Taurine was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). RPMI-1640, Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The anti-p53 upregulated modulator of apoptosis (PUMA) polyclonal antibody (cat. no. 55120-1-AP) was purchased from ProteinTech Group, Inc. (Chicago, IL, USA); the anti-Bcl-2 monoclonal antibody (cat. no. sc-783), anti-Bax monoclonal antibody (cat. no. sc-526), anti-β-actin monoclonal antibody (cat. no. sc-130300) and anti-HA monoclonal antibody (cat. no. sc-393579) were all purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (cat. no. ZDR-5307), HRP-labeled goat anti-rabbit IgG (cat. no. ZDR-5306), trypsin, SDS and TEMED were all purchased from Origene Technologies, Inc. (Beijing, China). Cisplatin (DDP) was purchased from Shandong Qilu Pharmaceutical Co., Ltd. (Jinan, China).

The human non-small cell lung cancer A549 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). A total of 40 4-week-old BALB/C nude mice (half from each sex), weighing ~20 g (SPF grade; certificate no. HNASLKJ20121369), were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China), and maintained in individually ventilated cages (20–26°C and 40–70% humidity), in the Animal Science Center of Nanchang University (Nanchang, China). The mice were fed with SPF grade aseptic mice feed purchased from Beijing Keao Xieli Feed Co., Ltd. (Beijing, China) and sterilized water, 12/12 h light/dark cycle. All animal experiments were approved by the Ethics Committee of Nanchang University.

The human non-small cell lung cancer cell line A549 were cultured in RPMI-1640 complete culture solution supplemented with 10% FBS and stored at 37°C in a 5% CO₂ incubator. A549 cells at the logarithmic growth phase were randomly divided into 6 groups: Control, taurine 20 mM, taurine 40 mM, taurine 80 mM, taurine 160 mM and the positive control group, DDP (10 mg/ml). The cells were then treated with the respective agents for 24, 48 and 72 h.

A549 cells at the logarithmic growth phase were collected, and the concentration of the cell suspension was adjusted based on the Trypan blue method. In total, 200 µl of cell suspension was added to each well of a 96-well plate, to a final density of 3,000-8,000 cells per well. Each assay point was performed in four replicates, and the empty wells at the edges of the plates were filled with sterile PBS. The cells were cultured at 37°C in a 5% CO₂ incubator. The following day, the cells were treated with taurine at the various concentrations or with DDP 10 g/ml. After 48 h incubation, 20 µl of MTT solution (5 mg/ml) was added to each well for and incubated for 4 h. The supernatant in each well was then gently aspirated, and 150 µl dimethyl sulfoxide was added to each well to dissolve the crystals, and the plate was shaken on a horizontal shaker for 10 min. The optical density (OD) at 490 nm were measured using a microplate reader, and the inhibition ratio was calculated using the following equation: Inhibition ratio (%) = (1 - OD value of the experimental group/OD value of the control group) ×100.

A549 cells at the logarithmic growth phase were collected and seeded into 12-well plates, at a density such that the adherent cells reached 70–90% confluence by the following day. The cells were then divided into five groups: Control, taurine 40 mM, taurine 80 mM, taurine 160 mM and DDP 10 g/ml, with 3 replicates in each group. The cell lines were treated for 48 h at 37°C in a 5% CO₂ incubator. The medium was then removed, the cells were washed twice with PBS and 500 µl Hoechst 33342 stain solution (5 µg/ml) was added into each well to completely cover the cells. The cells were then incubated in the dark at 37°C in a 5% CO₂ incubator for 20–30 min. Subsequently, the Hoechst 33342 stain was removed, the cells were washed twice with PBS and apoptotic morphology was observed under a fluorescence microscope (×200 magnification) and images were captured. A total of ten random high magnification fields were selected to calculate cell apoptosis ratio as follows: Cell apoptosis ratio = apoptotic cell number/total cell number ×100.

