The NSCLC NCI-H1299 cell line [American Type Culture Collection (ATCC), Manassas, VA, USA; ATCC number, CRL-5803™] was grown in RPMI-1640 medium (cat. no. 01-100-1ACS; Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 10% fetal bovine serum (FBS; cat. no. 04-001-1ACS; Biological Industries), 100 IU/ml penicillin and 100 µg/ml streptomycin (cat. no. SV30010; HyCone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in humidified atmosphere containing 5% CO₂.

The primary antibodies comprised of rabbit polyclonal anti-stathmin (1:3,000; cat. no. 569391; EMD Millipore, Billerica, MA, USA), anti-extracellular signal-regulated kinase 1 (ERK1; 1:1,000; cat. no. sc-93), anti-cyclin-dependent 2 (CDC2; 1:1,000; cat. no. sc-954), anti-nuclear factor-κB (NF-κB; 1:1,000; cat. no. SC-109), anti-B-cell lymphoma-2 (Bcl-2; 1:1,000; cat. no. sc-492), mouse monoclonal anti-β-actin (1:2,000; cat. no. sc-47778), anti-phospho(p)-ERK1 (1:1,000; cat. no. sc-7383), anti-caspase-3 (1:1,000; cat. no. sc-7272), anti-p53 (1:1,000; cat. no. sc-126), anti-Bcl-2-associated X protein (1:1,000; Bax; cat. no. sc-7480) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), mouse monoclonal anti-caspase-8 (1:1,000; cat. no. 9746), rabbit monoclonal anti-caspase-9 (1:1,000; cat. no. 9502), rabbit polyclonal anti-p-thr161-CDC2 (1:1,000; cat. no. 9114) (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-stathmin Ser25 (1:1,000; cat. no. ab194752), anti-p-stathmin Ser63 (1:1,000; cat. no. ab76583), anti-interleukin-10 (IL-10; 1:1,000; cat. no. ab34843) and anti-IL-6 (1:1,000; cat. no. ab6672) (Abcam, Cambridge, MA, USA).

The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (which was diluted at 1:3,000 for the detection of stathmin, NF-κB, IL-6 and IL-10, and at 1:2,500 for detecting caspase-9, Bcl-2, p-stathmin-Ser25, -Ser63, phospho-Thr161-CDC2, CDC2 and ERK; cat. no. sc-2004) and rabbit anti-mouse IgG (which was diluted at 1:3,000 for the detection of β-actin and p53, and at 1:2,500 for analyzing caspases-3, −8, Bcl-2-associated X and phosphor-ERK; cat. no. sc-358914) (Santa Cruz Biotechnology, Inc.).

The NF-κB inhibitor ammonium pyrrolidine dithiocarbamate (PDTC; cat. no. 5108-96-3; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) (13) and Taxol (cat. no. sc-201439; Santa Cruz Biotechnology, Inc.) were separately dissolved in dimethyl sulfoxide (DMSO), stored at −20°C and diluted to appropriate concentrations (Taxol, 100 nM; or PDTC, 5 or 10 µM) prior to use.

Nearly confluent cells at the logarithmic growth phase were divided into three groups (blank, C3 and p53^wt) for transfection as follows: Blank control without any plasmid (blank); empty vector pEGFP-C3 (C3); and pEGFP-C3-p53 (p53^wt). The plasmid pEGFP-C3-p53 was constructed by inserting the p53^wt gene into the pEGFP-C3 vector between the two cleavage sites of XholI and KpnI, and PEGFP-C3 was labeled with neomycin and kanamycin resistance genes. G418 (400 µg/ml; Sigma-Aldrich; Merck KGaA) was used to screen cells following transfection for 5 h. The PEGFP-C3 and pEGFP-C3-p53 plasmids were provided by Professor Cao Ya (Cancer Research Institute, Central South University, Changsha, China).

