Celecoxib (CBX), one type of COX-2 inhibitor, was purchased from Pfizer Inc. (New York, NY, USA). Sorafenib (SOR) was purchased from Bayer Pharma AG (Wuppertal, Germany), and both drugs were dissolved in dimethyl sulfoxide (DMSO). For western blot analysis, the following antibodies were used: mouse monoclonal anti-β-actin (Sigma Aldrich, St. Louis, MO, USA), mouse monoclonal anti-Bcl2, mouse monoclonal anti-survivin, mouse monoclonal anti-MEK, mouse monoclonal anti-phosphorylated (p-)MEK, mouse monoclonal anti-ERK, mouse monoclonal anti-p-ERK and horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Nonidet P-40 lysis buffer, chemiluminescent peroxidase substrate, propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI)and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) were from Sigma Aldrich; stock solutions of PI, DAPI and MTT were prepared by dissolving 1 mg of each compound in 1 ml of phosphate-buffered saline (PBS). The solution was protected from light, stored at 4°C and used within 1 month.

The A549 human lung adenocarcinoma cells obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS) in a 5% CO₂ incubator and passaged with 0.25% trypsin and 0.03% ethylenediamine tetraacetic acid (EDTA) solution.

A549 cells grown in monolayers were harvested and dispensed in 96-well culture plates in 100 μl of RPMI-1640 at a concentration of 5×10³ cells/well. After 24 h, differential drug concentrations of SOR (0–20 μM), CXB (0–40 μM) or both (0–10 μM SOX plus 20 μM CXB) were added to the cells. Then, the cells were incubated for another 4 h. At the end of the treatment, 200 μl of DMSO was added to each well after removing the supernatant. Then, cell viability was obtained by measuring the absorbance at a wavelength of 490 nm by Thermo Multiskan MK3 microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). This assay was carried out in triplicate. The growth inhibition rate was calculated according to the following formula: Inhibition rate (%) =[1 − (average absorbance of experimental group/average absorbance of blank control group)] × 100%.

A549 cells were cultured in 6-well plates in RPMI-1640 with 10% FBS medium and were treated with their respective half maximal inhibitory concentration (IC₅₀) values of SOR, CXB or both for 48 h. The coverslips were washed three times with PBS, and single cell suspensions were fixed in 1% PBS. Cells were stained with 100 μg/ml acridine orange (AO) and 100 μg/ml ethidium bromide (EB) for 1 min. Then cells were observed under a fluorescence microscope. At least 200 cells were counted, and the percentage of apoptotic cells was determined. Triplicates were performed in all experiments, and the experiments were performed on five occasions. In addition, we also evaluated survivin and Blc-2 protein expression by western blotting as an additional indicator of apoptosis.

To determine the cell cycle distribution, 5×10⁵ A549 cells were plated in 60-mm dishes and treated with the respective IC₅₀ values of SOR, CXB or both for 48 h. After treatment, the cells were collected by trypsinization, fixed in 70% ethanol, and kept at −20°C overnight for fixation. Cells were washed in PBS, resuspended in 1 ml of PBS containing 100 μg/ml RNase and 40 μg/ml PI and incubated in the dark for 30 min at room temperature. The distribution of cells in the cell cycle phases was analyzed from the DNA histogram with a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) and CellQuest software (San Jose, CA, USA).

PGE2 synthesis was determined by competitive enzyme-linked immunosorbent assay (ELISA) as previously described (14). In brief, A549 cells were treated with their respective half maximal inhibitory concentration (IC₅₀) values of SOR, CXB or both for 48 h in 12-well plates, and then these culture media were centrifuged to remove cell debris. Cell-free culture media were collected at the indicated times, and PGE2 levels were determined by competitive ELISA as described by the kit manufacturer (Cayman Chemical, Ann Arbor, MI, USA) using an ELISA reader (μQuant; Bio-Tek Instruments, Inc., Winooski, VT, USA).

All animal experiments were performed in accordance with the institutional guidelines, following a protocol approved by the Ethics Committees of the Disease Model Research Center, The First Hospital of Jilin University. Female BALB mice, 6–7 weeks of age, were maintained under specific pathogen-free (SPF) conditions and provided with food and water ad libitum. All the animals were fed with a normal pellet diet one week prior to the experimentation.