Following a 48 h treatment, as aforementioned, 1–5×10⁵ cells were collected and resuspended in 500 µl Binding Buffer (Annexin V-FITC/PI double staining cell apoptosis detection kit; Jiangsu Keygen Biotechnology Co., Ltd., Nanjing, China). Annexin V-fluorescein isothiocyanate (FITC; 5 µl) and 5 µl propidium iodide (PI) were added successively into the cell suspension, mixed well and incubated for 5–15 min at room temperature in the dark. The cells were then analyzed for apoptotic status using a FACSCalibur flow cytometer with the BD CellQuest™ Pro Analysis system (version 6.1; BD Biosciences, Franklin Lakes, NJ, USA).

Following the aforementioned 48 h treatments, A549 cells were collected and total protein was extracted with cell lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology, Haimen, China) and denatured. Standard bovine serum albumin solution was used to produce the standard curve of protein content to measure the concentration of protein. A total of 30 µg total protein per lane from each sample was subjected to 10% SDS-PAGE. Following electrophoresis, protein was transferred to a polyvinylidene fluoride membrane using a current of 330 mA for 60 min. The membrane was blocked by 1% bovine serum albumin (cat. no. ST023; Beyotime Institute of Biotechnology) at room temperature for 2 h, and then incubated with specific antibodies against PUMA, Bax or Bcl-2 (1:200) at 4°C overnight, followed by incubation with the appropriate HRP-conjugated secondary antibody (1:2,000) for 1.5 h. The western blots were developed using pierce™ ECL western blotting substrate (Pierce; Thermo Fisher Scientific, Inc.), and the protein bands analyzed for protein density using Image-Pro Plus 6 software (Media Cybernetics, Inc., Rockville, MD, USA). We used the ratio of the gray value of the target protein to the gray value of the β-actin as the final result, to produce a semi-quantitative analysis.

A549 cells were inoculated subcutaneously in the left and right flanks of the abdomen of 40 5-week-old nude mice. Once the diameter of the tumors reached between 2–3 mm, the mice were randomly divided into four groups: i) Control group, treated with 0.2 ml saline was injected at multiple sites inside and around the tumor; ii) taurine treatment group, treated with 0.2 ml taurine solution (100 mg/kg) injected at multiple sites inside and around the tumor; iii) exogenous PUMA treatment group, treated with 0.2 ml mixture of pCEP4-(HA)₂-PUMA plasmid (100 µg; Institute of Cancer Research, University of Pittsburgh, Pittsburgh, PA, USA) and liposome (Invitrogen; Thermo Fisher Scientific, Inc.) injected at multiple sites inside and around the tumor which have been sterilized with 75% alcohol; iv) taurine and exogenous PUMA treatment group (combined treatment group), treated with 0.1 ml taurine solution (100 mg/kg) and 0.1 ml mixture of pCEP4-(HA)₂-PUMA plasmid (100 µg) and liposome injected simultaneously at multiple sites inside and around the tumor. The treatment was applied seven times, with an interval of 72 h between each treatment. The diameter of the tumors was measured every 72 h for the calculation of tumor volumes, and tumor growth curves were plotted accordingly. The mice were euthanized on day 27 and the tumors were photographed and weighed. The tumor suppression ratio was then calculated as follows: Tumor volume (cm³) V_T=1/2× a × b² (a is the longer arm, b is the shorter arm); Tumor suppression ratio (%) = (average tumor weight of control group-average tumor weight of treatment group)/average tumor weight of control group ×100.

