DDP was obtained from Sigma- Aldrich; Merck (Shanghai, China). XPC-siRNA (si-XPC) and the negative control (siNC) were purchased from Shanghai GeneChem Co., Ltd. (Shanghai, China). The FITC Annexin V Apoptosis Detection kit 1 (BD Biosciences, Franklin Lakes, NJ, USA) was used for detecting cell apoptosis by flow cytometry. The anti-Akt (dilution 1:1,000; cat. no. 4691S), anti-p-Akt (dilution 1:1,000; cat. no. 4060S), anti-caspase-3 (dilution 1:1,000; cat. no. 9662S), anti-cleaved-caspase-3 (dilution 1:1,000; cat. no. 9661S) and anti-caspase-9 (dilution 1:1,000; cat. no. 9502P) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); and the anti-p-mTOR (dilution 1:1,000; cat. no. 13152-1) antibody was purchased from Signalway Antibody LLC (College Park, MD, USA). The anti-PI3K (dilution 1:1,000; cat. no. A0982) and anti-mTOR (dilution 1:1,000; cat. no. A11928) antibodies were purchased from ABclonal Technology (Wuhan, China), and the anti-XPC (dilution 1:2,000; cat. no. ab203693) antibody was purchased from Abcam (Cambridge, UK). The anti-Bax (dilution 1:1,000; cat. no. 50599-1-AP) and anti-Bcl-2 (dilution 1:1,000; cat. no. 12789-1-AP) antibodies were purchased from ProteinTech Group, Inc. (Rosemont, IL, USA). The actin (dilution 1:1,000; cat. no. TA-09) and GAPDH (dilution 1:1,000; cat. no. TA-08) antibodies were obtained from OriGene Technologies, Inc. (Beijing, China). The secondary antibody horseradish peroxidase conjugated goat anti-rabbit IgG (dilution 1:5,000; cat. no. ZB-2306), also was purchased from OriGene Technologies, Inc.

A549 and A549/DDP human lung carcinoma cells were obtained from the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI-1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences), 100 U/ml penicillin and 100 U/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified atmosphere of 5% CO₂ at 37°C. In addition, A549/DDP cells were cultured in medium containing 2 mg/l DDP in order to retain the drug resistant phenotype. The cells were divided into 3 groups as follows: The blank group (without transfection), the group transfected with the small interfering RNA targeting the XPC (si-XPC) group and the negative control (si-NC) group. The target sequence of si-XPC was: CTCTGACCTGTTACAAGTA. A total of 2×10⁵ A549/DDP cells were added into a 6-well plate. The cells were transfected at 70% confluence with si-XPC and/or si-NC using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The transfection reagent was mixed with si-RNA or si-NC in a ratio of 2 µl:1 µg. We determined the transfection efficiency using western blotting. Following 24 h of transfection, each group of cells that were in the logarithmic growth phase was collected for the next experiment.

Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Rockville, MA, USA) was used to detect cell viability following different treatments. Briefly, A549 and A549/DDP cells were transfected with si-XPC and si-NC and were incubated in a 96-well plate (3,000 cells/well) overnight. Following DDP treatment at the indicated doses (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/l) for 24 h, 10 µl of CCK-8 solution was added to each well and the plates were incubated for 1 h. The optical density (OD) of each well was detected at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instructions. The half inhibitory concentration (IC₅₀) of the cells was determined by CCK-8 assay. The half-maximal inhibitory concentration (IC₅₀) of the drugs was calculated on GraphPad Prism using the log(inhibitor) vs. response-variable slope (4 parameters) equation under the non-linear regression dialogue.

Following transfection of A549/DDP cells, the cells were allowed to grow for 24 h. The monolayer was scratched with a 200-µl pipette tip. The attached cells were washed 3 times with phosphate-buffered saline (PBS) in order to remove floating cells and debris. Subsequently, serum-free medium was added. Moreover, DDP was added in the petri dishes as described above. The cells were continuously incubated for 48 h. The wounds were visualized every 24 h, and the lines that were aligned with the wounds of each group were photographed in each experiment under an inverted microscope (TS100; Nikon Corp., Tokyo, Japan). In each well, at least 8 regions of each condition was captured randomly at a magnification of ×100. The images were analyzed using ImageJ v2.1.4.7 software (National Institutes of Health, Bethesda, MD, USA).

