Osthole was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). A 50 mM stock solution of osthole was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. RPMI-1640, trypsin, penicillin and streptomycin were purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Fetal bovine serum (FBS) and Giemsa were purchased from Solarbio Science and Technology (Beijing, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DMSO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Matrigel and antibodies were purchased from BD Biosciences (San Jose, CA, USA). All other reagents were procured locally.

The A549 human lung cancer cell line was obtained from the China Center for Type Culture Collection (Wuhan, China) and maintained in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

The proliferation of A549 cells following treatment with osthole was measured using the MTT assay. Briefly, A549 cells were plated at a density of 1×10⁴ cells/well in 96 well plates overnight and then treated with various concentrations of osthole (0, 20, 40, 60 and 80 μmol/l). Following a 24 h treatment, 20 μl MTT solution (2 mg/ml in PBS) was added to each well and the cells were cultured for another 4 h at 37°C. The medium was then totally removed and 150 μl DMSO was added to solubilize the MTT formazan crystals. Finally, the plates were shaken and the optical density was determined at 570 nm (OD570) using an ELISA plate reader (Model 550, Bio-Rad, Hercules, CA, USA). At least three independent experiments were performed.

For the cell migration assay, Transwell chambers were used. Briefly, A549 cells (1×10⁵ cells/well) were placed in the upper chambers of 8 μm Transwells and treated with various concentrations of osthole (0, 40 and 80 μmol/l). The bottom chambers of the Transwells were filled with 0.6 ml RPMI-1640 with 10% FBS as a chemoattractant. After 24 h, non-migratory cells were carefully removed with a cotton swab. The filter membrane was fixed with cold methanol and acetic acid (3/1, v/v) for 30 min, then stained with Giemsa. Images were captured using an Olympus inverted microscope using ×200 magnification and cell migration was quantified by counting the number of cells in 5 random fields. The percentage inhibition of migratory cells was quantified and expressed in relation to the untreated control cells. All experiments were repeated three times.

The invasion assay was performed using the same Transwells as were used in the migration assay. Briefly, A549 cells (1×10⁵ cells/well) were placed in the upper chambers of matrigel-coated 8 μm Transwells and treated with various concentrations of osthole (0, 40 and 80 μmol/l). The bottom chambers of the Transwells were filled with 0.6 ml RPMI-1640 with 10% FBS as a chemoattractant. Following incubation for 24 h, non-invading cells were carefully removed with a cotton swab. Cells that had penetrated through the matrigel located on the underside of the filter were fixed with cold methanol and acetic acid (3/1, v/v) for 30 min, then stained with Giemsa. The degree of invasiveness was quantified by counting the number of cells in 5 random fields. All experiments were repeated three times.

The expression of cellular proteins was evaluated by western blotting. A549 cells were plated onto 6 well plates and starved overnight, then treated with various concentrations of osthole (0, 40 and 80 μmol/l). Following treatment for 24 h, the total proteins were solubilized and extracted with lysis buffer (20 mM HEPES, pH 7.9, 20% glycerol, 200 mM KCl, 0.5 mM EDTA, 0.5% NP-40, 0.5 mM DTT and 1% protease inhibitor cocktail). Protein concentrations were determined by bicinchoninic acid (BCA) protein assay. All samples were separated by SDS-PAGE to determine the proteins associated with cell invasion and migration, MMP-2 and MMP-9.

All experiments were conducted three times. Data were expressed as the mean ± SD. Statistical correlation of data was checked for significance by ANOVA and the Student’s t test. P<0.05 was considered to indicate a statistically significance result. The analyses were performed using SPSS 13.0 software.

In order to investigate the growth inhibitory effects of osthole, A549 cells were treated with various concentrations of osthole for 24 h and the rate of inhibition was determined by MTT assay. We observed that the growth of the A549 cells was suppressed in a dose-dependent manner (Fig. 2).

Transwell assays were performed to investigate the effects of osthole on lung cancer cell migration and invasion. A549 cells were treated with various concentrations of osthole (0, 40 and 80 μmol/l) in order to perform the Transwell migration and matrigel-based Transwell invasion assays. As shown in Fig. 3, the A549 cells migrated from the upper to the lower chamber and this was inhibited by osthole. As shown in Fig. 4, the penetration of the A549 cells through the matrigel to the lower surface of the filter was also inhibited by osthole. These inhibitory effects were higher at an osthole concentration of 80 μmol/l than of 40 μmol/l. Our results indicate that osthole significantly inhibits lung cancer cell migration and invasion in a dose-dependent manner, suggesting a crucial role for osthole in the suppression of lung cancer metastasis.

The levels of migration- and invasion-associated proteins during the treatment with osthole were examined by western blotting. As shown in Fig. 5, the levels of MMP-2 and MMP-9 in the osthole-treated cells were lower than those in the control cells. MMP-2 and MMP-9 are significant in lung cancer cell invasion and migration. The inhibitory effects on MMP-2 and MMP-9 may be responsible for the inhibition of the invasion and migration of A549 cells following exposure to osthole.