When the mice were euthanized, the tumors were collected and any blood was washed off with PBS. Immediately following photography, the tumors were fixed with 4% paraformaldehyde for 24 h at 4°C, and examined for expression levels of PUMA, Bax and Bcl-2 by immunohistochemistry. Tissue samples were paraffin embedded, sectioned (3 µm), heated at 65°C for 2 h, dewaxed with conventional xylene (xylene I for 15 min, the xylene II 15 min), and dehydrated with a gradient alcohol series (100% ethanol for 2 min, 100% ethanol for 2 min, 95% ethanol for 2 min, 95% ethanol for 2 min, 80% ethanol for 2 min then 70% ethanol for 2 min), and then washed 1 time with tap water for 3 min. Antigen retrieval was performed using citric acid buffer (0.01 M, pH 6.0, 100°C) and boiled for 20 min in a microwave oven (wattage, 800 W; time, 20 min). Endogenous peroxidase was inactivated with 3% H₂O₂ at 37°C for 10 min in a water bath. Sections were incubated with specific antibodies against PUMA, Bax or Bcl-2 (1:200) at 4°C for 6 h, followed by incubation with a horseradish peroxidase-conjugated secondary antibody at 37°C for 1 h (dilution, 1:2,000; cat. no. ZDR-5307) and stained with 3,3N-diaminobenzidine tertrahydrochloride (0.5 mg/ml) and hematoxylin (4 mg/ml) for 5 min at 25°C. Under an Olympus BX-41 binocular light microscope (at ×40 magnification), images from five fields on each section of two non-consecutive sections of each tumor were captured. The images were then analyzed using HMIAS-2000 image analyzing software (version no. V1.0; Wuhan Qianping Imaging Technology Co., Ltd., Wuhan, China) for automatic calculation of integrated optical density to determine protein expression.

SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used to analyze the experimental data, and expressed as the mean ± standard deviation. One-way analysis of variance followed by LSD post hoc test or unpaired t-test was performed for comparisons between multiple groups, and a Q-test was used to compare two groups among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of taurine on cell proliferation, A549 cells were treated with various taurine concentrations. As shown in Fig. 1, the inhibitory effect of taurine on the proliferation of A549 cells significantly increased in a time- and concentration-dependent manner when compared with the control group.

A549 cells were treated with different concentrations of taurine (range, 40–160 mM) for 48 h, and then double-stained with anti-Annexin V-FITC antibody and PI. The apoptosis ratio (Annexin V-FITC⁺/PI⁻, the lower right quadrant) of A549 cells corresponded with increasing concentrations of taurine (Fig. 2), and the apoptosis ratios of all taurine groups were significantly different when compared with the control group (P<0.05, P<0.01). In addition, Hoechst 33342 fluorescence staining revealed that the numbers of apoptotic cells were also increased as taurine concentration increased, and that the number of apoptotic cells were significantly higher than in the control group (Fig. 3; P<0.01).

Western blot analysis revealed that the expression of PUMA and Bax was significantly upregulated, whereas that of Bcl-2 was significantly downregulated in A549 cells. This trend continued with increasing concentrations of taurine, compared with the control group (Fig. 4; P<0.05). Taurine altered the expression levels of PUMA, Bax and Bcl-2 in a dose-dependent manner.

As shown in Fig. 5, treatment with exogenous PUMA alone, taurine alone and combined treatment with exogenous PUMA and taurine gave an average tumor suppression ratio of 42.94±1.99, 50.09±2.35 and 78.52±1.46%, respectively. These ratios all differed significantly from that of control group (P<0.01). The average tumor volumes and weights of all treatment groups were significantly different from that of control group (P<0.01). In addition, the tumor suppression ratio of the combined-treatment group was much higher than that of the taurine or PUMA single-treatment groups, indicating that combined treatment with taurine and exogenous PUMA act synergistically to suppress the growth of xenograft tumors in nude mice.

The results of immunohistochemistry (Figs. 6–8) revealed that the levels of PUMA and Bax were significantly elevated in the cytoplasm of tumor cells treated with taurine, PUMA and taurine and exogenous PUMA, when compared with the control group (P<0.01). Moreover, the levels of proteins in the cytoplasm were significantly higher in the taurine and exogenous PUMA group, than in the PUMA or taurine single-treatment groups (P<0.01). Compared with the control group, the level of cytoplasmic Bcl-2 was significantly decreased in the taurine, exogenous PUMA and taurine and exogenous PUMA treatment groups (P<0.01). In addition, the level of cytoplasmic Bcl-2 was the lowest in the taurine and exogenous PUMA group (Fig. 9), and was significantly different to that of the taurine or PUMA single-treatment groups (P<0.01).