One identified general human lung cancer cell line, H460, was cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco by Life Technologies™, Grand Island, NY, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in the presence of 5% CO₂.

Ten participants were recruited at the Xiangya Hospital, Central South University (Changsha, Hunan, China). Consent forms were obtained from individual patients, and experimental protocols were approved by the Institutional Review Board of Xiangya Hospital. All subjects enrolled in the study were Chinese. All clinical and biological data were available for the samples (Table I). Lung cancer and corresponding non-tumor normal tissues were collected, and each biopsy sample was divided into two sections; one was submitted for routine histological diagnosis, and the remaining section was used for qPCR, immunohistochemistry and western blot experiments.

Total RNA was extracted from the lung cancer and corresponding non-tumor normal tissues using TRIzol reagent, and cDNA synthesis was carried out using the RevertAid First Strand cDNA Synthesis kit (both from CWBio, Beijing, China) according to the manufacturer's recommendations. Quantitative real-time PCR (qRT-PCR) was carried out with GoTaq qPCR Master Mix (Promega, Madison, WI, USA). For detection of Fra-1 mRNA expression levels, GAPDH was amplified in parallel as an internal control. The sequences of the primers used for qPCR were as follows: Fra-1 forward, 5′-gcatgggctaaggatttgaa-3′and reverse, 5′-tcccaaatttagcctgttgg-3′; and GAPDH forward, 5′-cga ccactttgtcaagctca-3′ and reverse, 5′-actgagtgtggcagggactc-3′. The expression of mRNA was assessed by evaluated threshold cycle (CT) values. The CT values were normalized with the expression levels of GAPDH, and the relative amount of mRNA specific to each of the target genes was calculated using the 2^−ΔΔCT method (17–22). qPCR was carried out with the Bio-Rad CFK96™ Real-Time system (Bio-Rad, Hercules, CA, USA). The data were analyzed by Bio-Rad CFK manager software (Bio-Rad). Expression of mRNA was assessed by the evaluated threshold cycle (CT) values and GAPDH was used as an internal control.

Immunohistochemistry was conducted using the peroxidase anti-peroxidase technique following a microwave antigen retrieval procedure. The antibody for Fra-1 was purchased from Boster Biotechnology (Wuhan, China). The antibody against Fra-1 (1:100) was overlaid on lung cancer and corresponding non-tumor normal tissue sections and incubated overnight at 4°C. Secondary antibody incubation (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was performed at room temperature for 30 min. Color reaction was developed by using 3,3′-diaminobenzidine tetrahydrochloride (DAB) chromogen solution. All slides were counterstained with hematoxylin. Positive control slides were included in every experiment in addition to the internal positive controls. The specificity of the antibody was determined with a matched IgG isotype antibody as a negative control.

Sections were evaluated in a blinded manner by two investigators in an effort to provide a consensus on staining patterns by light microscopy (Olympus). Fra-1 staining was assessed according to the methods described by Hara and Okayasu (23) with minor modifications. Each case was rated according to a score that added a scale of intensity of staining to the area of staining. At least 10 high-power fields were chosen randomly, and >1,000 cells were counted for each section. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1+, <30% of tissue stained positive; 2+, between 30 and 60% stained positive; 3+, >60% stained positive. The minimum score when summed (extension + intensity) was, therefore, 0, and the maximum, 6. A combined staining score (extension + intensity) of ≤2 was considered to be a negative staining (low staining); a score between 3 and 4 was considered to be moderate staining; whereas a score between 5 and 6 was considered to be strong staining. An optimal cut-off level was identified as follows: a staining index score of 0–2 was used to define tumors with negative expression and 3–7 indicated positive expression of these two proteins. Agreement between the two evaluators was 95%, and all scoring discrepancies were resolved through discussion between the two evaluators.

The coding region of the Fra-1 gene was generated by PCR with the primer pair 5′-atactcgaatgaacctggccatcagcat-3′ and 5′-gcggaattctcacagggacatgaaatccg-3′. PCR was performed under the following conditions: 1 cycle for 5 min at 94°C; 30 cycles for 45 sec at 94°C, 45 sec at 55°C, and 90 sec at 72°C, and ended with 10 min at 72°C. The fragments were cloned into the TA vector (Promega) and used to transform E. coli JM109 (Takara, Dalian, China). Following selection and propagation, the pure plasmid DNA was prepared by standard methods. The DNA fragments were removed from the TA vector by restriction enzyme digestion with XhoI and EcoR1 (Promega) to subclone into the pEGFP-N1 vector. The fusion sequences were verified by DNA sequencing using ABI 3730.

