Cal (C₁₆H₁₂O₅) was obtained from Standard Biotech Co., Ltd (Shanghai, China). The A549 human lung adenocarcinoma cell line was purchased from American Type Culture Collection (Manassas, VA, USA). TPA and bovine serum albumin (BSA) were provided by Sigma-Aldrich (St. Louis, MO, USA). Transwell chambers were purchased from Corning Incorporated (Corning, NY, USA). MMP2, MMP-9, E-cadherin (E-Cad), integrin β1, PKC-α antibodies were obtained from Boster Systems, Inc. (Pleasanton, CA, USA). AEB071, a PKC-α inhibitor, was provided by Sellek Chemicals (Houston, TX, USA). The ERK1/2 inhibitor (PD98059), penicillin/streptomycin, trypsin, EDTA, RNase, 1% Triton X-100, SDS-PAGE gel (10%) and polyvinylidene difluoride (PVDF) membranes were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). AnnexinV/ propidium iodide (PI) was provided by Immunotech (Marseille, France). Basal Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). Crystal violet, acetic acid and pure methanol were purchased from Aladdin Shanghai Biochemical Technology Co., Ltd.(Shanghai, China). Other reagents used were of analytical grade and obtained from commercial sources.

DMEM supplemented with 10% FBS, 1% nonessential amino acids, 100 U/ml penicillin and 100 µg/ml streptomycin were used as the medium for A549 cell cultivation. The medium was replaced every 2 days. The incubation conditions were maintained at 37°C with a humidified atmosphere of 5% CO₂.

The cell viability inhibiting effect of Cal on the A549 cells was determined using an MTT assay. Briefly, the logarithmic growth phase A549 cells were digested in 0.1% trypsin, DMEM supplemented with 10% FBS was added, and the cells were plated in 96-well plates at a final density of 0.6×10⁴ cells/well. The cells were treated with a series of diluted concentrations of Cal (10, 20, 30, 40, 50, 60, 70, 80 or 90 µM) and incubated at 37°C for 24 h. Following incubation, MTT (5 mg/ml, 10 µl) solution was added to each well and incubated for 4 h at 37°C. Subsequently, the supernatant in each well was discarded and dimethylsulfoxide (DMSO; 100 µl) was added. The optical density (OD) value at 570 nm was measured using a Spectra Max 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability was determined by the OD value, and was calculated as the percentage of viable cells. The measurement was performed in three independent experiments.

The A549 cells (6×10⁵/well) were seeded into 6-well plates and treated with different concentration of Cal (20, 30 and 40 µM), which were then cultured at 37°C for 24 h. The cells were collected and digested with 0.25% trypsin and 0.02% EDTA (1:1) at 37°C for 3-4 min. The cells were then pipetted gently and collected and centrifuged at 112 g at room temperature for 5 min. Subsequently the cells were washed with cold phosphate-buffered saline (PBS; 0.01 M; pH 7.4) twice and resuspended in the residual PBS. Following the addition of 1 ml pre-chilled (−20°C) 80% ethanol, the cells were stored at −20°C overnight. Following washing twice with PBS, 60–80 µl RNAase (1 mg/ml) was added, and the cells incubated at 37°C for 30 min. Following chilling on ice for 2 min, Annexin V-fluorescein isothiocyanate (FITC)/PI solution (100 mg/l PI, 0.1% TritonX-100) was added, and the sample was incubated in the dark at room temperature for 30 min. Cell apoptosis was analyzed using an FC 500 flow cytometer (Beckman Coulter, Brea, CA, USA). Data were acquired by the RXP software of the machine.

A cell adhesion experiment was performed in 96-well plates coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and air dried in a Logic⁺ laminar hood (Labconco, Kansas City, MO, USA) overnight. The wells were blocked with 2% BSA (50 µl/well) and incubated at 37°C for 2 h. The A549 cells were stimulated with 80 nM TPA, as described previously (28). Subsequently, the cells were treated with different concentrations of Cal (20, 30 and 40 µM) for 24 h. Then cells were inoculated into the 96-well plate at a concentration of 1×10⁴ cells/well and incubated at 37°C for 30 min. The non-adherent cells were removed with 200 µl PBS (pH 7.4) following incubation. Subsequently, the adherent cells in each well were stained with 0.1% crystal violet and lysed with 30% acetic acid, and measured optical density at 550 nm on the Spectra Max 190 microplate reader. The adhesion rate was calculated from the OD values of triplicate experiments.

