Human lung carcinoma cell line A549 was purchased from ATCC. These cells were cultured with the RPMI-1640 complete medium (Gibco-BRL, Grand Island, NY, USA) containing 10% fetal calf serum (FCS), supplemented with 2.2% (w/v) sodium bicarbonate, 0.03% (w/v) L-glutamine, as well as 100 U/ml penicillin and 100 mg/ml streptomycin, in a 37°C, 5% CO₂ incubator.

Totally 343 patients with NSCLC (squamous carcinoma or adenocarcinoma) were screened in this study, who had admitted to the Affiliated Tumor Hospital, Xinjiang Medical University and received standard lung cancer resection (i.e., lobectomy and systematic mediastinal lymph node dissection), from January 1, 2010 to December 31, 2013. Inclusion criteria were as follows: i) patients with complete follow-up data; ii) patients who had not received chemotherapy before surgery; iii) patients with cancer at stage II or III who received systematic platinum-based two-drug combination chemotherapy for four cycles or radiotherapy after surgery; iv) patients with cancer at stage I who did not receive systematic chemotherapy until tumor recurrence during the follow-up period; and v) patients from whom the tumor and adjacent tissues were obtained. After screening, 140 patients were finally included in this study. Prior written and informed consent were obtained from every patient and the study was approved by the ethics review board of the Affiliated Tumor Hospital, Xinjiang Medical University.

These 140 patients with NSCLC were followed up by telephone, which begun from the date of surgery and ended at Jun 1, 2015. Endpoint events included tumor recurrence and patient death. Evaluation indexes included the 1-, 2-, and 3-year disease-free survival (DFS) and OS rates.

The expression of ERCC1 was detected with immunohistochemistry, according to Hubner et al (18). The tumor and adjacent tissues from NSCLC patients were obtained and cut into 5-µm sections. After dewaxing and rehydration, these sections were treated with 3% H₂O₂ for 20 min, followed by antigen retrieval for 10 min. After blocked with serum blocking solution at room temperature for 30 min, these sections were incubated with mouse anti-human anti-ERCC1 monoclonal antibody (1:100 dilution; ab2356; Abcam, Cambridge, MA, USA) at 4°C overnight. Then the sections were incubated with biotinylated secondary antibody at 37°C for 1 h. After washing, these sections were treated with SABC agent and subjected to DAB colorization. After hematoxylin staining, dehydration, xylene clearing, and neutral resin sealing, the sections were observed under microscope.

Immunohistochemical assessment was performed by two independent senior physicians from the Department of Pathology, according to the evaluation and scoring criteria from Planchard et al (19). Five fields with high magnification (×400) were randomly selected from each section, and totally 100 cells were counted. Semi-quantitative H-score indicating the relative protein expression level was obtained as the product of the staining intensity score and the positive tumor cell percentage. For the staining intensity score: 0, negative (no staining); 1, weak positive (light yellow staining); 2, positive (dark yellow staining); and 3, strong positive (brown staining). For the positive tumor cell percentage: 0, no positive tumor cells; 0.1, 1–9%; 0.5, 10–50%, and 1.0, >50%.

Cell proliferation was assessed with the MTT assay (20). Briefly, cells were seeded onto 96-well plates and cultured overnight. The cells were pre-incubated with or without the p38MAPK inhibitor BIRB796 (SelleckChem, Houston, TX, USA) for 1 h and then with diamminedichloroplatinum (DDP; Sigma-Aldrich, St. Louis, MO, USA) at indicated concentrations. After 68 h, these cells were treated with 20 µl MTT (4 mg/ml) for 4 h. After the medium was discarded, 120 µl dimethylsulfoxide (DMSO) was added into each well. The absorbance at 655 nm was read by the Model 550 microplate reader (Bio-Rad, Hercules, CA, USA).

