4′,6-Diamino-2-phenylindole (DAPI) and antibodies against TNF-α (MK1169) and GSK-3β (27C10) were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). Other reagents were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The MCO-5AC CO₂ thermostat incubator was obtained from Sanyo Electrical Biomedical Co., Ltd. (Osaka, Japan).

A549 lung carcinoma cells were provided by the School of Pharmaceutical Sciences of Jilin University (Changchun, China). The A549 cells were maintained in plastic dishes (150 mm) with RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Shanghai BaiJin Chemical Group Co., Ltd., Shanghai, China), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. The cells were divided into five groups: Control, cisplatin and rutin (low, medium and high) groups. Cells were seeded into 96-well plates (5×10⁻⁵ cells/well) in Dulbecco's modified Eagle's medium (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) supplemented with 10% fetal bovine serum and incubated with 1×10⁻⁸ (low), 2×10⁻⁸ (medium) or 4×10⁻⁸ mol/l (high) rutin or 1×10⁻⁹ mol/l cisplatin (both from Sigma-Aldrich; Merck KGaA) for 24 h. Control cells were treated with PBS under the same culture conditions.

Cells were lysed using tissue Total Protein Lysis Buffer (Yi Li Biotechnology Co., Ltd., Shanghai, China) and centrifuged at 10,000 to 14,000 × g for 15 sec at 4°C. Lysates were subsequently analyzed according to the instructions of a TNF-α ELISA kit (EH3TNFA; Nanjing Zhi Bai Cui Biology Technology Co. Ltd., Nanjing, China), with measurement of the absorbance value at 450 nm.

A549 cells were homogenized in radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA). Cell suspensions were centrifuged at 10,000 to 14,000 × g for 4°C at 15 sec. The total protein concentration of the homogenates was measured using a bicinchoninic acid assay reagent. Equal amounts of protein extract (20 µl) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes via electroblotting. Separated proteins were transferred to polyvinylidene difluoride membranes (Sigma-Aldrich; Merck KGaA). After blocking with 5% non-fat milk (4°C, 2 h), the membranes were probed with primary antibodies against TNF-α (1:500; MK1169; Wuhan Boster Biological Technology, Ltd.) and GAPDH (1:500; SAB2100894; Sigma-Aldrich; Merck KGaA) at 20–27°C for 2 h, followed by incubation with anti-mouse immunoglobulin G secondary antibodies (1:400; AP130P; Sigma-Aldrich; Merck KGaA) at 37°C for 20 min. Immunocomplexes were visualized using an enhanced chemiluminescence detection system (EMD Millipore, Billerica, MA, USA). Immunoreactive bands were analyzed using Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature, then washed three times with PBS for a total of 10 min. Cells were incubated with GSK-3β antibody (1:200) at 4°C overnight. Following this, the cells were washed three times for 5 min and subsequently incubated with anti-mouse immunoglobulin G secondary antibodies (1:500; AP130P; Sigma-Aldrich; Merck KGaA) at 4°C for 2 h. DAPI was then used for nuclear staining. Cells were mounted and images were captured using a Nikon Eclipse 80i fluorescence microscope (Nikon Corporation, Tokyo, Japan). The protein expression of GSK-3β and the number of apoptotic cells (DAPI) were analyzed using Image Pro Plus 6.0 software.

All data were obtained from at least three separate experiments and are presented as the mean ± standard error of the mean. Statistical comparisons were made using the Student's t-test. Statistical analysis of the data was performed using SPSS version 11 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

An ELISA kit was used to assess the expression of TNF-α in different groups. The results of the ELISA demonstrate that the expression of TNF-α protein in the cisplatin group was significantly increased compared with that in the control group (P<0.01; Fig. 1, Table I). Furthermore, the expression of TNF-α in the low, medium and high rutin groups was also increased significantly compared with that in the control group (P<0.01; Table I).

Western blotting was conducted to further investigate the expression of TNF-α. The results were analyzed semi-quantitatively according to the grayscale value. TNF-α levels were significantly increased in the cisplatin group compared with the control group (Fig. 2; P<0.05). Additionally, the expression of TNF-α in the low, medium and high rutin groups was also significantly higher compared with that in the control group (Fig. 2; P<0.05).

DAPI staining results (Fig. 3) demonstrated that cells in the control group had uniform chromatin, a large nucleus and integrity of the nuclear envelope. By contrast, apoptotic morphological changes of A549 cells after 48 h rutin or cisplatin treatment were observed. Relative to the control group, cells groups treated with rutin and cisplatin exhibited higher numbers of detached cells with round and shrunken morphologies and condensed nuclei.

To validate the expression pattern of GSK-3β in lung cancer cells, GSK-3β specific fluorescent staining was performed on A549 cells (Fig. 4). The results of the staining assay revealed that the cells in the control group had uniform chromatin and a large nucleus. Relative to the control group, levels of GSK-3β protein were markedly increased in the cisplatin group. In addition, GSK-3β protein expression was increased in the low, medium and high rutin groups compared with the control group.