A scratch assay was performed, as described previously. Briefly, the A549 cells were detached using 0.1% trypsin and resuspended in serum-free DMEM, seeded at a concentration of 5×10⁶ cells/cm² into 6-well plates in the medium containing 10% FBS. Following overnight incubation at 37°C, a cell-monolayer was yielded for a wound healing assay. To introduce the wound, three wound tracks were scored in the monolayer, ~5 mm in distance, in each well using a 200 µl pipette tip. The suspended cells were washed twice with DMEM, and the wounded cell monolayer was incubated in FBS-free medium containing different concentrations of Cal (20, 30 and 40 µM), stimulated with 80 nM TPA, at 37°C for 24 h. Images of the wound area were captured at 0 and 24 h using an IX73 microscope (Olympus Corporation, Tokyo, Japan). The areas in the scratch wound, which were not covered in cells were quantified using ImageJ 2.1.4.7 software (Media Cybernetics, Inc., Rockville, MD, USA). The closure rate was determined as the percentage of the area at 0 h. The experiments were performed in triplicate.

To determine cell migration, experiments were performed using Transwell chambers. The A549 cells were stimulated with TPA and incubated in the presence or absence of various concentrations of Cal (20, 30 and 40 µM) for 24 h. Following incubation, the cells were detached using trypsin and resuspended in serum-free medium. Medium containing 10% FBS was added to the lower chamber as a chemotactic factor, and the cells were seeded in the upper chamber at a density of 1×10⁴ cells/well in 50 µl serum-free medium. Following incubation for 8 h at 37°C, any A549 cells, which did not penetrate the polycarbonate membrane were removed using cotton swabs. The cells, which had penetrated through membrane were fixed with methanol and stained with 0.1% crystal violet for 10 min. The chambers were visualized in six randomly-selected visual fields under an IX73 microscope, in which the number of cells were counted. Each experiment was performed in triplicate.

To determine cell invasion, experiments were performed in Transwell chambers coated with Matrigel, as described previously (29). Briefly, 50 µl Matrigel was coated on the membrane at the base of the Transwell chamber and air dried in a Logic⁺ laminar hood (Labconco) overnight. Following blocking with 2% BSA (50 µl/well), the chambers were incubated at 37°C for 2 h and were then rinsed with PBS. The cells, which had been exposed to different concentration Cal (20, 30 and 40 µM) were placed into the upper layer of the Transwell chamber at a concentration of 2×10⁴ cells/well. The medium (600 µl/well) containing chemotactic factor (10% FBS) was added to the lower layer of the Transwell chamber. The cells were cultured at 37°C for 24 h. Cotton swabs were used to remove the cells, which did not penetrate the polycarbonate membrane. The cells, which penetrated through and adhered to the membrane were fixed with methanol and stained with 0.1% crystal violet for 10 min. Following the crystal violet staining, the cells were rinsed with distilled water to removing excess dye. Subsequently six randomly-selected visual fields in each well were selected and visualized under an IX73 microscope. The number of cells that penetrated the membrane were counted, and the invasion rate was quantified by the number of permeated cells associated with the OD value. Each experiment was repeated three times.

To analyze the expression levels of migration-associated proteins in the A549 cells affected by Cal, western blot analysis was performed. Briefly, analyses were performed following stimulation with TPA and treatment with Cal (20, 30 or 40 µM), and with or without the PKC inhibitor (AEB071) or ERK1/2 inhibitor (PD98059), following TPA stimulation and Cal (30 µM) treatment. The A549 cells were suspended in 250 µl lysis buffer, containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate and protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). The cell lysate was centrifuged at 10,000 g for 20 min at 4°C. Equal quantities of proteins from each sample (50 µg) were subjected to 10% sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The proteins were then transferred onto PVDF membranes and the membranes were blocked with 5% (w/v) BSA for 2 h and washed in Tris-buffered saline with Tween 20 (TBST) three times. Subsequently, the membranes were incubated with the following primary detection antibodies: E-Cad, integrin β1, MMP-2, MMP-9, PKC-α, ERK1/2 and phosphorylated (p)-ERK1/2 (1:1,000) overnight at 4°C. The membranes were then washed and incubated with horseradish-peroxidase-conjugated IgG for 1 h at room temperature and then washed in TBST three times. Chemiluminescence reagents of western blotting were added for visualization of the protein bands, and quantification of the proteins bands was performed using ImageJ software.