After treatment, cells were lysed with lysis buffer. The protein concentration was determined, and protein sample was subjected to SDS-PAGE and then transferred onto the nitrocellulose membrane. After blocked with 5% non-fat milk in TBST at room temperature for 2 h, the membrane was incubated with primary antibodies against p38 (ab170099), p-p38 (ab178867), β-tubulin (ab6046) and ERCC1 (ab2356) (all from Abcam) at 4°C overnight. The membrane was then incubated with HRP-conjugated secondary antibody (1:5,000 dilution; Abcam) at room temperature for 2 h. After washing with TBST, the protein bands were visualized by the enhanced Phototope™-HRP Detection kit (Cell Signaling Technology, Beverly, MA, USA) and exposed to Kodak medical X-ray processor (Carestream Health, Rochester, NY, USA). The p38 was used as loading control.

After treatment, total RNA was extracted with the TRIzol reagent RNA Extraction kit (Molecular Research Center, Cincinnati, OH, USA), following the manufacturer's instructions. The first-strand cDNA was synthesized by RevertAid™ Premium First-Strand cDNA Synthesis kit (Fermentas International Inc., Burlington, ON, Canada). The PCR primer sequences were as follows: ERCC1 forward, 5′-TGTCCAGGTGGATGTGAAAGAT-3′ and reverse, 5′-GGCCTTGTAGGTCTCCAGGTA −3′; and GAPDH forward, 5′-TGTTGCCATCAATGACCCCTT-3′ and reverse, 5′-CTCCACGACGTACTCAGCG-3′. PCR was performed on the Gene Amp PCR system 9700 (PE Applied Biosystems, Foster City, CA, USA), with the following conditions: Denaturation at 94°C for 2 min; then 95°C for 30 sec, 61°C for 30 sec, and 72°C for 1 min, for totally 30 cycles; followed by extension at 72°C for 10 min. Products were resolved and examined by 1.0% agarose gel electrophoresis. Quantitative real-time PCR was performed on the Bio-Rad CFX96™ real-time system (Applied Biosystems, Framingham, MA, USA). Relative mRNA expression levels of ERCC1 were determined with the 2^−ΔΔCt method.

SPSS20.0 software was used for statistical analysis. The χ² test and non-parametric Wilcoxon rank sum test were performed for the comparison of clinical data. The Kaplan-Meier analysis with Breslow test was used for the survival analysis. Cox regression model was applied for the multivariate analysis. P<0.05 was considered as statistically significant.

To investigate the expression levels of ERCC1 in the NSCLC tumor and adjacent tissues, immunohistochemistry was performed. Positive and negative staining of ERCC1 in the tissues from NSCLC (squamous carcinoma and adenocarcinoma) was shown in Fig. 1A. The positive staining of ERCC1 was mainly located in the nucleus. The H-score assessment showed that for the tumor tissues, the median H-score was 2, which was higher than the median H-score for the adjacent tissues (0) (P<0.01) (Fig. 1B and C). When H-score ≥2.0 in the tumor tissue was considered as positive (i.e., the staining intensity score >2 while the positive tumor cell percentage >50%), the positive staining percentage for the tumor tissue was 46.4% (65/140). These results suggest that the expression level of ERCC1 is upregulated in the NSCLC tumor tissue.

The association between the ERCC1 expression and clinicopathological features of NSCLC was next investigated. As shown in Table I, the expression of ERCC1 is associated with the NSCLC pathological type. The expression level of ERCC1 in the squamous carcinoma (59.3%, 35/59) was significantly higher than the adenocarcinoma (37%, 30/81) (P<0.01). Moreover, significant differences were observed in the ERCC1 expression levels between different histological stages (P<0.05), and the expression level of ERCC1 showed an increasing trend along with the decreasing differentiation degrees. Furthermore, the expression level of ERCC1 for the smoking patients (60.3%, 41/68) was significantly higher than the non-smokers (33.3%, 24/72) (P<0.01). On the other hand, the ERCC1 expression was not significantly associated with the other investigated clinicopathological features, including age, sex, pathological staging, T staging, N staging, and history of radio- and/or chemotherapy. These results suggest that the expression of ERCC1 is significantly associated with the NSCLC pathological type, histological grade, and patient smoking status.

