare the differences among ≥3 groups. All statistical analyses were performed with SPSS v.20.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

A549 and A549/DDP cells were treated with different concentrations of DDP for 48 h. The results from the MTT assay demonstrated that the IC₅₀ value of DDP in normal A549 cells was 3.8 µM, whereas it reached 47.67 µM in A549/DDP cells, which is more than 10 times higher compared with that in normal A549 cells (Fig. 1A). Furthermore, DDP had almost no effect on A549/DDP cells at concentrations ranging from 5–30 µM (Fig. 1B). This finding indicated that A549/DDP cells were successfully induced. In addition, A549 and A549/DDP cells were treated with different concentrations of DMC. The IC₅₀ values of DMC in A549 and A549/DDP cells were 19.88 and 20.83 µM, respectively (Fig. 1C and D), indicating that induced A549/DDP cells were not resistant to DMC. It has been reported that targeting ERCC1 may mediate therapeutic sensitivity to platinum-based drugs (25). Subsequently, to further explore the resistance of A549/DDP cells to DDP, western blotting was performed. The results demonstrated that the expression of ERCC1 was significantly increased in A549/DDP cells compared with that in A549 cells (Fig. 1E).

The efficiency of DMC and DDP combined treatment in A549/DDP cells was determined and showed that all DMC concentrations (5, 10 and 20 µM) could restore the cell sensitivity to DDP in drug-resistant A549/DDP cells (Table I). Based on the concentration screening results, the lowest DMC concentration (5 µM) was chosen for further experiments. A549/DDP cells were treated with DDP (3.8 µM) in combination with DMC. The DDP concentration of 3.8 µM corresponded to the IC₅₀ value obtained in normal A549 cells. However, the combined drug group did not significantly inhibit cell proliferation compared with the single drug groups (Fig. 2A; combined drug group vs. DDP, P=0.112; combined drug group vs. DMC, P=0.053). Therefore, cells were subsequently treated with elevated concentrations of DDP. The results demonstrated that combination treatment with 10 and 20 µM DDP significantly inhibited the proliferation of A549/DDP cells (P<0.05; Fig. 2B and C). Subsequently, normal human lung fibroblasts (MRC-5 cells) were treated with high concentrations of DMC. The results from the MTT assay revealed that DMC exhibited low toxicity in MRC-5 cells, even at concentrations up to 80 µM (Fig. 2D). Furthermore, combination treatment with DMC and DDP did not induce significantly greater toxicity in MRC-5 cells compared with DDP monotherapy (Fig. 2D).

The MTT assay demonstrated that the drug combination significantly attenuated the proliferation of the A549/DDP cells. Furthermore, following treatment with the drug combination for 48 h, the results from the TUNEL assay revealed that the apoptosis rate of the A549/DDP cells in the combined drug group was significantly elevated compared with control and monotherapy groups (DDP or DMC) (Fig. 3A). The apoptosis rates in the single drug groups were 5.30±0.95 and 7.57±0.76% in DDP and DMC groups, respectively, whereas it reached 13.13±2.29% in the combined drug group. In addition, DMC increased the sensitivity of A549 cells to DDP (P<0.01; Fig. 3B). To investigate the mechanism underlying A549/DDP cell apoptosis, the expression of apoptosis-related proteins was detected by western blotting. ERCC1 expression was significantly decreased in the DMC treatment group (Fig. 3D and E). Bax and Bcl-2 proteins belong to the Bcl-2 family of genes, whereas Bcl-2 is involved in the inhibition of apoptosis. By contrast, Bax not only antagonizes the inhibitory effect of Bcl-2 but also promotes apoptosis (26,27). CMN co-treatment enhanced the effects on apoptosis in A549/DDP cells. The results demonstrated that Bcl-2 and Bax expression was decreased and increased, respectively, in the combination groups (Fig. 3C and F-G). In addition, expression of cleaved caspase-3 was increased in the combination group compared with control and single drug groups (Fig. 3C and H). Poly ADP-ribose polymerase (PARP), a cleavage substrate of caspase, is a major contributor to apoptosis and cleaved PARP is considered an important indicator of apoptosis and an activator of caspase 3 cleavage (28). The results demonstrated that cleaved PARP expression was increased in the combined drug group, indicating that the caspase-mediated apoptosis pathway was activated (Fig. 3C-I).

The present study investigated whether the drug combination could exert the same effects on inhibiting proliferation in vivo. To do so, a subcutaneous ectopic tumor formation model was established by injecting A549/DDP cells into nude mice, which were then treated with DMSO (Control group), single drug (DDP or DMC) and a combination treatment. The results revealed that the tumor size in the combination group was significantly lower compared with that in the single drug groups (Fig. 4A). Following tumor growth in nude mice for 20 days, tumors were removed and weighed. Consistent with the tumor size measurements, tumor weight in the combination group was also lower than that in the other groups (Fig. 4B and C). In addition, immunohistochemical staining indicated that the Ki-67 proliferation index was significantly decreased in the combined drug group compared with that in the other groups (Fig. 4D and E). These findings confirmed the in vitro effects of DDP treatment combined with DMC.