A549 cells were purchased from the American Type Culture Collection and were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Sigma-Aldrich, Merck KGaA). Cells were maintained at 37°C and 5% CO₂.

Total RNA was extracted from A549 cells using the RNeasy Mini kit (Qiagen, Inc.) according to the manufacturer's protocol. The mRNA expression levels of BCL2-like 2 (BCL2L2), BCL2 apoptosis regulator (BCL2), BCL2 associated agonist of cell death (BAD) and BCL2 associated X apoptosis regulator (BAX), COX-2, Wnt and β-catenin in A549 cells were measured by RT-qPCR with β-actin as an endogenous control as previously described (17). The forward and reverse primers used for qPCR were synthesized by Invitrogen, Thermo Fisher Scientific, Inc., and are presented in Table I. qPCR was performed using SYBR-Green Master Mix (Takara Bio, Inc.) according to the manufacturer's instructions and an ABI 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used: 95°C for 90 sec, followed by 45 cycles of 95°C for 30 sec, 57.5°C for 20 sec and 72°C for 30 sec. mRNA expression levels were calculated using the 2^−ΔΔCq method (18) and normalized to β-actin levels.

The human COX-2 gene was cloned into a pcDNA3.1 plasmid (Invitrogen; Thermo Fisher Scientific, Inc.) to produce the pcDNA3.1-COX-2 vector. A549 cells (1×10⁵ cells/well) were cultured in six-well plates until 90% confluence was reached and subsequently transfected with the pcDNA3.1-COX-2 vector (100 nM) or empty pcDNA3.1 (100 nM) plasmid using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

A549 cells (1×10³ cells/well) were incubated in 96-well plates with 2.5, 5.0 and 7.5 mg/ml TF (purity ≥95%, Sigma-Aldrich; Merck KGaA) for 24, 48 and 72 h at 37°C. TF were originally extracted from Daphne genkwa and dissolved in 40% ethanol. For the control group, cells were incubated with PBS instead of TF. A total of 20 µl MTT (5 mg/ml) solution in PBS was added to the wells at each time point, and the cells were incubated for an additional 4 h. Subsequently, 100 µl dimethyl sulfoxide were added to the wells to dissolve the formazan crystals and the optical density was measured at wavelength of 490 nm using a plate reader. Each experiment was performed in triplicate.

A549 cells were incubated with 5 mg/ml TF for 24 h at 37°C based on the maximum inhibitory effect on cell growth. A total of 1×10⁵ A549 cells in 500 µl serum-free DMEM were pipetted into the upper chamber of Transwell inserts (8-µm pore size; Corning Life Sciences) and incubated for 48 h at 37°C. For the invasion assay, the Transwell inserts were precoated with Matrigel (1 mg/ml; BD Biosciences) for 48 h at 37°C. The cells were subsequently fixed with 4% paraformaldehyde for 5 min at 37°C and stained with 0.1% crystal violet for 30 min at 37°C. The numbers of migratory or invading A549 cells were counted in at least three randomly selected fields of view using a phase contrast microscope (Olympus Corp; magnification, ×100). Each experiment was performed at least three times.

A549 cells (1×10⁶ cells/well) were cultured until 90% confluence was reached. Apoptosis was assessed after incubation with TF (5 mg/ml) for 24 h at 37°C. A549 cells were subsequently trypsinized, washed in cold PBS, and adjusted to 1×10⁶ cells/ml in PBS. Cells were stained with annexin V-fluorescein isothiocyanate and propidium iodide (Annexin V-FITC kit; cat. no. 556547; BD Biosciences) and analyzed using a flow cytometer (FACScan; BD Biosciences). Cell apoptosis was measured using BD FACSuite software (version 2; BD Biosciences).

A total of 60 specific pathogen-free male BALB/c nude mice (age, eight weeks; weight, 32–35 g) were purchased from Shanghai Slack Experimental Animals Co., Ltd. Mice received an inguinal injection of 1×10⁸ A549 cells in 150 µl PBS and were randomly divided into two groups (n=10 per group). Mice received treatment 6 days after tumor implantation when the tumor diameter reached 5–8 mm. Mice were intravenously injected with 20 mg/kg/day TF (dissolved in 40% ethanol) or vehicle (ethanol) as a control (19). The treatment was continued for 10 days (20). The tumor volumes were calculated as previously described using the following equation: Volume (mm³)=axb²/2, where a and b represent the longest and shortest diameters, respectively (21). On day 30, three mice per group were sacrificed for further analysis. The remaining mice were kept to investigate the survival time over a 100-day period. Tumor growth, animal health and behavior including self-centered behavior, motivation, anhedonia, anxiety and despair behavior were monitored every five days as described previously (22). Mice were sacrificed by cervical dislocation when the tumor diameter reached 15 mm. Experiments were performed in triplicate. Tumor inhibition rate was calculated using the following formula: Inhibitory rate (%)=(mean tumor volume in PBS-mean tumor volume in TF)/mean tumor volume in PBS ×100%. The present study was approved by the Ethics Committee of Mudanjiang Medical University.