All data in the present study were obtained from three independent experiments and are expressed as the mean ± standard deviation. One-way analysis of variance was used for multiple comparisons and Student's t test was used to evaluate the differences between two groups. All analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of Cal on cell viability was assessed using an MTT assay. The A549 cells were treated with increasing doses (0–90 µM) of Cal for 24 h. As shown in Fig. 1B, following exposure to Cal, the viability of A549 cells decreased in a dose-dependent manner. No significant change in cell viability were observed, compared with the 0 µM (DMSO treatment only) group, following 24 h treatment with Cal at concentration between 0 and 40 µM, indicating that Cal was not toxic to the A549 cells at these concentrations. Following treatment with Cal at concentrations >40 µM, cell viability reduced significantly at 24 h. These results indicated that treatment with Cal at doses >50 µM for 24 h resulted in the dose-dependent loss of cell viability in the A549 cells, however, doses <40 µM for 24 h did not cause cytotoxicity. Therefore, concentrations of Cal<40 µM was selected for the subsequent experiments.

To understand whether the effect of Cal on A549 cell proliferation had any association with apoptotic rates, the binding of Annexin V to phosphatidylserine, exposed on the cell membrane, was measured, which is generally recognized as an early indicator of apoptosis. As shown in Fig. 1C and D, the total percentages of Annexin V+/PI-cells (right lower quadrant representing early apoptosis) and Annexin V+/PI+ cells (right upper quadrant representing late apoptosis and necrosis) increased between 23.39 and 43.77% following treatment of A549 cells with Cal at 20, 30 and 40 µM for 24 h, compared with 3.44% apoptosis in the control group. These data indicated that Cal induced A549 cell apoptosis in a dose-dependent manner, which was associated with the inhibition of proliferation.

To investigate the inhibition of Cal on TPA-treated A549 cell adhesion, a cell matrix adhesion assay was performed. As shown in (Fig. 2A), following treatment with Cal at concentrations of 20, 30 and 40 µM, the cell adhesion rates of the A549 cells were 86.58, 75.40 and 62.38% of that in the TPA-induced group, respectively (P<0.01). These data suggested that Cal inhibited the adhesion ability of the A549 cells to the cell matrix.

The effect of Cal on TPA-induced A549 cell migration capability was estimated by a wound-healing assay and Transwell chamber assay. In the wound-healing assay (Fig. 2B and C), the cells treated with TPA covered 49.91% of the wound area, which was significantly higher than the untreated cells following incubation (13.31%). The wound closure rates were 41.81, 36.62 and 22.98% following treatment with Cal at 20, 30 and 40 µM, respectively, which were significantly lower than that of the TPA-treated group (P<0.05).

Following 8 h Transwell chamber migration, the percentage of A549 cells that penetrated the membrane in the non-TPA-stimulated group was only 28%. The percentages of cells that penetrated through the membrane following TPA treatment were significantly decreased when exposed to Cal concentrations of at 20, 30 and 40 µM to 72.46, 52.46 and 48.23%, respectively, compared with the TPA-treated only group (Fig. 2D and E; P<0.01).

A Transwell coated with Matrigel was used to determine the suppression of Cal on A549 cell invasion. As shown in Fig. 2F and G, the relative percentages of penetrated cells in the non-induced cell group increased between 26.69 and 100% when exposed to TPA. This results indicated that the invasion capability of the A549 cells induced by TPA was increased significantly, compared with control group. Compared with the TPA-treated only group, the increased number of penetrated cells innduced by TPA was significantly suppressed by Cal at concentrations of 20, 30 and 40 µM (62.72, 52.99 and 41.70%, respectively) in a dose-dependent manner. (P<0.05).

The increase in tumor cell mobility is important in the metastasis processes. The expression levels of E-Cad and integrin β1, associated with cell migration, were detected in the present study using western blot analysis As shown in Fig. 3A and B, the relative expression level of E-Cad decreased between 221 and 100% when treated with TPA. However, the expression level was increased to 106.00, 137.09 and 185.94% following exposure to different concentrations of Cal for 24 h. The relative activity of integrin β1 activitiy was increased between 57.34 and 100% following stimulation with TPA. Cal inhibited this TPA-induced integrin β1 activity to 84.50, 71.04 and 61.28% following exposure to 20, 30 and 40 µM Cal for 24 h. These results indicated that Cal suppressed the migration ability of the A549 cells by regulating the expression of E-Cad and reducing the expression of integrin β1.

