All experiments were approved by the Huazhong University of Science and Technology Committee and the Tongji Medical College Ethics Committee, Tongji Hospital (Wuhan, China).

Dimethyl sulfoxide (DMSO), angelicin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich, Invitrogen Life Technologies (Carlsbad, CA, USA). Polyclonal antibodies against Bax, Bcl-2, caspase-3, caspase-9, cyclin B1, cyclin E1, Cdc2, E-cadherin, MMP2, MMP9, p-ERK1/2, ERK1/2, p-JNK1, JNK1, P-p38 MAPK, p38 MAPK, Akt and p-Akt were purchased from Cell Signaling Technology, Inc. (Danvers, MA USA). GAPDH, goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco, Invitrogen Life Technologies (Carlsbad, CA, USA). A protease inhibitor cocktail was purchased from Roche Diagnostics (Basel, Switzerland). A BCA protein assay reagent kit and an enhanced chemiluminescence (ECL) plus reagent kit were obtained from Pierce Biotechnology, Inc. (Rockford, IL, USA).

Human lung cancer A549 cells were purchased from the Chinese Academy of Sciences Cell Bank (CBP60084; Shanghai, China) and were maintained in RPMI-1640 with 10% FBS and antibiotics (penicillin and streptomycin) in an incubator with a humidified atmosphere (5% CO₂, 37°C).

An MTT colorimetric assay was performed according to the manufacturer's instructions. In brief, exponentially growing cells were seeded in 96-well plates at a density of 5×10³ cells/well. Following an overnight incubation, the cells were treated with various doses of angelicin for 24 h at 37°C. Then, the medium was discarded, and MTT (0.5 mg/ml) was added into each well and incubated for 4 h at 37°C. Subsequently, the MTT-containing medium was removed and replaced with 150 µl of DMSO. The absorbance at 570 nm was then determined using a Bio-Rad Model 680 microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The IC₅₀ was calculated from the MTT dose-response curves of cell viability against drug concentration. Three replicate wells were used for each analysis.

A TUNEL assay was utilized to analyze the pro-apoptotic effect of angelicin in A549 cells. After being appropriately treated with angelicin, cells were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.05 % Triton X-100 on ice for 5 min. The cells were subjected to TUNEL while being incubated in a humidified chamber at 37°C for 60 min in the dark. The cells were washed 3 times with phosphate-buffered saline (PBS; pH 7.4); anti-fluorescence quenching solution was then added and allowed to react for 5 min. Finally, the cells were examined using a confocal laser scanning microscope.

To detect the apoptotic effects of angelicin on A549 cells, an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was used. In brief, A549 cells were seeded in a 6-well plate and incubated for 24 h; the cells were then treated with the DMSO control or angelicin (10, 25 or 50 µmol) for 24 h. Next, the cells were collected, washed with PBS, and resuspended in 100 µl of 1X binding buffer. Annexin V-FITC/PI were added to each group, and the cells were incubated for 15 min at room temperature in the dark. The cells were then analyzed by flow cytometry (BD Calibur; BD Biosciences, San Jose, CA, USA). Each experiment was performed 3 times.

Flow cytometry used to examine the effect of angelicin on the cell cycle. After being treated with the DMSO control or angelicin (10, 25 or 50 µmol) for 24 h, A549 cells were harvested, washed twice with PBS (pH 7.4) and fixed with 70% ethanol for 20 min. Then, the cells were centrifuged (300 × g, 5 min) to eliminate the ethanol, washed twice with PBS (pH 7.4) and stained with PI in the dark for 30 min. Finally, the cell cycle distribution was assessed by flow cytometry (BD Calibur; BD Biosciences). Each experiment was performed 3 times.

The anti-migratory effects of angelicin on A549 cells were examined through a wound-healing assay. After attached cells had grown to 90% confluence, a wound in the monolayer was created using a pipette tip, and the cells were washed twice with PBS (pH 7.4). Then, the cells were treated with the DMSO control or angelicin (10, 25 or 50 µmol) for 24 h. The number of migrated cells was determined using an inverted microscope. Five randomly chosen fields were analyzed in each well.

Transwell chambers (Corning Costar, Cambridge, MA, USA) were used for the cell migration assays. A549 cells (1×10⁵) were seeded in the top chamber with FBS-free medium. Culture medium containing the DMSO control or angelicin (10, 25 or 50 µmol) was added to the bottom chamber. After the cells were incubated for 24 h at 37°C, the upper side of the membrane was removed, and the cells in the lower chamber were fixed in 4% paraformaldehyde for 15 min. The fixed cells were washed with PBS (pH 7.4) 3 times and then stained with 0.25% crystal violet for 5 min. Cell migration was evaluated using an inverted microscope (×200). Six randomly chosen fields were analyzed in each group and presented as the mean of 3 independent experiments.