Tumor tissues were harvested and fixed using 4% formaldehyde for 30 min at 25°C. Tissues were embedded in paraffin and cut into 4-µm serial sections. Antigen retrieval was performed by incubating the tumor sections with citrate buffer (pH 6.0) for 7 min at 100°C. Tumor sections were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) overnight at 4°C and incubated with rabbit anti-human COX-2 (1:1,000; ab15191; Abcam), Wnt (1:1,000; ab15251; Abcam) and β-catenin (1:1,000; ab32572; Abcam) primary antibodies overnight at 4°C. Following the primary incubation, sections were incubated with horseradish peroxidase-conjugated polyclonal anti-rabbit IgG secondary antibodies (1:10,000; ab6721; Abcam) for 1 h at room temperature. A Ventana Benchmark automated staining system was used to perform the staining (Bio-Rad Laboratories, Inc.). The staining was observed in six randomly selected fields of view (magnification, ×100) under a Zeiss immunofluorescence microscope (DMI5000M; Carl Zeiss AG). Densitometric quantification of the protein expression was performed using Quantity-One software (version 1.2; Bio-Rad Laboratories, Inc.)

TUNEL staining was used to analyze apoptotic cells in lung tumor tissues. Briefly, paraffin-embedded tumor sections were labeled with BrdU (cat. no. MAB4072; 1:1,000; Sigma-Aldrich; Merck KGaA) as previously described (23) and TUNEL-positive cells were identified using the ApopTag kit (EMD Millipore) according to the manufacturer's instructions. The staining was observed in at least three randomly selected microscopic fields under a fluorescence microscope (Carl Zeiss AG; magnification, ×100). Statistical quantification of TUNEL-positive tumor cells was performed to evaluate the pro-apoptotic effects of TF using six randomly selected fields of view to count the total number of cells and TUNEL-positive cells.

All data are expressed as the mean ± SD of three independent experiments and analyzed using the Student's t-test or one-way ANOVA followed by the Tukey post hoc test. The Kaplan-Meier method and the log-rank test were used to evaluate overall survival rate. All data were analyzed using SPSS software (version 19.0; IBM Corp.) and GraphPad Prism software (version 5.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The inhibitory effects of TF on the proliferation, migration and invasion of A549 cells were analyzed in vitro. Compared with the control group, TF-treated cells exhibited a significant dose-dependent decrease in proliferation (P<0.01; Fig. 1A and B). A concentration of 5 g/ml TF inhibited the proliferation of A549 cells to the greatest extent. Furthermore, treatment with 5 mg/ml TF for 24 h significantly inhibited the migration and invasion of A549 cells compared with the control group (P<0.01; Fig. 1C and D). These results indicated that TF treatment significantly inhibited the proliferation, migration and invasion of A549 cells.

The effects of TF (5 mg/ml) on the apoptosis of A549 cells were investigated in vitro. TF treatment significantly increased the apoptosis of A549 cells compared with the control (P<0.01; Fig. 2A). RT-qPCR revealed that TF treatment decreased the expression levels of the anti-apoptotic genes BCL2L1 and BCL22 compared with control A549 cells (P<0.01; Fig. 2B). Furthermore, TF treatment increased the expression levels of the pro-apoptotic genes BAX and BAD compared with control A549 cells (P<0.01; Fig. 2C). These results indicated that TF treatment significantly affected apoptosis of A549 cells by regulating the expression of apoptosis-associated genes.

The potential mechanism underlying the anti-cancer effects of TF was analyzed by investigating the COX-2/Wnt/β-catenin signaling pathway in A549 cells. TF treatment significantly decreased the mRNA expression levels of COX-2, Wnt and β-catenin in A549 cells compared with the control group (P<0.01; Fig. 3A). Furthermore, COX-2 overexpression inhibited the TF-mediated COX-2, Wnt and β-catenin downregulation in A549 cells (P<0.01; Fig. 3B). Additionally, COX-2 overexpression inhibited the TF-mediated decrease in proliferation and apoptosis of A549 cells (P<0.01; Fig. 3C and D). The transfection efficiency was determined by RT-qPCR after 72 h (Fig. S1). These results suggested that TF treatment significantly suppressed proliferation and promoted apoptosis of A549 cells via the COX-2/Wnt/β-catenin signaling pathway.

The anti-cancer efficacy of TF treatment was investigated in mouse xenograft tumor model. No animals presented with multiple tumors in the present study. As shown in Fig. 4A, TF treatment significantly inhibited tumor growth in mice compared with controls (P<0.01), with a tumor inhibition rate of 64.07%. Results demonstrated that TF treatment increased the percentage of apoptotic cells in tumor tissue compared with control (P<0.01; Fig. 4B). Immunohistochemistry revealed that COX-2, Wnt and β-catenin expression levels in tumor tissues were significantly decreased compared with the controls (P<0.01; Fig. 4C). Furthermore, TF administration significantly prolonged the survival of the animals compared with controls over a 100-day period (P<0.01; Fig. 4D). These results suggested that TF may serve as an efficient anti-cancer agent for lung cancer in vivo.