Exponentially growing A549 cells were harvested, and a tumorigenic dose of 2.5×10⁶ cells was injected intraperitoneally into the 6- to 7-week old female BALB mice. Tumors were allowed to grow in the mice for 10 days, after which the animals were randomly assigned into one of four treatment groups (10 mice per group). The control group received 1% polysorbate resuspended in deionized water. The other three groups were treated with CXB (4.56 mg/kg body weight), SOX (80 mg/kg body weight) or CXB plus SOX (2.5 and 40 mg/kg body weight, respectively) intraperitoneally on alternative days for 3 weeks. The doses were selected based on previous experiments (15,16). Tumor weights were measured after the mice were sacrificed, and tumor volumes were measured before the treatment injections were administered and on day 7, 14 and 21 of the treatment. On day 22, the animals were euthanized using chloroform, and their spleen tissues were collected and cultured for a splenocyte surveillance study. Furthermore, A549 cells were collected from the site of the treatment injection for in vivo and ex vivo cell cycle phase distribution studies.

Spleens from the treated mice were collected, and single-cell spleen suspensions were pooled in serum-free RPMI-1640 by filtering the suspension through a sieve mesh with the aid of a glass homogenizer to exert gentle pressure on the spleen fragments. Samples were washed twice in PBS 0.1% (w/v) and bovine serum albumin (BSA). After centrifugation at 200 × g for 10 min, the cells were placed into 96-well flat-bottom microplates in triplicate at 3×10³ cells/well in RPMI-1640 supplemented with 10% FBS. The cells were then incubated in a total volume of 100 μl/well. Serum-free RPMI-1640 was used as a control. After 24 h, cell proliferation was measured by the MTT assay.

A549 cells were treated with their respective IC₅₀ values of SOR, CXB or their combination for 48 h. The cells were then homogenized in a lysis buffer (Tris-HCl 50 mmol/l, EDTA 5 mmol/l, NaCl 150 mmol/l, sodium deoxycholate 1%, Na₃VO₄ 500 μmol/l, Triton X-100 0.5%, AEBSF 10 μmol/l, NaF 10 mmol/l) on ice. The homogenates were then centrifuged at 14,000 rpm at 4°C for 30 min, and the supernatants were collected for protein concentration determination using the Bradford reagent (Sigma). Cell extracts (50 μg of protein) were separated on a sodium dodecyl sulfate-polyacrylamide electrophoretic gel (SDS-PAGE) and transferred to nitrocellulose membranes, which were blocked in 3% BSA for 2 h. After blocking, the membranes were incubated with primary antibodies overnight at 4°C for 2 h, and then with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Proteins were visualized by exposing the chemiluminescence substrate (Sigma) to X-OMAT AR autoradiography film (Eastman Kodak, Rochester, NY, USA). Blots were stripped and reprobed with anti-β-actin to control for loading variations. Quantity One software (Bio-Rad Laboratories) was used for quantification of the protein bands.

Statistical analyses were undertaken using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA) and the SPSS^® Statistical Package, version 19.0 (SPSS Inc., Chicago, IL, USA) for Windows^®. Data are expressed as the mean ± SD. The statistical significance was determined using one-way analysis of variance (ANOVA). P<0.05 was considered to indicate a statistically significant result.

To evaluate the effect of SOR, CXB and their combination on the cell viability of NSCLC cells in vitro, A549 cells were treated with increasing concentrations of CXB (0–40 μM) or SOR (0–20 μM). SOR inhibited cell proliferation of A549 cells dose-dependently with an IC₅₀ of 4.4±0.18 μM. CXB also reduced cell viability of A549 cells in a dose-dependent manner with an IC₅₀ of 25.5±0.79 μM. Combination treatment (0–10 μM in the presence of 20 μM CXB) reduced the cell viability of A549 cells in a dose-dependent manner with an IC₅₀ of 2.8±0.79 μM. Based on these results, we chose the respective IC₅₀ values of the drugs for further treatments throughout the study.

