The normal human bronchial epithelial (HBE) cell line and 8 human lung cancer cell lines (95-D, A549, NCI-H1299, NCI-H1975, NCI-H661, NCI-H446, NCI-H1395 and Calu-3) were purchased from the American Type Culture Collection (Manassas, VA, USA). SPCA-1 and LTEP-α-2 cell lines were obtained from Shanghai Institutes for Biological Sciences (Shanghai, China). Primary lung adenocarcinoma and corresponding non-cancerous tissues were obtained during surgery from 24 patients who were treated at Kunshan First People’s Hospital affiliated to Jiangsu University (Kunshan, China). The materials to be analyzed were selected by a pathologist to ensure that macroscopically the samples were entirely cancerous and from an area devoid of necrotic tissue. Cancerous and non-cancerous tissues were stored at −80°C until protein extraction. Patients provided written informed consent, and the study was approved by the ethics committee of Jiangsu University.

The human lung cancer cell lines, 95-D, A549, NCI-H1299, NCI-H1975, NCI-H661, NCI-H446, NCI-H1395, Calu-3, SPCA-1, LTEP-α-2, and the HBE cell line were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI-1640 supplemented with 10% fetal calf serum, L-glutamine (5 mmol/l), non-essential amino acids (5 mmol/l), penicillin (100 U/ml) and streptomycin (100 U/ml) (Invitrogen, Carlsbad, CA, USA), at 37°C in a humidified 5% CO₂ atmosphere.

The HMGB1 eukaryotic expression vector construction and plasmid transfection using Lipofectamine were performed as previously described (11). The expression of HMGB1 was determined by western blot analysis.

Cell proliferation was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, the most common assay for determining cell growth and death. The MTT survival assay has been described in detail in previous studies (12). Briefly, exponentially growing cells were recultured (5,000 cells/well) overnight in 96-well tissue culture plates. Up to 20 μl of MTT (Sigma Aldrich, St. Louis, MO, USA) was added to the medium in each well, with a final concentration of 2 mg/ml. After 4 h of incubation, the medium containing MTT was discarded, and 120 μl of DMSO were added followed by incubation for 10 min. The absorbance was measured using an ELISA reader at 570 nm, with the absorbance at 630 nm taken as the background correction. Cell viability was expressed as a percentage of the untreated controls. All experiments were performed at least 3 times.

The invasion assay was performed using Transwell filters (Millipore, Billerica, MA, USA) as previously described (13). The Transwell filter surfaces (8 μm pore size) were uniformly coated with 25 mg Matrigel overnight at 4°C prior to the experiment. The lower chamber was filled with culture medium containing 10% fetal calf serum, and the subconfluent proliferating cells were carefully transferred onto the coated upper surface of the chamber. After 24 h of incubation, the filter was gently removed, and the upper surface of the filter was wiped using a cotton swab to remove all attached cells. The cells that invaded the Matrigel and attached to the lower surface of the filter were fixed with 4% paraformaldehyde, stained with Giemsa, and then counted in 15 randomly selected microscopic fields per filter. Finally, the invasion rate was calculated and compared. Experiments were performed independently at least 3 times.

Western blot analysis was performed as previously described (14). The following primary antibodies were used for immunoblotting: β-actin (C-4), HMGB1 (E-1), nuclear factor (NF)-κB (P65A) and secondary antibody horseradish peroxidase (HRP)-labeled goat anti-mouse (GAM-007), goat anti-rabbit (SC-2004) IgG, as well as phospho-ERK1/2 (T202/Y204), ERK1/2, phospho-p38 (T180/Y182) and p38 (Cell Signaling Technology, Danvers, MA, USA, 1:1,000 dilution). The protein bands were quantified by densitometry using QuantityOne software (Bio-Rad, Hercules, CA, USA), and the values were expressed relative to β-actin (control for loading and transfer). At least 3 independent experiments were performed for each cell type examined.

mRNA expression was determined by RT-PCR. PCR reaction conditions and cycle numbers were rigorously adjusted so that each reaction occurred within the linear range of amplification. Detailed methods for RNA isolation, cDNA synthesis and RT-PCR analyses have been previously described (15). The primers for gene-specific PCR were as follows: GAPDH sense, 5′-CAA CTA CAT GGT CTA CAT GTT CC-3′ and antisense, 5′-CAA CCT GGT CCT CAG TGT AG-3′; and HMGB1 sense, 5′-TTA GAT AGC CCT GTC CTG GTG G-3′ and antisense, 5′-TGA ATG TGG CAT CTT TGT TTG A-3′. PCR products were analyzed by electrophoresis using 1% agarose gels containing 0.1 mg/ml ethidium bromide and the gels were photographed under ultraviolet light. mRNA expression levels were quantified by densitometry of the cDNA bands using QuantityOne software (Bio-Rad). At least 3 independent experiments were performed for each cell type examined.