Cell transfection was accomplished using Lipofectamine, according to the manufacturer's instructions (Invitrogen, USA). Cells (2×10⁵) were plated into each well of a 6-well plate 24 h prior to the transfection. For each transfection, 2 µg of pEGFP-N1-Fra-1 plasmid and pEGFP-N1 vector plasmid were transfected into the H460 cells, respectively. The plasmids were diluted with 100 µl of serum-free media, and 4 µl Lipofectamine was added into 100 µl serum-free media. The two solutions were combined, mixed gently and incubated at room temperature for 30 min. Then 200 µl of the mixture and 200 µl of serum-free media were added into each well. The cells were then incubated at 37°C for 24 h, followed by replacing the transfection media with fresh complete culture media. After an additional 48 h of culture, the cells were harvested for the following cell apoptosis, intracellular reactive oxygen species (ROS) measurement, and western blot experiments.

Cell apoptosis was analyzed by flow cytometric analysis using a MoFlo™ XDP High-Performance Cell Sorter (Beckman Coulter, Brea, CA, USA), propidium iodide (PI) and Hoechst 33342 double staining (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). Briefly, H460 cells were transfected transiently with pEGFP-N1-Fra-1 plasmid and the pEGFP-N1 vector plasmid, respectively. After 48 h, the cells were collected in an Eppendorf tube and washed twice with PBS by centrifugation. The supernatants were discarded. To detect apoptosis, 500 µl PBS, 5 µl Hoechst 33342 and 5 µl PI were added to each tube, and the contents of the tube were mixed in the dark at room temperature for 15 min, followed by FCM testing (Beckman Coulter). Data were acquired and analyzed with Summit v5.2 software (Beckman Coulter).

The impact of Fra-1 was measured by flow cytometry using the sensitive and relatively mitochondrion-specific lipophilic cationic probe fluorochrome JC-1. JC-1 accumulates to form J-aggregates and emits red fluorescence in the mitochondria with a high membrane potential, yet dissociates into monomers and emits green fluorescence in those that lose cross-membrane electrochemical gradient. The cells were suspended in 1 ml of warm staining buffer at ~1×10⁶ cells/ml and incubated at 37°C for 5 min. Then 1 µl of 2 mM JC-1 was added (2 µM final concentration) and the cells were incubated at 37°C in 5% CO₂, for 15 to 30 min. The cells were pelleted by centrifugation, resuspended by gently flicking the tubes, and 500 µl PBS was added to each tube. Cells were analyzed with MoFlo™ XDP High-Performance Cell Sorter. Data were acquired and analyzed with Summit v5.2 software.

The production of intracellular ROS was measured by performing flow cytometry using the oxidation-sensitive probe, 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Applygen, Beijing, China). Briefly, 10 mM DCFH-DA stock solution (in methanol) was diluted 4,000-fold in cell culture medium without serum or other additives to yield a 2.5-mM working solution. After exposure of human umbilical vein endothelial cells (HUVECs) to silica nanoparticles for 3 and 24 h, respectively, the cells in 6-well plates were washed twice with PBS and incubated in 2 ml working solution of DCFH-DA at 37°C in the dark for 30 min. Then the cells were washed twice with cold PBS and resuspended in the PBS for analysis of intracellular ROS by FACS (Beckman Coulter).

Intracellular Ca²⁺ concentration was measured by means of the fluorescent Ca²⁺ chelator Fura-2 AM, which permeates into cells where it is cut into Fura-2, resorting within cells. Fura-2 combines with intracellular Ca²⁺ to form a fluorescent compound, whose fluorescent intensity was determined at an excitation wavelength of 340 nm and an emission wavelength of 510 nm in FACS (Beckman Coulter). After treatment, the cells were harvested and rinsed with PBS. The harvested cells were suspended in PBS and incubated with 5 µM Fura-2 AM for 60 min at 37°C. During the session of incubation with Fura-2 AM, the cell cultures were mildly shaken at intervals of 10 min aimed to facilitate the combination of Fura-2 and Ca²⁺ to form the fluorescent compound. Then, the cells were washed twice and resuspended in PBS for FACS measurement (Beckman Coulter). Data were acquired and analyzed with Summit v5.2 software.

The lung cancer tissues, corresponding non-tumor normal tissues and H460 cells were lysed in RIPA buffer (CWBio), and total protein concentration was determined using the Pierce^® BCA protein assay kit (Thermo Scientific, Inc., Rockford, IL, USA). Extracts containing 50 µg of proteins were separated on 10% SDS-PAGE gels and electroblotted onto nitrocellulose membranes (Hyclone Laboratories). The membranes were inhibited using Tris-buffered saline/Tween-20 (25 mM Tris-HCl, 150 mM NaCl, pH 7.5 and 0.05% Tween-20) containing 5% non-fat milk followed by overnight incubation at 4°C with the primary antibodies (rabbit anti-MDM2 antibody, 1:200 and rabbit anti-P53 antibody, 1:200; Boster Biotechnology). Following three washes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (catalog no. sc-2491, dilution 1:5,000; Santa Cruz Biotechnology, Inc.) and the specific signals were visualized using an ECL detection system. The anti-GAPDH antibody (1:3,000; Santa Cruz Biotechnology, Inc.) was used as a loading control.