The association between the ERCC1 expression and postoperative prognosis of NSCLC was next investigated. Our results showed that in the term of DFS, for the ERCC1-positive NSCLC patients, the 1-, 2-, and 3-year DFS rates were 79.8, 69.1, and 53.4%, respectively, with the median DFS of 22.17 m (ranging from 9.85 to 34.49 m). On the other hand, the 1-, 2-, and 3-year DFS rates for the ERCC1-negative NSCLC patients were 85.9, 76.8, and 66.8%, respectively, with the median DFS of 28.4 m (ranging from 19.34 to 37.46 m) (Table II). The Kaplan-Meier survival analysis showed that the DFS for ERCC1-negative patients was superior to the ERCC1-positive patients (Fig. 2A). In the term of the OS, for the ERCC1-positive NSCLC patients, the 1-, 2-, and 3-year OS rates were 88.4, 67.9, and 64.7%, respectively, with the median OS of 51.93 m (ranging from 41.69 to 62.17 m). On the other hand, the 1-, 2-, and 3-year OS rates for the ERCC1-negative NSCLC patients were 94.2, 87.1, and 84.2%, respectively (Table II). The survival analysis showed that the OS for ERCC1-negative patients was superior to the ERCC1-positive patients (Fig. 3A).

Considering the impact of other factors from the adjuvant chemotherapy, subgroup analysis was performed for the combined data of NSCLC patients at stages II–III. As shown in Table III, no significant effects of the ERCC1 expression on DFS or OS were observed for the NSCLC patients at stage I (Figs. 2B and 3B). For the NSCLC patients at stages II–III, no significant association was observed between the ERCC1 expression and DFS (Fig. 2C), while the 3-year OS for ERCC1-negative patients (74.2%) was significantly higher than the ERCC1-positvie patients (56.1%) (P<0.05) (Fig. 3C). These results suggest that the elevated expression of ERCC1 in the NSCLC patients who need the postoperative adjuvant chemotherapy may indicate the tumor resistance.

Since the expression level of ERCC1 for the squamous carcinoma was higher than the adenocarcinoma, according subgroup analysis was further conducted. As shown in Table IV, the 3-year DFS and OS rates for the patients with ERCC1-negative squamous carcinoma were 40.7 and 68.4%, respectively, with the median DFS of 13.63 m (ranging from 2.73 to 24.53 m). On the other hand, the 3-year DFS and OS rates for the patients with ERCC1-positie squamous carcinoma were 14.8 and 57.7%, respectively, with the median DFS of 33.63 m (ranging from 13.95 to 53.31 m). The Kaplan-Meier survival analysis showed that the DFS and OS rates of the patients with ERCC1-negative squamous carcinoma were superior to the ERCC1-positive patients (both P<0.05) (Figs. 2D and 3D). For the adenocarcinoma, the 3-year DFS and OS rates for the ERCC1-negative patients were 27.8 and 83.4%, respectively, with the median DFS of 27.93 m (ranging from 17.55 to 38.31 m). The 3-year DFS and OS rates for the patients with ERCC1-positive adenocarcinoma were 31.4 and 72.4%, respectively, with the median DFS of 26.27 m (ranging from 13.95 to 53.31 m). No significant effects of ERCC1 expression on the DFS and OS were observed for the patients with adenocarcinoma (Figs. 2E and 3E). In addition, the COX regression multivariate survival analysis showed that the pathologic staging was the only independent factor affecting the patient DFS, while the pathologic staging, T staging, and ERCC1 expression were independent factors affecting the OS of NSCLC patients (Table V).

Both p38MAPK and ERCC1 are closely related to the tumor resistance. Studies have shown that positive correlation could be observed between the ERCC1 and p-p38 levels in the NSCLC tissues (21). The relationship between p38MAPK and ERCC1, and its effect on the cell tumor resistance, were then investigated. Our results from the MTT assay showed that the treatment of p38 inhibitor, BIRB796, significantly enhanced the sensitivity of A549 cells to cisplatin (Fig. 4A). The mRNA and protein expression levels of ERCC1, and the performance of the p38 signaling pathway, were then investigated. Our results showed that along with the increasing treatment concentrations of cisplatin, the mRNA and protein expression levels of ERCC1, and the level of p-p38, were gradually elevated, which could be significantly declined by the treatment of 10µmol/LBIRB796 (Fig. 4B and C). These results suggest that the p38 signaling pathway could exert direct or indirect regulatory effects on the expression of ERCC1.