The data are presented as the means ± SD. Statistical comparisons of the experimental results between the treated group and the control group were made using the two-tailed Student’s t-test. All statistical tests were performed using SPSS version 17.0 software. P≥0.01 was considered to indicate a statistically significant difference.

In order to elucidate the role of HMGB1 in lung cancer, we examined the level of HMGB1 expression in lung adenocarcinoma cells by western blot analysis using clinical samples. A total of 24 lung adenocarcinoma cases were examined. In each patient, the level of HMGB1 expression normalized to β-actin expression in cancerous tissue was compared to that observed in non-cancerous tissue. In 87.5% (21/24) of the specimens, HMGB1 expression increased in the cancerous tissues compared to the non-cancerous tissues. The protein bands were quantified using QuantityOne software, and the augmentation was deemed to be statistically significant (P<0.01) (Fig. 1). Therefore, we hypothesized that the overexpression of HMGB1 contributes to the development of human lung adenocarcinoma. However, due to the fact that we analyzed only a small number of specimens, future analysis with a much larger number of specimens is required.

To determine the effect of HMGB1 on the proliferation and metastasis of lung cancer cells, the HMGB1 expression levels in the 10 lung cancer cell lines and the HBE cell line were analyzed by RT-PCR. As shown in Fig. 2, the HMGB1 protein level increased in 8 of the lung cancer cell lines compared to the HBE cell line and there was little or no change in the expression levels of GAPDH in these cell lines. In vitro data confirmed that TOB1 overexpression is an important event in lung cancer.

Cell proliferation and invasion are indispensable for cancer metastasis. Due to the fact that HMGB1 expression was increased in the lung cancer tissues and cell lines, we hypothesized that HMGB1 regulates the progression of lung cancer. According to gain-of-function approaches, A549 and NCI-H1975 cells were selected as model systems, as they are lung adenocarcinoma cell lines that express almost undetectable levels of HMGB1. Moreover, A549 cells are epidermal growth factor receptor (EGFR) wild-type and NCI-1975 cells are EGFR mutated, and EGFR status is known to modulate proliferation and metastasis. Using Lipofectamine and G418-mediated plasmid stable transfection, multiple clones transfected with HMGB1 were selected and confirmed by RT-PCR (Fig. 3A and B). The dynamics of A549, A549/PC3 and A549/HMGB1 cell growth were determined by MTT assay. Following a 6-day period, the overexpression of HMGB1 enhanced the proliferative ability of the A549 cells, compared to the control groups. The marked decrease in cell viability was observed on day 3 (Fig. 3C). We examined the effect of HMGB1 expression on the invasive ability of A549 cells using a Transwell assay. The results demonstrated that, compared to the parental cells, the overexpression of HMGB1 enhances the ability of the A549 cells to invade through the Matrigel-coated filter. As shown in Fig. 3E, the invasion rate of A549/HMGB1 cells increased by >60% corresponding to the vector-transfected cells (P<0.01). In the EGFR-mutated NCI-H1975 cells, the overexpression of HMGB1 also promoted cell proliferation and invasion (Fig. 3D and F).

To determine whether plasmid transfection induces the activation of Erk1/2 and p38 MAPK, we transfected the A549 cells with HMGB1 recombinant plasmid and then immunoblot analysis was performed to determine the phosphorylation levels of Erk1/2 and p38. The results demonstrated that HMGB1 overexpression enhanced the expression levels of phosphorylated Erk1/2 and p38; however, the levels of total Erk1/2 and p38 remained unaffected. These data suggest that Erk1/2 and p38 MAPK are activated in HMGB1-transfected lung cancer cells. The MAPKs integrate a wide range of upstream signals to determine patterns of downstream gene expression through the regulation of transcription factors. NF-κB has been well characterized as a molecule regulated by the MAPK signaling pathway (16). In this study, the expression of NF-κB was upregulated in HMGB1-overexpressed A549 cells (Fig. 4A and C). As for EGFR-mutated NCI-H1975 cells, the overexpression of HMGB1 activated the ERK and p38 MAPK signaling pathways when compared with the control cells, implying that HMGB1 activated the ERK and p38 MAPK signlaing pathways through an EGFR-independent pathway (Fig. 4B and D).