We next examined whether the combination of relatively low concentrations of SOR and CXB could additively or synergistically inhibit HCC cell growth in vitro using the respective IC₅₀ values of SOR and CXB. As shown in Fig. 1, the inhibitory rate of the combination treatment was higher than the rates after single drug treatments (P<0.01). There was no significance different between the SOR-treated group and CXB-treated group (P>0.05).

To investigate whether SOR and CXB alone or their combination could induce apoptosis, we analyzed the apoptosis after treatment with SOR and/or CXB. Treatment with the combination of these drugs led to a marked increase in apoptotic cells when compared to the extent of apoptosis following treatment with the single drugs (P<0.05) (Fig. 2A). In addition, there was no significance different between the SOR-treated group and CXB-treated group in regards to induction of NSCLC cell apoptosis.

The effects of SOR and CXB on the cell cycle distribution of A549 cells were then analyzed by flow cytometry. A549 cells treated with SOR or CXB demonstrated an increased percentage of apoptotic cells (cell cycle arrest at the G0/G1 phase) compared with the untreated cells (Fig. 2B). The low-dose combination resulted in an even greater percentage of apoptotic cells when compared with the percentage of apoptosis following treatment with higher doses of either drug alone (P<0.01). These data are consistent with the results from the acridine orange (AO) staining assay. In conclusion, these results indicate an additive mechanism of SOR and CXB in inducing cell death through apoptosis.

In order to explore the possible mechanism of the pro-apoptotic effect of the combination treatment with SOR and CXB, expression patterns of survivin and Bcl-2 were determined by western blot analysis. The results (Fig. 2C and D) showed that treatment with the combination of CXB and SOR significantly decreased the expression of inhibitor of apoptosis genes, survivin and Bcl-2, in A549 cells, when compared to the expression levels in cells treated with CXB or SOR alone (P<0.01).

To examine the effect of SOR and CXB on PGE2 production in A549 cell, ELISA was performed. As shown in Fig. 3, CXB inhibited PGE2 production; however, CXB in combination with SOR or SOR alone did not inhibit PGE2 production, suggesting that the synergy between CXB and SOR did not extend to COX-2-dependent PGE2 production in NSCLC cells.

The RAF/MEK/ERK pathway is downstream of Ras activation, and tyrosine phosphorylation of these proteins is essential for cancer cell proliferation (17). Therefore, we evaluated the effect of SOR and CXB alone and in combination on the phosphorylation of these proteins by western blotting (Fig. 4). We compared the phosphorylation of these proteins in cells treated with the respective IC₅₀ values of SOR and CXB alone and in combination for 2 h. It was found that SOR and CXB alone or in combination inhibited the tyrosine phosphorylation of MEK and ERK (Fig. 4). In addition, following treatment with the combination of SOR and CXB, a greater reduction in phospho-MEK and ERK was caused than what was observed by either agent alone.

We assessed the in vivo therapeutic efficacy of SOR and CXB in female BALB mice bearing A549 cell tumors. Mice were sacrificed and tumor tissue was obtained 21 days after treatment. The tumor weight of each animal was measured. It was found that the tumor weight in the mice treated with the combination of SOR and CXB was lower than the tumor weight of the untreated group and single drug-treated groups (P<0.01) (Fig. 5A). In addition, we found that the tumor growth rate after treatment with the combination of SOR and CXB was significantly reduced when compared with the single drug-treated groups and the untreated group (P<0.01) (Fig. 5B). These results indicate that the combination treatment of SOR and CXB suppressed the tumorigenicity of A549 cell-derived tumors in mice.

To assess the efficacy of SOR and CXB in modulating splenocyte proliferation, spleen cells of the treated A549 mice were isolated and cultured in RPMI-1640 containing 10% fetal bovine serum for 24 h and subjected to in vitro proliferation assays using MTT assay. As shown in Fig. 6, the inhibitory growth rate of cell proliferation following treatment with the combination of SOR and CXB was higher than this rate in the single drug-treated groups, which demonstrated that the combination treatment inhibited A549 cell proliferation.