Cell transfection was performed with Lipofectamine^® 2000 (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Additionally 4 µg plasmids in 10 µl Lipofectamine 2000 were transiently transfected into cells at a transfection efficiency of >80% in 250 µl incomplete RPMI-1640 medium.

Protein was extracted with lysis buffer consisting of 50 mM pH 8.0 Tris-HCl, 1 mM ethylenediaminetetraacetic acid, 2% SDS, 5 mM dithiothreitol and 10 mM phenylmethylsulfonyl fluoride. The protein concentration was determined using BCA Protein Assay Reagent (Pierce; Thermo Fisher Scientific, Inc.). Total proteins (50 µg) were then separated by 10% SDS-PAGE and electro-transferred onto nitrocellulose membranes. The membranes were blocked with phosphate-buffered saline (PBS) containing 5% non-fat milk overnight at 4°C, incubated with the aforementioned primary antibodies overnight at 4°C, and then with the aforementioned HRP-conjugated secondary antibodies for 2 h at room temperature. An enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Inc.) was used for immunoblotting. Protein belts were exposed on the films in a dark room, then scanned and saved for the edition by Photoshop 7.0 software (Adobe Systems Inc., San Jose, CA, USA).

Cells (5×10³ cells/well) were seeded in a 96-well plate and each sample was placed in 6 parallel wells. A total of 10 µl 5 mg/ml MTT (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was added to each well for 4 h for 24, 48 or 72 h. The supernatant was then discarded, 100 µl DMSO was added and the plate was shaken with a Transference Decoloring Shaker (ZD-2008; Haimen Kylin-Bell Instrument Manufacturing Co., Ltd., Shanghai, China) at a fixed low speed at room temperature for 10 min. The optical density (OD) value was measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 490 nm. The relative proliferative ratio of the cells per well was calculated according to the following formula: Relative proliferation (%) = (OD_transfection/OD_control) × 100%. The relative proliferative ratio of the blank control was set as 100% at all time points.

Cells were seeded in media at a density of 2×10³ cells/well in 6-well plates (2 parallel wells/sample) for ~2 weeks at 37°C in an incubator with an atmosphere containing 5% CO₂. When clear colonies were observed by the naked eye, the plates were washed three times with PBS and fixed with 100% methanol for 15 min at room temperature. The cells were then stained with crystal violet for 15 min at room temperature and washed with water to remove excess dye. Colonies containing >50 cells were counted under a light inverted microscope (Leica Microsystems GmbH, Wetzlar, Germany) at ×100 magnification. Colony formation rate (%) = (means number of colonies/2000) × 100%. The experiment was repeated three times.

Cells were cultured overnight at 37°C in an atmosphere containing 5% CO₂ to yield a monolayer in 6-well plates, and then the monolayer was scratched using 200-µl pipette tips, washed twice with PBS and RPMI-1640 medium was added. Wound healing status was monitored at 0, 12, 24 and 36 h, and images were captured with an inverted light microscope at ×4 magnification in order to analyze the capability of cell migration in the 2-dimensional plane.

The upper chambers of a Transwell plate with 8.0 µm pore size of polycarbonate membrane of polystyrene plates (cat. no. 3422; Costar, Corning Incorporated, Corning, NY, USA) were pretreated with heated serum-free RPMI-1640 medium for 30 min at 37°C and then the medium was removed. Cells were adjusted to a concentration of 2×10⁵ cells/ml in RPMI-1640 medium with 0.2% FBS. A total of 200 µl sample was added to the upper chamber, and the lower chamber was filled 800 µl RPMI-1640 medium with 10% FBS. The plates were incubated at 37°C for 24 h.

The upper chamber was removed, the medium was discarded and a cotton swab was used to remove cells on the bottom of the upper chamber. The remaining migrated cells on the back of the membrane in the bottom of upper chamber were fixed with 100% methanol for 10 min at room temperature and stained with 0.1% crystal violet for 10 min at room temperature for Transwell analysis in 3-dimensional space. Images were captured in 5 random visual fields under an inverted light microscope at ×40 magnification. Experiments were repeated three times.

