All chemicals used were reagent grade or higher. DDP and GEN were purchased from Sigma-Aldrich (St. Louis, MO, USA). The nonidet P-40 lysis buffer, chemiluminescent peroxidase substrate, propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), pyrogallol and H₂O₂ were obtained from Merck (Whitehouse Station, NJ, USA). Dimethyl sulfoxide (DMSO) was purchased from Merck (Darmstadt, Germany). The stock solutions of PI, DAPI and MTT were prepared by dissolving 1 mg of each compound in 1 ml phosphate-buffered saline (PBS). The solutions were protected from light, stored at 4°C and used within one month.

The A549 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO-BRL) at 37°C in a 5% CO₂ atmosphere and at 95% humidity.

The A549 cells were grown in monolayers and were harvested and dispensed in 96-well culture plates in 100 μl DMEM at a concentration of 5×10³ cells per well. After 24 h, the assigned concentrations of DDP, GEN or a combination of the two were added to the cells. Wells containing medium alone were used as the negative controls, while the wells containing cells but without any treatment were used as the positive controls. The cells were then incubated for an additional 4 h. At the end of the treatment, 200 μl DMSO was added to each well following the removal of the supernatant. The cell viability was determined by measuring the absorbance of the wells at a wavelength of 490 nm using the Thermo Multiskan MK3 microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). This assay was performed in triplicate. The inhibition rate was calculated according to the following formula: Inhibition rate (%) = [1 - (average absorbance of experimental group / average absorbance of blank control group)] × 100.

The A549 cells were cultured in six-well plates in DMEM supplemented with 10% FBS medium and were treated with DDP, GEN or DDP and GEN in combination for 48 h. The cover slips were washed three times with PBS and single cell suspensions were fixed in 1% PBS. The cells were stained with 100 μg/ml acridine orange (Sigma-Aldrich) and 100 μg/ml ethidium bromide (Sigma-Aldrich) for 1 min. The cells were then observed under a fluorescence microscope (BX43, Olympus Corporation, Tokyo, Japan). In total, ≥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.

The activity of caspase-3, -8 and -10 was measured using caspase colorimetric protease assay kits (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. In brief, the A549 cells were treated with DDP and GEN alone or in combination for 24 h. Following treatment, the cells were washed twice with ice-cold PBS and harvested by centrifugation at 1,000 × g for 10 min. The cell pellets were then lysed in 150 μl buffer. Protein concentrations in the lysate were determined using the Lowry method (16). An 80-μl aliquot of the lysate was incubated with 10 μl of substrate for each caspase at 37°C for 2 h. The samples were analyzed at 405 nm in a microplate reader (Thermo Fisher Scientific Inc.). The relative caspase activity of the control group was set as 100.

Female BALB/c nude mice, 4–5 weeks old, were obtained from the Experimental Animal Center of the Jilin University (Changchun, Jilin, China). All the animal experiments were performed in accordance with institutional guidelines, following a protocol approved by the Ethics Committees of the Disease Model Research Center (The First Hospital of Jilin University, Changchun, Jilin, China). Female BALB mice, 6–7 weeks old, were maintained under specific pathogen-free conditions and provided with food and water ad libitum. All the animals were fed with a normal pellet diet for one week prior to the experiments.

The exponentially growing A549 cells were harvested and a tumorigenic dose of 2.5×10⁶ cells was injected intraperitoneally into the 4–5 week-old mice. When the tumors reached 100–200 mm³ in size, the mice were divided randomly into four groups, with 10 mice per group. The control group received 1% polysorbate resuspended in deionized water. In the DDP group, DDP was administered at a dose of 9 mg/kg as an intraperitoneal bolus injection, while in the GEN group, GEN was administered at a dose of 800 μg/kg each day orally for five days. In the combination group, DDP (5 mg/kg) was administered with GEN on day one, followed by four days of GEN (500 μg/kg) treatment. The tumor weight was measured subsequent to the mice being sacrificed by cervical dislocation, and the tumor volume was measured prior to the treatment injections being administered and on days seven, 14 and 21 of treatment.