The rate of apoptosis was analyzed by flow cytometry using an Annexin V-FITC/PI kit (BD Biosciences, Franklin Lakes, NJ, USA). A549/DDP cells were seeded in a 6-well plate and incubated overnight. Following transfection, the cells were incubated with DDP in growth media for 24 h. The cells were collected and resuspended in 500 µl of 1X binding buffer. Subsequently, staining was conducted as described by the manufacturer's protocol.

Total RNA was extracted with TRIzol reagent (Axygen, Inc.; Corning Inc., Corning, NY, USA). The cDNA was generated with a cDNA Synthesis kit (Toyobo Life Science, Osaka, Japan). qRT-PCR was performed using a SYBR-Green assay kit (Roche Diagnostics, Basel, Switzerland) on an Applied Biosystems thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR reactions were run on the ABI 7500 real-time PCR system using the following conditions: 95°C for 30 sec, followed by 35 cycles at 62°C for 30 sec and 72°C for 30 sec, final extension at 72°C for 5 min. The relative mRNA expression levels of GAPDH were calculated and quantified with the 2^−∆∆Cq method (15). GAPDH was used as an internal control. The primers for XPC and GAPDH were as follows: 5′-GACAAGCAGGAGAAGGCAAC-3′ and 5′-GGTTCGGAATCCTCATCAGA-3′ for the XPC sense and reverse primers, respectively; and 5′-TGGACCTGACCTGCCGTCTA-3′ and 5′-AGGAGTGGGTGTCGCTGTTG-3′ for the GAPDH sense and reverse primers, respectively.

The expression levels of various proteins were detected by western blot analysis. The total protein in the cells was collected by RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The protein concentration was determined using a BCA protein concentration assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and protein products (8–12 µg/µl; 40–50 µg total) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA). Subsequently, the membranes were blocked with BSA for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. Following washing with PBST (Beijing Solarbio Science & Technology Co., Ltd.), the PVDF membranes were subsequently incubated with the secondary antibodies for 1 h. Following additional washing with PBST to remove the secondary antibodies, the protein signals were detected by the ChemiDoc XRS gel documentation system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using ECL Western Blotting Substrate (Beijing Solarbio Science and Technology Co., Ltd.). The results were scanned and quantified using the ImageJ software v2.1.4.7.

Each experiment was conducted at least 3 times and the results were presented as the mean values ± standard deviation (SD). One-way analysis of variance (ANOVA) was carried out to compare the differences among multiple groups, and the Bonferroni test was performed followed ANOVA. In addition, the independent samples t-test was performed to compare the differences between two groups. Statistical analysis was performed using the GraphPad Prism 5 statistical software (GraphPad Software, Inc., San Diego, CA, USA). A P-value of <0.05 was considered to indicate a statistically significant difference.

The cytotoxic effects of different concentrations of DDP (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/l) were examined in A549/DDP and A549 cells. A CCK-8 assay was used to assess cell viability. The results revealed that the growth inhibitory rate was increased following the increase in the concentration of DDP, while the inhibitory effect of DDP on A549 cells was significantly higher than that noted in A549/DDP cells (Fig. 1A). Furthermore, we calculated the IC₅₀ values of A549 and A549/DDP cells using a CCK-8 assay, and the results indicated that it was 6.43±0.89 mg/l in A549 cells and 14.6±2.87 mg/l in A549/DDP cells. A549/DDP cells were more resistant to DDP treatment in vitro than A549 cells and 10 mg/l of DDP was selected as the optimal concentration in the follow-up experiments (Fig. 1B).

Western blot assays indicated that the expression levels of XPC were significantly increased in A549/DDP cells compared with those noted in A549 cells (P<0.05) (Fig. 1C). Moreover, to determine whether upregulation of XPC expression was a result of increased transcription, qRT-PCR was performed to analyze the mRNA levels of XPC. The results demonstrated that the mRNA levels of XPC were also upregulated (Fig. 1D). DDP treatment induced an increase in XPC protein levels in A549/DDP cells (Fig. 1E). These results indicated that the expression levels of XPC were upregulated in A549/DDP cells compared with those in A549 cells.