A total of 1×10⁶ cells/well were incubated in 24-well plates containing serum-free incomplete RPMI-1640 medium without the special indicator phenol red. After 24 h at 37°C, the supernatant was collected for detection of autocrine IL-10 and IL-6 levels from the tumor cells using ELISA.

The absorbance value was measured at a wavelength of 450 nm with the Human IL-10 (cat. no. EK1102) and Human IL-6 (cat. no. EK1062) ELISA kits [Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China], according to the manufacturer's protocols.

Cells were washed with ice-cold PBS and digested with 0.25% trypsin. Cells were collected for centrifugation at 697 × g for 5 min at room temperature, and the pellet was suspended with PBS as a single-cell solution, which was centrifuged at 697 × g at room temperature and washed twice with PBS. Cells were resuspended with 300 µl PBS and fixed with 700 µl 70% ethanol at 4°C overnight for the detection of cell cycle distribution and apoptosis.

Cell cycle distribution and apoptosis were detected using FACS, which was performed by a specialized institute (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China).

Statistical analysis was performed using the statistical software SPSS, version 17.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Analysis of variance and least significant difference method were applied to perform multiple comparisons between the groups. P<0.05 was considered to indicate a statistically significant difference.

Western blotting for p53 demonstrated that the p53^wt lane exhibited a band while the blank and the C3 lanes did not exhibit any trace of p53, which demonstrated that the NCI-H1299 cells were originally p53-deficient, and that exogenous p53^wt was successfully introduced and expressed in the cells. There were no differences in the expression between the blank and the C3 controls, but the expression of Op18/stathmin was notably impaired in the p53^wt group, compared with the two control groups (Fig. 1A).

Similarly, the levels of Op18/stathmin phosphorylation at the Ser25 and Ser63 sites were notably decreased in the p53^wt group, compared with the two control groups, and there were no evident changes in the expression of Op18/stathmin at these two sites of phosphorylation between the blank and C3 control groups (Fig. 1B).

Cell proliferation curves demonstrated that the relative proliferation ratios were 96.5, 96.3 and 96.2% in the C3 control group at the 24, 48 and 72 h time points, respectively, which were similar to the blank group, that was set as 100% at all time points. Compared with the two approximately parallel curves for the blank and C3 control groups, the representative curve for the p53^wt group descended steeply with proliferation ratios of 83.5, 72.9 and 59.9% at the three time points, respectively (Fig. 2A). The histograms demonstrated that no statistical difference existed between the blank and C3 control groups at the three time points, but the relative proliferation ratio of the p53^wt group was significantly reduced, compared with the two control groups at all time points (P<0.01; Fig. 2B).

Colony formation analysis demonstrated that a number of large colonies composed of several small colonies with merging borders, as depicted in the blank and C3 control groups. By contrast, there were only a few sparsely distributed small colonies in the p53^wt group (Fig. 2C). The mean colony formation ratios, which implied the mean percentage of forming colonies (>50 cells) in 2,000 cells, were 10.65 and 10.23% in the two control groups, respectively, and 6.62% in the p53^wt group, which was significantly reduced, compared with the other groups (P<0.01). The difference between the ratios of the two control groups was not significant (Fig. 2D).

The wound healing assays demonstrated that a large proportion of the scratched area remained empty among all three groups at the 12 h time point. After 24 h, the wound width of the C3 group was reduced, compared with the blank group, but the difference was not notable by the naked eye. The wounds had almost healed at 36 h in the blank and C3 control groups; however, a large area of the scratched region remained empty in the p53^wt group. p53^wt introduction inhibited the migration of NCI-H1299 cells in the 2-dimensional plane (Fig. 3A).

