The NSCLC cell lines A549, H520 and PC-9 were obtained from Tianjin Medical University Cancer Institute. The primers for HMGB1, RAGE, MMP-9, PCNA, P53 and ACTIN were all obtained from Invitrogen; Thermo Fisher Scientific, Inc. HMGB1 (cat. no. ab227168), RAGE (cat. no. ab3611), MMP-9 (cat. no. ab38898) antibodies were all purchased from Abcam. ACTIN (cat. no. sc-58673), GAPDH (cat. no. sc-365062), P53 (cat. no. sc-126), pCNA (cat. no. sc-9857), Bax (cat. no. sc-4239), Bcl-2 (cat. no. sc-7382), Mcl-1 (cat. no. sc-12756) and STAT3 (cat. no. sc-8019) antibodies were purchased from Santa Cruz Biotechnology, Inc. The antibodies against NF-κB (cat. no. 8242), p-NF-κB (cat. no. 3033 and p-STAT3 (cat. no. 9131) were all purchased from Cell Signaling Technology, Inc.

MTT was purchased from Roche Molecular Biochemicals. EP was purchased from Sigma-Aldrich; Merck KGaA and RPMI-1640 from Thermo Fisher Scientific, Inc. Fetal bovine serum (FBS) was obtained from Gibco; Thermo Fisher Scientific, Inc. and TRIzol reagent was purchased from Invitrogen; Thermo Fisher Scientific, Inc. The PrimeScript RT Reagent kit (Perfect Real-Time) and SYBR Premix Ex Taq (Tli RNaseH Plus) were obtained from Takara Biotechnology Co., Ltd. The cell apoptosis kit with propidium iodide and Annexin V-FITC was from BD Pharmingen.

The cell lines A549, H520 and PC-9 were all cultured in RPMI-1640 medium. All three cultures were supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml). The cell lines were all maintained at 37°C in a humidified atmosphere containing 5% CO₂.

The MTT assay was used to analyze cell growth. Following treatment with EP, the cells were incubated in a 96-well-plate at a density of 3×10⁴ cells/well. MTT (10 µl, 5 mg/ml) was added to the wells at 0, 1, 2, 3 and 4 days of culture. After incubation for 2 h, the crystals were dissolved by adding DMSO (150 µl/well) and mixed well with a multichannel pipette. The optical density (OD) of the soluble formazan in each well was measured at 490 nm with a microplate reader (Thermo Fisher Scientific, Inc.). All experiments were performed in triplicate.

mRNA expression levels were determined by RT-qPCR. Total RNA was extracted with TRIzol according to the manufacturer's specifications. Reverse transcription was carried out using a Prime Script RT Reagent kit (Perfect Real-Time) and cDNA amplification was conducted using SYBR Premix Ex Taq (Tli RNaseH Plus) according to the manufacturer's instructions. Target genes were amplified using oligonucleotide primers. The ACTIN gene was used as endogenous control. The PCR primer sequences are listed in Table I. Data were analyzed using the comparative Cq method (2^−ΔΔCq) (23). Each experiment was conducted in triplicate.

The cells of each group were collected and treated with EP (0, 5, 10, 20 and 30 mmol/l). Subsequently, proteins were extracted with CellLytic M cell Lysis Reagent with protease inhibitor and phosphatase inhibitor cocktails (all from Sigma-Aldrich; Merck KGaA). The proteins were quantified using a BCA protein assay kit. Proteins (30 µg) were separated by 10% SDS-PAGE. Polyvinylidene difluoride (PVDF) membrane were blocked with a blocking reagent [Tris-buffered saline-0.1% Tween-20 (TBST) containing 5% polyvinyl pyrrolidone (PVP), 5% FBS] at room temperature for 1 h and then incubated with primary antibody (dilution 1:1,000) overnight at 4°C. After washing the membranes three times, PVDF membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology; dilution 1:2,000) for 1 h at normal temperature. Immunoreactive bands were visualized by an Imaging System (Tanon Science and Technology) after adding chemiluminescent HRP substrate (Millipore Corp.). Each experiment was conducted in triplicate.

The A549, H520 and PC-9 cells were treated with EP and seeded in 6-well plates with 800 cells/clone. The culture medium was replaced every 3 days. Colonies were counted when visible with the naked eye. The cells were then fixed with methanol and stained with 5% crystal violet solution. Each experiment was conducted in triplicate.

The percentage of apoptotic cells was calculated by means of an Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (BD Biosciences) according to the manufacturer's instructions. The percentage of apoptotic cells (Annexin⁺/PI⁺) was analyzed via flow cytometry (BD Biosciences). Each experiment was conducted in triplicate.

