RPMI-1640, trypsin, penicillin and streptomycin were purchased from Biological Industries (Kibutz Beit Haemek, Israel). Fetal bovine serum (FBS) was purchased from Solarbio Science&Technology (Beijing, China). 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Propidium iodide (PI), and Hoechst 33342 were purchased from Sigma-Aldrich (St. Louis, USA). Annexin V-FITC and PI double staining kit were purchased from Key Gene (Nanjing, China). 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 DMSO and stored at -20°C. Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were procured locally.

The human lung cancer cell line A549 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.

Cell proliferation was measured using the MTT assay. A549 cells were plated at a density of 1 × 10⁴cells per well in 96-well plates overnight and then treated with different concentrations of Osthole (0, 25, 50, 100, 150, and 200 μM). After 24, 48 and 72 h treatment, 20 μl of MTT solution (2 mg/ml in PBS) were added to each well and the cells were cultured for another 4 h at 37°C. Then the medium was totally removed and 150 μl DMSO was added to solubilize MTT formazan crystals. Finally, the plates were shaken and the optical density was determined at 570 nm (OD570) using a ELISA plate reader (Model 550, Bio-Rad, USA). At least three independent experiments were performed.

Cell cycle was evaluated using DNA flow cytometry analysis. A549 cells were plated at a density of 1 × 10⁶cells per well in 6-well plates overnight and then treated with different concentrations of Osthole (0, 50, 100, 150 μM). After 48 h treatment, the cells were harvested and washed twice with PBS, then centrifuged at 1200 ×g for 5 min, fixed in 70% ethanol at 4°C. Before flow cytometry analysis, the cells were washed again with PBS, treated with RNase(50 μg/ml), and stained with PI(100 μg/ml) in the dark for 30 min. The samples were analyzed by FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Apoptotic rates were determined by flow cytometry analysis using an Annexin V-FITC Apoptosis Kit. A549 cells were plated at a density of 1 × 10⁶ cells per well in 6-well plates overnight and then treated with different concentrations of Osthole (0, 50, 100, 150 μM) for 48 h. Staining was performed according to the manufacturer's instructions, and flow cytometry was conducted on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The percentage of the early apoptosis was calculated by annexin V-positivity and PI-negativity, while the percentage of the late apoptosis was calculated by annexin V-positivity and PI-positivity.

A549 cells were treated with different concentrations of Osthole (0, 50, 100, and 150 μM) for 48 h. Cells were washed twice with PBS and fixed with cold methanol and acetic acid (3/1, v/v) before being stained with Hoechst 33342(1 mg/ml) for 30 min at 37°C. Stained cells were observed with a fluorescence microscope(×400, Nikon, Japan).

The expression of cellular proteins was evaluated by Western blotting. After treatment for 48 h, the cells were washed twice with ice-cold PBS. 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% NP40, 0.5 mM DTT, 1% protease inhibitor cocktail). Protein concentration was determined by bicinchoninic acid (BCA) protein assay. Equal amounts of protein (50 μg) from each sample were subjected to seperate on a SDS-PAGE. After electrophoresis, proteins were electroblotted to polyvinylidene difluoride membranes. The membranes were blocked at room temperature and then incubated at 4°C overnight with the first antibodies of Cyclin B1, p-Cdc2, Bcl-2, Bax, t-Akt and p-Akt seperately. After being washed three times with TBST(20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 g/L Tween20), membranes were incubated with secondary antibodies. After incubation, the membranes were washed three times with TBST, and visualization was made using an ECL kit.

The data are expressed as mean ± SD. Statistical correlation of data was checked for significance by ANOVA and Student's t test. Differences with P < 0.05 were considered significant. These analyses were performed using SPSS 11.0 software.

To investigate the growth inhibition effects of Osthole, the cells were treated with different concentrations of Osthole for 24, 48 and 72 h, and the rate of inhibition was determined by MTT assay. We observed that growth of A549 cells was suppressed in a dose- and time-dependent manner(Figure 2).

To determine whether Osthole inhibits the cell cycle progression of A549 cells, the cells were treated with different concentrations of Osthole (0, 50, 100, and 150 μM) for 48 h and the cell cycle distribution was analyzed by flow cytometry. As shown in Figure 3, the percentage of cells in G2/M phase with Osthole treatment were 4.9%, 8.8%, 14.1% and 19.5% after 48 h, respectively.

A549 cells were treated with different concentrations of Osthole (0, 50, 100, and 150 μM) for 48 h and were analyzed by flow cytometry. As showed in Figure 4A, B, the numbers of early and late apoptotic cells were significantly increased compared to control group. The proportion of early and late apoptotic cells in the 150 μM treatment group was about six times higher than in the drug-free group. The proportion of apoptotic cells in treated cells were increased in a dose-dependent manner.

After incubation with different concentrations of Osthole (0, 50, 100, and 150 μM) for 48 h, the cells were examined by fluorescent microscopy analysis. As shown in Figure 4C, condensation of chromatin, nuclear fragmentations and apoptotic bodies were found clearly in treated cells. The results showed that when exposed to Osthole, A549 cells underwent the typical morphologic changes of apoptosis in a dose-dependent manner.

To investigate the mechanism underlying cell cycle arrest induced by Osthole, we tested the effect of this compound on p-Cdc2, Cyclin B1 levels. As shown in Figure 5, Western blotting analysis revealed that Osthole decreased the protein levels of Cyclin B1 and p-Cdc2 via a dose-dependent manner.

To investigate the mechanism underlying apoptosis induced by Osthole, we tested the effect of this compound on Bcl-2, Bax levels. As shown in Figure 6, Western blotting analysis revealed that Osthole treatment leads to decrease in Bcl-2 levels and increase in Bax levels as compared to control cells. These results indicated that Osthole up-regulation of the Bax/Bcl-2 ratio in a dose-dependent manner.

In order to better understand the molecular basis of Osthole induced G2/M arrest and apoptosis, we investigated the expression of p-Akt and t-Akt after treatment with Osthole(0, 50, 100, and 150 μM) for 48 h. As shown in Figure 7, the levels of p-Akt are dose-dependently decreased in response to Osthole, while the total Akt protein levels remained constant during Osthole treatment.