After the cells were treated with the DMSO control or angelicin, proteins from the A549 cell lysates were extracted and 12% SDS-PAGE was used to separate the protein samples. Then, the proteins were transferred to a PVDF membrane, which was blocked with 5% skim milk and incubated with different antibodies overnight at 4°C. The antibodies were diluted to the following concentrations: Bax (1:1,000), Bcl-2 (1:2,000), caspase-3 (1:1,000), caspase-9 (1:800), cyclin B1 (1:1,000), cyclin E1 (1:1,000), Cdc2 (1:2,000), E-cadherin (1:2,000), MMP2 (1:800), MMP9 (1:800), p-ERK1/2 (1:2,000), ERK1/2 (1:2,000), p-JNK1 (1:1,000), JNK1 (1:1,000), P-p38 MAPK (1:1,000), p38 MAPK (1:1,000), Akt (1:2,000) and p-Akt (1:1,000). After being washed 3 times in TBST, the membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. An ECL kit was used to develop the immuno-reactive bands.

For the A549 xenograft studies, female nude mice aged 5 weeks were used. A549 cells (5×10⁶) were subcutaneously implanted in the right flank of the mice. The tumor-bearing mice were randomly divided into two groups: the PBS (pH 7.4) control group and the angelicin group. Mice in the angelicin group were treated with angelicin for 4 consecutive weeks by oral gavage (100 mg/kg), while mice in the control group received PBS (pH 7.4). Tumor volume and body weight were monitored once every 2 days. At the end of 4 weeks, the mice were sacrificed and the tumor xenografts were removed and measured.

Male nude mice (SIPPR-BK Laboratory Animal Co., Ltd., Shanghai, China) aged 5 weeks were used. A549 cells (5×10⁶) were injected via the tail vein. Two weeks later, the mice were randomly divided into two groups: the PBS (pH 7.4) control group and the angelicin (100 mg/kg/day) group. Mice in the angelicin group received an intragastric administration of angelicin dissolved in PBS (pH 7.4) every day for 4 weeks. The animals were sacrificed after this period. The lungs were then removed and fixed in Bouin's solution for 4 h, and the number of metastatic lesions was determined macroscopically.

Sections of the lung specimens were deparaffinized and rehydrated. Then, the sections were rinsed in PBS (pH 7.4) 3 times and incubated in 3% hydrogen peroxide for 15 min at room temperature. After being blocked in 10% goat serum for 30 min, the tissue sections were incubated overnight at 4°C with polyclonal antibodies (MMP2, 1:100; MMP9, 1:80; and E-cadherin, 1:50). The sections were subsequently incubated with peroxidase-conjugated goat anti-rabbit secondary antibody (1:100) for 1 h at room temperature, washed 5 times with PBS (pH 7.4), and visualized with the peroxidase substrate diaminobenzidine (DAB) under a microscope.

The data shown in this study were obtained from at least 3 independent experiments and analyzed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). Statistical significance was determined using an unpaired Student's t-test. All results are presented as the mean ± standard deviation (SD). P<0.05 was considered statistically significant vs. the control group.

The chemical structure of angelicin is shown in Fig. 1A. To examine the cytotoxic effects of angelicin on A549 cells, an MTT assay was performed. A549 cells were treated with different concentrations of angelicin (0, 2.5, 5, 10, 25, 50, 100, 150 and 200 µmol/l) for 24 h. As shown in Fig. 1B, the proliferation of angelicin-treated A549 cells was markedly suppressed at 24 h compared with that of cells in the control group. These results showed that the angelicin treatment suppressed A549 cell proliferation in a dose-dependent manner. As the IC₅₀ value for angelicin was 50.14 µmol, doses of 10, 25 and 50 µmol angelicin were selected for use in subsequent experiments to assess A549 cell growth suppression.

Additionally, the morphological changes of angelicin-treated cells were assessed using an inverted microscope (Fig. 1C). The results showed that the angelicin treatment caused marked morphological alterations, including the adherence of cells in poor condition, reduced cell volume, chromatin condensation, karyopyknosis and nuclear fragmentation. Furthermore, as the dose of angelicin increased, the morphological alterations became more apparent.

To assess whether angelicin-induced cell growth suppression was associated with cell apoptosis, the effects of angelicin on apoptosis were evaluated by flow cytometry using Annexin V-FITC/PI double staining. As shown in Fig. 2A and B, the percentage of early- and late-stage apoptotic cells increased in a dose-dependent manner. This result showed that the angelicin treatment caused a significant increase in apoptosis.

Moreover, the extent of A549 cell apoptosis was examined using TUNEL staining. The TUNEL assay revealed only 2±2% TUNEL-positive nuclei in the DMSO control cells, and in accordance with the flow cytometric analysis, a significantly increased percentage of TUNEL-positive nuclei was observed in the cells incubated with angelicin (Fig. 2C and D). In addition, angelicin-induced apoptosis occurred in a dose-dependent manner.