Transwell chambers (8 µm) were used for cell migration and invasion assays (Corning, Inc.). For the migration assay, 1×10⁵ cells were harvested and seeded on the upper chamber. For the invasion assay, 3×10⁵ cells were harvested and placed in the upper chamber coated with Matrigel (300 µg/ml, 100 µl). RPMI-1640 (600 µl) supplemented with 20% FBS was added to the lower chamber. The migrating or invading cells were fixed with 100% methanol for 30 min and stained with 5% crystal violet solution for 8 min post 24 h. Cells in 5 random fields (×4) were calculated using Nikon MA100 optical microscope (Nikon Corp., Tokyo. Japan). Each assay was performed in triplicate.

All statistical analyses were carried out using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Results are presented as mean ± standard deviation. The capability of cell migration and invasion between 20 mmol/l EP and 0 mmol/l EP in Fig. 3 (bar graph) were calculated using independent sample t-tests. Furthermore, the statistical analysis method used for the data of mRNA expression level and cell apoptosis index in Figs. 1, 2 and 4 (bar graphs) was the Kruskal-Wallis followed by Dunnett's post hoc test when the 0 mmol/l EP group was compared with the 5, 10, 20 and 30 mmol/l EP groups. P<0.05 was considered to indicate a statistically significant difference.

RAGE, also known as AGER, is a transmembrane receptor that binds to a variety of ligands, including HMGB1 (24,25). A549, H520 and PC-9 cells were treated with EP (0, 5, 10, 20 and 30 mmol/l) in order to investigate its effects on HMGB1 and RAGE protein as well as mRNA expression. The protein and mRNA expression levels of HMGB1 were significantly decreased with increasing EP dose (Fig. 1A and B). Similarly, the mRNA and protein expression levels of RAGE were also decreased with increasing EP dose (Fig. 1C and D).

NF-κB regulates DNA transcription, cytokine production and cell survival (26). In the present study, A549, H520 and PC-9 cells were treated with 30 mmol/l EP for 2, 4, 6 and 8 h in order to examine NF-κB and p-NF-κB levels. The protein expression of p-NF-κB was gradually decreased over time (Fig. 1E). STAT3 is a member of the STAT protein family (27). A549, H520, PC-9 cells were treated with 30 mmol/l EP for 2, 4, 6 and 8 h in order to examine STAT3 and p-STAT3 levels. The protein expression level of p-STAT3 was also decreased over the specified time period (Fig. 1F).

A hallmark of cancer is enhanced cell proliferation (28). A549 H520 and PC-9 cell growth was assessed via MTT and colony formation assays. It was observed that A549, H520 and PC-9 cell growth was markedly suppressed by EP in a dose/time-dependent manner (Fig. 2A). The colony formation rate of A549, H520 and PC-9 cells was significantly decreased in the EP-treated groups (Fig. 2B and C).

Proliferating cell nuclear antigen (PCNA) is a DNA clamp that is essential for replication (29) and tumor progression. Therefore, anticancer treatment effectiveness may be determined by PCNA (30). PCNA was investigated by western blot analysis and RT-qPCR. The data demonstrated that the expression of PCNA was decreased in the EP-treated groups (Fig. 2D and E).

A Transwell assay demonstrated that the migration and invasion potential of A549, H520 and PC-9 cells in the EP-treated groups was reduced compared with that of the control group (Fig. 3A-D).

Matrix metallopeptidase 9 (MMP-9) participates in the degradation of the extracellular matrix (31). MMP-9 may be associated with the development of various human malignancies by affecting invasion, metastasis, growth and angiogenesis (32,33). Western blot analysis revealed downregulation of MMP-9 (Fig. 3E). The results were consistent with those of RT-qPCR (Fig. 3F).

Flow cytometry was used to determine whether EP affects the apoptotic process of lung cancer cells. The results demonstrated that the apoptotic index of A549, H520 and PC-9 cells in the EP-treated groups was significantly higher compared with that of the control group (Fig. 4A and B).

P53, a tumor suppressor, directly participates in the endogenous apoptotic pathway by interacting with members of the Bcl-2 family (34,35). Western blot analysis and qPCR were used to determine P53 changes. The data demonstrated that the protein and mRNA levels of P53 increased with increasing EP concentration (Fig. 4C and D).

The Bcl-2 family plays an important role in the regulation of apoptosis, a type of programmed cell death. Bcl-2 family members may promote or inhibit apoptosis (36). Western blot analysis revealed that EP treatment increased the pro-apoptotic protein Bax and decreased the anti-apoptotic proteins Bcl-2 and Mcl-1 (Fig. 4E).