A549 human lung cancer cells were obtained from the Korean Cell Line Bank (Seoul, Korea). RPMI-1640 medium, fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Hoechst 33342 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Materials and chemicals used for electrophoresis were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Antibodies against Bcl-xL (#2762), Bax (#2772), caspase-3 (#9662), caspase-6 (#9762), caspase-8 (#9746) and caspase-9 (#9502), cleaved caspase-3 (#9661), poly (adenosine diphosphate-ribose) polymerase (PARP; #9542) and cleaved PARP (#9541), were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-cyclin B1 (#05-373), anti-cyclin-dependent kinase 1 (CDK1; #06-923), anti-cell division cycle 25c (cdc25c; #05-507) and anti-β-actin antibodies (#MABT825) were obtained from EMD Millipore (Billerica, MA, USA). Horseradish peroxidase (HRP)-coupled goat anti-mouse IgG ALX-211-205TS-C100 and anti-rabbit IgG ADI-SAB-301-J were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Muse™ Annexin V & Dead Cell kit was purchased from EMD Millipore.

The fruit of Korean Citrus platymamma hort. ex Tanaka (called Byungkyul in Korea) was obtained from the Animal Bio Resources Bank (Jinju, Korea). The flavonoids were isolated at the Department of Chemistry, Gyeongsang National University (Jinju, Korea) by Professor Sung Chul Shin. The sample was prepared according to a previously described method (24). Samples were stored at −20°C until used for further experiments.

A549 cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin in a humidified incubator with 5% CO₂ in air at 37°C. The stock solution of flavonoids was prepared in dimethyl sulfoxide (DMSO), and subsequent dilutions were prepared for treatment. Cells grown to 70–80% confluence were untreated (control) or treated with FCP at various concentrations (100, 200, 300, 400 and 500 µg/ml) for 24 h in complete growth medium.

A549 cells were seeded at 10×10⁴ cells/ml in a 12-well plate and incubated for 24 h. The cytotoxicity was measured by a standard MTT assay following treatment with FCP at the specified concentrations for 24 h at 37°C. After 24-h incubation, 100 µl MTT reagent (5 mg/ml) was added to each well, and incubated at 37°C for 3 h to form formazan crystals. Then, the supernatant was discarded, and 500 µl DMSO was added to each well to dissolve the crystals. The optical density of the cells at 540 nm was measured using an enzyme-linked immunosorbent assay plate reader. The morphology of flavonoids-treated A549 cells was observed under a phase contrast microscope (Olympus Corporation, Tokyo, Japan).

Cells were seeded at a density of 5×10⁵ cells/well into a 6-well plate and treated with FCP at various concentrations (0, 91, 182 and 364 µg/ml) for 24 h at 37°C. The floating and adherent cells were collected, washed twice with cold phosphate-buffered saline (PBS) and centrifuged at 300 × g for 5 min at room temperature. The cell pellet was fixed using cold 70% ethanol (v/v) for 3 h at −20°C. The cells were washed once with PBS, and 200 µl of the cell suspension was transferred to a fresh tube. Next, 200 µl Muse™ Cell Cycle kit reagent (EMD Millipore) was added to each tube and incubated for 30 min at room temperature in the dark. For the apoptosis assay, the floating and trypsinized cells were collected, washed once with cold PBS and centrifuged at 300 × g for 5 min at room temperature. The pellet was resuspended in 1 ml medium, and 100 µl of this suspension was transferred to a new tube. Then, 100 µl Muse™ Annexin V & Dead Cell kit reagent was added to each tube and incubated for 20 min at room temperature in the dark. Then stained samples were analyzed with a Mini Flow Cytometry Muse™ Cell Analyzer (EMD Millipore).

A549 cells were grown to 80% confluence on 10-mm diameter glass slides placed in 24-well plates and treated with FCP at the indicated concentrations for 24 h at 37°C. Cells were washed with cold PBS and fixed with 37% formaldehyde (1:4 dilution with 95% ethanol) for 10 min at room temperature. The fixed cells were washed with PBS and stained with DAPI and VECTASHIELD^® (catalog number H-1500; Vector Laboratories, Inc., Burlingame, CA, USA). Cells were mounted in Fluorescence Mounting Medium (Dako, Glostrup, Denmark), and the nuclear morphology of the cells was examined by fluorescence microscopy (magnification, ×400; Leica Microsystems GmbH, Wetzlar, Germany).

The protein content was quantified using a Bradford assay (Bio-Rad Laboratories, Inc.). Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Immunobilon-P, 0.45 mm; EMD Millipore) using the TE 77 semi-dry transfer unit (GE Healthcare Life Sciences, Chalfont, UK). The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (pH 7.4) at room temperature for 1 h. Membranes were incubated overnight at 4°C with the primary antibodies (dilution, 1:1,000), and a second incubation was conducted with HRP-conjugated secondary antibodies (dilution, 1:1,000) for 3 h at room temperature. The bound antibodies were visualized using an enhanced chemiluminescence kit (GE Healthcare Life Sciences), and images were acquired using a ChemiDoc™ XRS+ system with Image Lab™ software version 4.1 (Bio-Rad Laboratories, Inc.).