To further explore the role of XPC in the resistance of A549/DDP cells to chemotherapy, XPC knockdown in A549/DDP cells was performed with siRNA. Based on previous studies, the most effective siRNA (siRNA-178) was selected from the 3 knockdown sequences (siRNA-177/178/179) for the following experiments. A549/DDP cells were transfected with si-XPC or si-NC. Western blotting detection demonstrated that the expression levels of XPC were significantly downregulated compared with those noted in the negative control group (Fig. 2A). Furthermore, XPC mRNA levels were also inhibited following si-XPC transfection (Fig. 2B). The effects of XPC knockdown on the sensitivity of the resistant cells to DDP were subsequently investigated. A CCK-8 assay was employed to examine the cell viability of A549/DDP cells. The results revealed that the inhibitory potencies of DDP were higher in the si-XPC transfected group than those in the other 2 groups (Fig. 2C). In addition, the IC₅₀ values of DDP in the 3 groups were calculated and as shown in Fig. 2D, no significant differences were noted in the IC₅₀ values of DDP between the control (14.6±2.87%) and the negative control groups (14.81±2.36%). In contrast to these observations, the IC₅₀ value of DDP was significantly reduced in the si-XPC transfected group (6.5±0.82) compared with that noted in the other 2 groups (P<0.05). These results indicated that A549/DDP cells were more sensitive to DDP treatment following efficient silencing of XPC.

The effect of XPC knockdown on the induction of apoptosis using Annexin V-FITC/PI double staining was explored. As revealed in Fig. 3A and B, the apoptotic rate of the si-XPC group was increased to 19.6% in the A549/DDP cells, indicating that XPC knockdown induced apoptosis in A549/DDP cells. A CCK-8 assay was used to detect the proliferative ability of A549/DDP cells. The results revealed that the proliferation of A549/DDP cells was significantly reduced following transfection with si-XPC (Fig. 2C). Furthermore, a wound healing assay was used to detect the migratory ability of A549/DDP cells. The results demonstrated that the migratory distance noted in A549/DDP cells was significantly reduced following transfection with si-XPC (Fig. 3C and D). Subsequently, the expression levels of the apoptosis-related proteins, including caspase-3, caspase-9, Bax, Bcl-2 and active caspase-3 were also determined by western blot assays. XPC knockdown increased the expression levels of active caspase-3/caspase-3 and Bax/Bcl-2, while it decreased the expression levels of caspase-9 in A549/DDP cells that were treated with DDP for 24 h (Fig. 4). These results indicated that XPC knockdown inhibited cell proliferation and survival.

In order to investigate whether the PI3K/Akt/mTOR pathway is involved in the XPC-mediated cell apoptosis of A549/DDP cells, the expression levels of apoptosis-related proteins were detected. Initially, the expression levels of the phosphorylated form of the Akt protein (Ser473) were significantly increased in drug-resistant cells, while no significant change was noted in the expression levels of the total Akt protein (Fig. 5A). Subsequently, DDP-resistant cells were treated with PI3K inhibitors and western blotting was performed in order to detect the expression levels of XPC. The results revealed that XPC expression in the treatment group was significantly lower than that in the control group (Fig. 5B). This indicated a potential interaction between p-Akt and XPC.

XPC silencing using siRNA did not affect the protein levels of PI3K (Fig. 5C). Silencing of XPC caused a significant decrease in the expression levels of p-Akt and p-mTOR proteins in A549/DDP cells (Fig. 5D and E). However, transfection of si-XPC did not result in a significant change in the protein levels of Akt and mTOR. This transfection caused a decrease in the levels of the phosphorylated (activated) form of mTOR (Ser2448), a downstream target of PI3K/Akt, which has been revealed to promote cell growth. The results of the present study indicated the potential impact of XPC on the Akt/mTOR signaling pathway. Furthermore, silencing of XPC increased the levels of active caspase-3 protein following DDP treatment (Fig. 4B). Collectively, the data indicated the effects of XPC on the Akt/mTOR signaling pathway and confirmed that XPC plays a vital role in the regulation of apoptosis in A549/DDP cells.