The cultured cells were washed twice with PBS (pH, 7.2) and the cells were then lysed with Triton X-100 9Sigma-Aldrich) in Hepes buffer (Sigma-Aldrich), which consisted of 150 mm NaCl, 50 mm Hepes, 1.5 mm MgCl₂, 1% Triton X-100, 0.1% SDS, protease inhibitor cocktail, 100 mm NaF and 100 mm Na₃VO₄, for 30 min. The cell lysates were clarified using centrifugation at 10,000 × g for 15 min and the protein concentrations were determined using the Bradford reagent (Sigma-Aldrich). The protein samples were separated on an 8–15% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequent to blocking in Tris-buffered saline containing 5% skimmed milk and 0.1% Tween 20 for 2 h at room temperature, the protein samples were then immunoblotted with primary mouse monoclonal anti-human PI3K (1:3,000), anti-human phosphorylated(p) PI3K(p-PI3K; 1:2,000), anti-human AKT (1:3,000), anti-human pAKT (1:3,000) and anti-human β-actin (1:5,000) antibodies (Abs), which were all purchased from Cell Signaling Technology, Inc., (Beverly, MA, USA) for 2 h at room temperature. Next, the samples were incubated with horseradish peroxidase-conjugated polyclonal goat anti-mouse immunoglobulin G Abs (1:5,000, Amersham Biosciences, Uppsala, Sweden) for 2 h at room temperature. All the immunoblots were visualized using an enhanced chemiluminescence kit (Pierce Protein Biology, Rockford, IL, USA).

The data are expressed as the mean ± standard deviation. Statistical comparisons between more than two groups were performed using one-way analysis of variance followed by Tukey’s post-hoc test. Statistical analyses were undertaken using the SPSS^® statistical package, version 19.0 (SPSS Inc., Chicago, IL, USA) for Windows^® and GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To evaluate the effect of DDP and GEN alone or in combination on the cell viability of NSCLC cells in vitro, a MTT assay was performed for 48 h whilst the A549 cells were being treated with DPP and GEN alone or in combination. It was found that the inhibitory rates of DDP and GEN alone or in combination were higher compared with the control group (P<0.01). There was no significance different between the DDP and GEN groups (P>0.05). However, the inhibitory rate in the group treated with GEN and DDP in combination was higher compared with either agent alone (P<0.05; Fig. 1).

To investigate whether DDP and GEN alone or in combination could induce apoptosis, apoptosis was analyzed following treatment with DDP and GEN. It was found that the A549 cells treated with DDP or GEN had significant levels of cell apoptosis compared with the untreated cells (Fig. 2A). Treatment with a combination of DDP and GEN led to a marked increase in the level of apoptotic cells compared with either agent alone (P<0.01; Fig. 2A).

To explore the possible mechanism for the induction of cell apoptosis in cells treated with a combination of DDP and GEN, the activity of caspase-3, -8 and -10 was determined by ELISA. The results revealed that treatment with a combination of DDP and GEN could significantly increase the activity of caspase-3, -8 and -10 compared with treatment using DPP or GEN alone (P<0.01) (Fig. 2B, C and D).

The in vivo therapeutic efficacy of DDP and GEN was assessed in female BALB/c nude A549 tumor-bearing mice. The mice were sacrificed and the tumor tissue was removed 21 days after treatment. The tumor weight was then measured and it was found that the tumor weight in the mice treated with a combination of DDP and GEN was lower compared with the tumor weight in the untreated group and the groups treated with either agent alone (P<0.01) (Fig. 3A). In addition, it was also found that the tumor volume following treatment with DDP and GEN in combination was significantly lower compared with the untreated group and the groups treated with either agent alone (P<0.01; Fig. 3B). These results indicate that treatment with DDP in combination with GEN markedly suppresses the tumorigenicity of A549 cells in mice.

To clarify the molecular mechanisms involved in the effect of GEN in combination with DDP on A549 cell proliferation, the present study focused on the effects of GEN and DDP alone and in combination on the activation of PI3K/AKT pathway, which participates in the main intracellular signaling required for cell proliferation and survival. GEN and DDP alone or in combination were found to inhibit the tyrosine phosphorylation of AKT and PI3K (Fig. 4). In addition, treatment with a combination of GEN and DDP resulted in a greater reduction in p-AKT and PI3K compared with treatment with either agent alone. These results indicate that treatment with DDP in combination with GEN inhibits tumor cell growth, to a certain extent, by suppressing the PI3K/AKT pathway.