Data are expressed as the mean ± standard deviation of ≥3 independent experiments. Statistical analysis was performed with Student's t-test and one-way analysis of variance (ANOVA), using SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate as statistical significant difference. Significant differences were determined using ANOVA with post-test Neuman-Keuls method for comparisons of ≥3 treatment groups, while Student's t test was used for comparisons of two groups.

The HPLC chromatogram of the fruit of Korean Citrus platymamma hort. ex Tanaka, which was recorded at 280 nm, is shown in Fig. 1, where peaks corresponding to 11 different flavonoids were clearly distinguished. These peaks were identified based on their HPLC retention times, molecular ion masses and spectroscopic data. In total, 11 flavonoids were successfully identified from Korean Citrus platymamma hort. ex Tanaka, including neoeriocitrin, naringin, hesperidin, isosinensetin, sinensetin, tetramethyl-O-isoscutellarein, nobiletin, tetramethoxyflavone, heptamethoxyflavone, tangeretin and hydroxypentamethoxyflavone. The quantification is shown in Table I.

The anti-proliferative effect of FCP was investigated by MTT assay in A549 cells incubated for 24 h with different doses of FCP (0, 100, 200, 300, 400 and 500 µg/ml). As indicated in Fig. 2A, FCP significantly inhibited the viability of A549 cells in a dose-dependent manner. The results revealed that the half maximal inhibitory concentration (IC₅₀) of FCP was 364 µg/ml. According to their IC₅₀ value, the concentrations of flavonoids used in the following experiments were 0, 91, 182 and 364 µg/ml. In addition, microscopic examination revealed that morphological changes in cell shape such as cell shrinkage, and a gradual decrease in the living cell population, were observed with increasing concentrations of FCP (Fig. 2B). These results suggest that FCP have anti-proliferative effects on A549 cells.

To investigate the mechanisms responsible for the anti-proliferative effects of FCP, the cell cycle progression of A549 cells following FCP treatment at the specified concentrations was examined by flow cytometry. The sub-G1 apoptotic cell population and G2/M phase population were significantly increased in a dose-dependent manner, whereas the number of cells in the G0/G1 and S phases were remarkably decreased (Fig. 3A). Fig. 3B shows the quantitative representations of cell cycle progression in A549 cells treated with FCP, which were observed to be dose-dependent. These results indicate that FCP are able to induce G2/M arrest and apoptosis in A549 cells.

This phase is controlled by formation of a complex of cyclin B1 and CDK1, which is regulated by cdc25c (25). In the present study, the effect of FCP on the regulatory proteins involved in G2/M arrest was examined by immunoblotting, which revealed that FCP significantly decreased the expression levels of cyclin B1, CDK1 and cdc25c proteins in a dose-dependent manner (Fig. 3C). These data suggest that FCP induce G2/M arrest by downregulation of cyclin B1, CDK1 and cdc25c proteins in A549 cells.

Induction of apoptosis was examined in A549 cells following FCP treatment by Annexin V -fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-labelled flow cytometry and Hoechst 33342 staining. In FCP-treated A549 cells, the total apoptotic cell proportion was significantly increased in a dose-dependent manner (Fig. 4A and B), and the late apoptotic cell proportion was higher than the early apoptotic cell proportion, compared with the control. Furthermore, Hoechst 33342 staining of FCP-treated A549 cells revealed apoptotic changes in these cells, including condensed and fragmented nuclei (Fig. 4C). These data indicate that FCP are able to induce apoptosis in A549 cells.

It is well known that caspases play a key role in modulating apoptosis (26). Among them, caspase-3 is the major effector protein of apoptosis, and is activated by an initiator caspase such as caspase-9 (27). The activation of caspases and subsequent cleavage of PARP from the native 116-kDa form to the 89-kDa protein is one of the hallmarks of apoptosis (28). To clarify the mechanism of FCP-induced apoptosis, the expression of apoptosis-related proteins in A549 cells following FCP treatment was examined. Upon treatment with FCP, the expression levels of PARP and pro-caspases −3, −6, −8 and −9 were significantly decreased in a dose-dependent manner, which indicates caspase activation (Fig. 5A-D). FCP significantly increased the levels of cleaved PARP, a substrate of caspase-3, in A549 cells (Fig. 5E). The ratio of proapoptotic and antiapoptotic proteins is the determinant factor of apoptosis. In the present study, the Bax/Bcl-xL ratio was significantly increased by FCP in a dose-dependent manner (Fig. 5F). These results suggest that FCP induce apoptosis in A549 cells by the activation of caspases and subsequent PARP cleavage, at least in part through an increase in the ratio of Bax/Bcl-xL.