Furthermore, to gain insight into the potential pro-apoptotic mechanisms of angelicin, the protein expression levels of Bax, Bcl-2, caspase-3 and caspase-9, which are important apoptosis-associated proteins, were detected by western blotting. As shown in Fig. 2E-G, the angelicin treatment markedly induced Bax expression to a level comparable to that of the control cells and significantly reduced Bcl-2 expression in a dose-dependent manner. In addition, the angelicin treatment promoted the expression of cleaved caspase-3 and caspase-9 in a dose-dependent manner, demonstrating the pro-apoptotic effect of angelicin. All the results demonstrated that the pro-apoptotic effect of angelicin may be associated with the regulation of these proteins.

The effect of angelicin on the cell cycle distribution was determined using flow cytometry. Cells were or were not treated with angelicin at 10, 25 and 50 µmol for 24 h and were then stained with PI. The results showed that the proportion of cells in the G2/M and G0/G1 phase increased and decreased, respectively, in a dose-dependent manner (Fig. 3A and B).

To further elucidate the specific regulatory proteins responsible for the cell cycle arrest, we explored the effect of angelicin on the regulatory proteins cyclin B1, cyclin E1 and Cdc2. As shown in Fig. 3C and D, the levels of cyclin B1, cyclin E1 and Cdc2 were significantly downregulated in the angelicin-treated cells. These data revealed that alterations in the expression levels of cell cycle regulatory proteins and the arrest of cell growth in the G2/M phase are involved in angelicin-induced changes in cell cycle progression.

Tumor cell migration and invasion are key steps in cancer metastasis (16). To evaluate the potential effect of angelicin on A549 cell migration, we assessed alterations in cell mobility using a scratch test and a Transwell assay. The results showed that the angelicin treatment significantly decreased A549 cell migration capability in a dose-dependent manner (Fig. 4A and B). The Transwell assay results showed that the migration and invasion of the angelicin-treated A549 cells were significantly inhibited in a dose-dependent manner compared with that of the control cells (Fig. 4C and D).

E-cadherin, MMP2 and MMP9 are responsible for cell migration, invasion and cell-matrix adhesion, particularly in the process of cancer metastasis (17). Therefore, we detected the effects of angelicin on the expression of these proteins using western blotting. As shown in Fig. 4E and F, we observed that angelicin strongly increased E-cadherin expression but reduced MMP2 and MMP9 expression in A549 cells in a dose-dependent manner compared with that of the control cells. These results indicate that angelicin directly inhibits the migration and invasion of A549 cells.

The MAPK and Akt signaling pathways have important roles in regulating tumor cell apoptosis, cell cycle progression and metastasis (17). To further explore the mechanism underlying angelicin-induced apoptosis, cell cycle arrest and migration inhibition in A549 cells, we evaluated whether angelicin modulates the MAPK and Akt signaling pathways when affecting A549 cells. We first examined the activation status of p38 MAPK, JNK, ERK and AKT by western blotting with antibodies specific to the phosphorylated forms of these kinases. As shown in Fig. 5A-C, treating A549 cells with angelicin significantly increased the levels of phosphorylated JNK and ERK compared with the total protein expression levels in a dose-dependent manner. However, angelicin had no effect on the phosphorylation of p38 MAPK, AKT, or the total expression levels of these proteins. These results suggest that angelicin may activate the JNK and ERK pathways in A549 cells.

We next tested whether SP600125, a JNK inhibitor, or U0126, an ERK inhibitor, could reverse the angelicin-induced apoptosis, cell cycle arrest and migration inhibition in A549 cells. First, the levels of phosphorylated JNK and ERK were analyzed by western blotting. The SP600125 and U0126 treatments significantly attenuated angelicin-induced JNK and ERK activation (Fig. 6A and B). The results suggested that JNK and ERK activation may play a crucial role in angelicin-mediated effects on A549 cells. Next, the cell cycle, apoptosis and migration of A549 cells were examined with or without SP600125 and U0126 pretreatments. As shown in Fig. 6C-H, the SP600125 and U0126 treatments partially alleviated the angelicin-induced apoptosis, cell cycle arrest and migration inhibition of A549 cells. The results indicated that angelicin inhibits A549 cell growth and migration through both the ERK and JNK pathways.

We further evaluated the antitumor effect of angelicin in A549 cancer cells by utilizing nude mouse xenograft models. As anticipated, tumor size and weight decreased significantly in the angelicin group compared with the control group (Fig. 7A). The anti-metastatic effects of angelicin were further assessed with an in vivo model of lung metastasis. As shown in Fig. 7B, angelicin had an inhibitory effect on A549 tumor metastasis to the lungs compared with the control group. TUNEL labeling and the expression levels of MMP2, MMP9 and E-cadherin were evaluated immunohistochemically (Fig. 7C). Angelicin significantly increased the ratio of TUNEL-positive cells, which is indicative of apoptosis, in metastatic nodules of the lung metastasis model. Furthermore, the angelicin treatment resulted in decreased expression levels of MMP2 and MMP9 and increased expression of E-cadherin, which was consistent with the in vitro study. These results indicate that angelicin can significantly inhibit A549 cell growth and metastasis in vivo.