ECM degradation is crucial for tumor cell invasion, suggesting that MMPs are required. To clarify whether MMP-2 and MMP-9 were involved in the inhibition of invasion by Cal, the expression levels of TPA-induced MMP-2 and MMP-9 affected by Cal were investigated using western blot analysis. As shown in Fig. 3C, the relative activities of MMP-2 increased between 45.48 and 100%, compared with the TPA-only group. Cal inhibited the TPA-induced MMP-2 activities to 80.64, 57.40 and 48.14% following exposure to 20, 30 and 40 µM Cal, respectively for 24 h. Similar results were observed for MMP-9, in which MMP-9 activity increased between 46.13 and 100% following TPA stimulation, and decreased to 74.68, 65.47 and 58.10% with exposure to Cal at 20, 30 and 40 µM, respectively, compared with TPA treatment only (Fig. 3D). These results suggested that Cal suppressed MMP-2 and MMP-9 to prevent ECM degradation and inhibit metastasis of the A549 cells.

In order to further investigate the underlying mechanism, the effects of Cal on the expression levels of PKC-α and ERK1/2 were detected using western blot analysis. As shown in Fig. 3E and F, a significant increase in the expression of PKC-α following TPA induction, and suppression of PKC-α following Cal treatment, were observed, in a dose-dependent manner, in the A549 cells. The expression of PKC-α without TPA induction was 44.99%, compared with the A549 cells treated with TPA only, and the levels reduced to 66.30, 60.34 and 52.37% when the TPA-induced cells were exposed to 20, 30 and 40 µM Cal, respectively (P<0.01). The levels of p-ERK levels were 60.74% without TPA treatment and, following treatment with TPA, the phosphorylation of ERK1/2 increased significantly. These increased p-ERK levels were decreased to 79.27, 69.46 and 54.78% by 20, 30 and 40 µM Cal, respectively, in a dose-dependent manner (Fig. 3G and H; P<0.01). The results of the PKC-α and ERK1/2 analyses demonstrated that Cal inhibited the activation of PKC-α and the expression of p-ERK1/2.

To further verify whether Cal inhibited A549 cell migration and invasion through the PKC-α pathway, the A549 cells were pretreated with the PKC-α inhibitor, AEB071 (0.1 µM) for 30 min, and then stimulated with 80 nM TPA in the presence or absence of Cal (30 µM) for 24 h. As shown in Fig. 4A and B, the relative levels of PKC-α were reduced to 70.63 and 68.73% when treated with Cal or AEB071 alone, respectively, following induction by TPA. When the TPA-induced A549 cells were exposed to Cal combined with AEB071, the PKC-α level decreased significantly to 42.36%.

As shown in Fig. 4C and D, AEB071 and Cal increased the levels of E-Cad reduced by TPA. Significant increases were observed with their co-treatment. Treatment with AEB071 or Cal alone reduced the expression levels of integrin β1, MMP-2 and MMP-9. There were significant decreases in the expression levels of these proteins following co-treatment of AEB071 and Cal, compared with their treatment alone.

In the Transwell invasion experiment, when the A549 cells were treated with Cal or AEB071 alone, the cell invasion ability was decreased to 59.78 and 48.60%, compared with the TPA-induced group. The relative percentage of permeated cells was reduced to 37.92% when treated with Cal combined with AEB071 (P<0.01; Fig. 4E and F). The results suggested that Cal inhibited the invasion of A549 cells by downregulating the expression levels of integrin β1, MMP-2 and MMP-9, and elevating the expression of E-Cad, via suppression of the PKC-α pathway.

In order to investigate whether Cal affected the downregulation of ERK1/2 phosphorylation, the ERK1/2 inhibitor, PD98059 (25 µM), was used in the present study, and the protein expression levels of ERK1/2 and p-ERK1/2 in the A549 cells were detected using western blot analysis. The relative expression level of p-ERK1/2 was reduced to 51.42% and 38.73% following treatment with Cal or PD98059 alone, respectively, and the expression level was 26.13% following treatment with Cal and PD98059 combined (Fig. 5A and B).

As shown in Fig. 5C and D, the expression levels of integrin β1, MMP-2 and MMP-9 were decreased markedly following co-treatment with PD98059 and Cal. Notably, these expression levels were lower than those observed following treatment with either Cal or PD98059 alone. In the TPA-induced A549 cells, a significant increase in the expression of E-Cad was observed in the Cal and PD98059 co-treatment group, compared with treatment with either alone.

The results of the invasion assay indicated that the relative percentage of permeated cells were significantly inhibited by Cal and PD98059 co-treatment to 31.84%, compared with 61.29 and 48.80%, respectively, following treatment with Cal or PD98059 alone (P<0.01; Fig. 5E and F). These results suggested that Cal inhibited the invasion of A549 cells by downregulating the expression levels of integrin β1, MMP-2 and MMP-9, and elevating the expression of E-Cad via suppression of the ERK1/2 pathway.