The Transwell assays identified that the mean number of migrated cells was 28, 26 and 14 among the blank, C3 and p53^wt groups, respectively. Therefore, the number of migrated cells was notably decreased following p53^wt introduction (Fig. 3B). Histograms indicated that there was no apparent difference in the number of infiltrating cells between the blank and C3 control groups, but the number of migrated cells was significantly reduced in the p53^wt group, compared with the two control groups; therefore, p53^wt significantly reduced the motility of cells in 3-dimensional space (P<0.01; Fig. 3C).

Western blotting indicated that p53 expression successfully attenuated CDC2 phosphorylation at the thr161 site. The levels of ERK and p-ERK were also downregulated in the p53^wt group, but those of CDC2 were similar among the three groups (Fig. 4A). Additionally, introduction of exogenous p53^wt reduced the expression of NF-κB (Fig. 4B).

The NF-κB inhibitor PDTC was employed to block NF-κB signaling in p53-deficient NCI-H1299 cells. Western blot analysis demonstrated that the levels of p-thr161-CDC2, ERK and p-ERK, with ERK being slightly decreased at 5 µM, compared with at 0 µM and notably reduced at 10 µM, while p-ERK gradually decreased in a PDTC concentration-dependent manner. Blockage of NF-κB signaling also downregulated the activities of p-thr161-CDC2 and ERK, which is in agreement with the results of p53 introduction, and the concentration gradient of PDTC did not exert any effects on CDC2 expression (Fig. 4C).

ELISA confirmed that the mean autocrine IL-10 OD level in the culture supernatant was 0.109, 0.093 and 0.055 in the blank, C3 and p53^wt groups, respectively. The mean level of the p53^wt group was significantly decreased, compared with the two control groups (P<0.01), and there was no significant difference between those of the blank and C3 control groups (Fig. 5A). ELISA demonstrated that IL-6 autocrine OD levels were low in the three groups and p53 did not affect IL-6 (Fig. 5B).

Western blot analysis demonstrated that IL-10 was expressed normally in the blank and C3 control groups, which was similar between them, but was notably attenuated in the p53^wt group. Additionally, IL-6 was not expressed in the three groups (Fig. 5C).

Concentration gradients of the inhibitor PDTC downregulated the expression of IL-10 in a concentration-dependent manner in NCI-H1299 cells, which implied that blockage of NF-κB signaling induced the identical inhibitory effects on IL-10 expression as exogenous p53^wt induction (Fig. 5D).

The images of cell growth demonstrated that cells were nearly confluent in the blank and C3 control groups, but there was reduced quantity of cells in the p53^wt group. FACS analysis validated that the proportion of cell that entered the G₂ phase was 25.94, 24.58 and 2.63% among the blank, C3 and p53^wt groups, respectively, while the cellular apoptosis ratios were 4.58, 5.82 and 9.24%, respectively. By contrast, the majority of NCI-H1299 cells were arrested at G₁/S phases when transfected with exogenous p53^wt (Fig. 6A).

The expression levels of caspase-3 and −9 were increased in the p53^wt group, compared with the two control groups, while no evident changes were observed in caspase-8 expression levels in the three groups (Fig. 6B). Introduction of p53 attenuated the expression of the anti-apoptotic protein Bcl-2, and upregulated the levels of its counterpart Bax (Fig. 6C). The expression levels of all of these molecules in the blank control group were similar to those in the C3 group (Fig. 6B and C).

The images of cell growth demonstrated that there was a large amount of cells in the suspension in the three groups after 24 h treatment with 100 nM Taxol, and the adherent cells became significantly sparse in the p53^wt group, compared with the two control groups. FACS analysis indicated that the ratios of cell apoptosis were 11.23, 12.37 and 19.52% among the blank, C3 and p53^wt groups, respectively. The ratios of G₂ phase cells were 29.34, 28.59 and 15.79% among the blank, C3 and p53^wt groups, respectively. These results indicated that p53^wt introduction resulted in an increase in the cytotoxicity of Taxol in NCI-H1299 cells and a decrease in the ratios of cells at the G₂ phases (Fig. 7).