Differences in non-parametric variables were analyzed by the Mann-Whitney U test. Differences in the quantitative variables between groups were analyzed by Student's t-test using SPSS 11.0 program (SPSS, Chicago, IL, USA). A probability value (p) <0.05 was considered to indicate a statistically significant result.

To detect the mRNA expression levels of the Fra-1 molecule in lung cancer and the adjacent non-cancerous tissues, 10 samples of each were selected to perform qPCR of the Fra-1 gene. The data were analyzed using the 2^−ΔΔCT method, and the fold change in the expression of this gene relative to the internal control gene, GAPDH, was analyzed. The expression of the Fra-1 gene was higher in the lung cancer samples compared with the adjacent non-cancerous tissues and the normalized Fra-1 gene expression in lung cancer was upregulated by 3.76-fold (p=0.0094<0.01) (Table II, Fig. 1A).

To determine whether the Fra-1 gene is expressed at a higher level in lung cancer compared with adjacent non-cancerous tissues, the protein expression levels of Fra-1 were further examined by western blot analysis in 1 to 3 samples (Fig. 1B). In comparison with the adjacent non-cancerous tissues, the expression level was identified to be higher in the lung cancer tissues, which corresponded with the qPCR results.

To confirm the pattern of Fra-1 in lung cancer, immunohistochemistry (IHC) was carried out with antibodies against the Fra-1 protein in lung cancer and the adjacent non-cancerous tissues. Fra-1 was identified as being differentially expressed between lung cancer tissues vs. the adjacent non-cancerous tissues. IHC showed a similar pattern in protein expression with the western blot results. A total of 50.09% (13/22) of lung cancer tissues had high Fra-1 expression and 13.64% (3/22) of the adjacent non-cancerous tissues had a high score. The distribution of low scores was 9.09% (2/22) and 63.64% (14/22) in the lung cancer and the adjacent non-cancerous tissues, respectively (p=0.012<0.05) (Table III). This corresponded with the qPCR results.

We found that Fra-1 was highly expressed in the lung cancer tissues by qPCR, western blot and IHC technologies. To elucidate the function of Fra-1 in the apoptosis of lung cancer cells, the lung cancer cell line, H460, was transfected with the plasmid pEGFP-N1/Fra-1 or the control vector. After determining Fra-1 protein by qPCR technologies, we performed a Hoechst 33342/PI double-staining experiment to test the rate of apoptosis in the H460, H460/vector, and H460/Fra-1 cells. A considerable decrease in apoptotic cells was observed in the H460/Fra-1 cells (6.29±0.41%) compared with the H460/vector (11.73±0.85%) and H460 cells (12.04±0.92%) (Fig. 2A).

Cell apoptosis is closely related with a reduction in ΔΨm and an increase in intracellular ROS and Ca²⁺ concentrations. Thus, we tested the influence of Fra-1 on these three parameters. Our results showed that overexpression of Fra-1 in lung cancer H460 cells, led to an increase in ΔΨm and inhibited cell apoptosis (Fig. 2B). ROS results showed that overexpression of Fra-1 in lung cancer H460 cells, led to a decrease in intracellular ROS (Fig. 2C). Ca²⁺ experimental results showed that overexpression of Fra-1 in lung cancer H460 cells, led to a decrease in Ca²⁺ concentrations and suppressed the apoptosis of the lung cancer cells (Fig. 2D).

To uncover the possible mechanism of Fra-1 in lung cancer, we tested the expression levels of key molecules in the P53 signaling pathway by western blot technology. P53 was downregulated in the lung cancer tissues compared with the level in the adjacent non-cancerous tissues, while MDM2 was upregulated in the lung cancer tissues (Fig. 3). Combined with the above result for which Fra-1 was upregulated in lung cancer, we propose that Fra-1 is correlated with dysregulation of the P53 signaling pathway in lung cancer tissues in vitro.

To confirm whether Fra-1 affects the expression of P53 and MDM2 in vivo, the H460 cells were transfected with the plasmid pEGFP-N1/Fra-1 or control vector. We harvested the cells and tested the expression levels of P53 and MDM2 proteins in vivo. P53 was downregulated in the H460 cells in which Fra-1 was overexpressed, while MDM2 was upregulated (Fig. 4). Our results revealed that Fra-1 overexpression affected the expression of P53 and MDM2 in vivo.