Induction of the cell cycle arrest and apoptosis by flavonoids isolated from Korean Citrus aurantium L. in non-small-cell lung cancer cells

Food Chemistry 135 (2012) 2728–2735

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Induction of the cell cycle arrest and apoptosis by flavonoids isolated from Korean Citrus aurantium L. in non-small-cell lung cancer cells Kwang Il Park a, Hyeon Soo Park a, Arulkumar Nagappan a, Gyeong Eun Hong a, Do Hoon Lee a, Sang Rim Kang b, Jin A. Kim c, Jue Zhang d, Eun Hee Kim e, Won Sup Lee f, Sung Chul Shin g, Young Sool Hah h, Gon Sup Kim a,⇑ a

Research Institute of Life Science and College of Veterinary Medicine, Gyeongsang National University, Gazwa, Jinju 660-701, Republic of Korea Department of Biological Engineering, School of Natural Science, Kyonggi University, Yeongtong, Suwon 443-760, Republic of Korea Korea National Animal Research Resource Center and Korea National Animal Bio-Resource Bank, Gyeongsang National University, Gazwa, Jinju 660-701, Republic of Korea d Key Laboratory of Nuclear Medicine of Ministry of Health, Jiangsu Institute of Nuclear Medicine, China e Department of Nursing Science, International University of Korea, Jinju 660-759, Republic of Korea f Department of Internal Medicine, Institute of Health Sciences, Gyeongsang National University School of Medicine, Gyeongnam Regional Cancer Center, Gyeongsang National University Hospital, Jinju 660-702, Republic of Korea g Department of Chemistry and Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea h Clinical Research Institute, Gyeongsang National University Hospital, Jinju 660-702, Republic of Korea b c

a r t i c l e

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Article history: Received 25 April 2012 Received in revised form 14 June 2012 Accepted 28 June 2012 Available online 6 July 2012 Keywords: Korean Citrus aurantium L. Flavonoid Human lung cancer cells Cell cycle arrest Apoptosis

a b s t r a c t This study investigated the anti-proliferative and apoptotic effect of flavonoids isolated from Korean Citrus aurantium L. using A549 lung cancer cells. Flavonoids potently inhibited of A549 cells in a dosedependent manner, whereas flavonoids had a weak inhibitory effect on proliferation of WI-38 cells. Flow cytometry and Western blot analysis showed that flavonoids induced cell cycle arrest at the G2/M checkpoint by controlling the proteins expression level of cyclin B1, cdc2, cdc25c and p21WAF1/CIP1. Also, flavonoids induced apoptosis through the regulation of the expression of caspases, cleaved PARP and Bax/Bcl-xL ratio. The activity of caspase-3 on A549 cells increased in a dose-dependent manner. These results clearly indicated that the anti-cancer effect of flavonoids on A549 cells follows multiple cellular pathways through G2/M arrest and the induction of apoptosis. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Globally, lung cancer is one of the most common cancers and the leading causes of cancer-related mortality. Non-small-cell lung cancer (NSCLC) accounts for more than 80% of all lung cancers. The most widely-used therapies for NSCLC are chemotherapy, radiation and surgery. Despite advances in therapies, approximately 85% of adenocarcinoma patients die within 5 years of diagnosis and one third of patients with stage IV disease have a 2-year survival rate of <20% (Douillard, Eckardt, & Scagliotti, 2002; Erridge, Moller, Price, & Brewster, 2007). In 2007 there were 17,846 lung cancer cases in Korea, accounting for 11% of all cancer cases in Korea. Although the survival rate for lung cancer has increased gradually, new therapeutic agents are needed to improve this rate. The flavonoids comprise a family of naturally-occurring polyphenol compounds of plants. They have a common benzoc-pyrone (phenylchromone) chemical structure. The main sources ⇑ Corresponding author. Tel.: +82 55 772 2346; fax: +82 55 751 2349. E-mail address: [email protected] (G.S. Kim). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.06.097

containing flavonoids are tea, vegetables and fruits including Citrus spp. (family Rutaceae) (Nogata et al., 2006). In particular, Citrus spp. contains abundant flavonoids, including hesperidin, naringin and nobiletin (Benavente-Garcia & Castillo, 2008). Flavonoids are a commonly used traditional medicine in several Asian countries because of their antioxidant, antiviral, antiinflammatory and anticancer properties (Havsteen, 1983). The intake of flavonoids reduces the risk of squamous cell oesophageal and colorectal cancer (Rossi et al., 2007; Theodoratou et al., 2007). Cell cycle and control of apoptosis are the important regulatory mechanisms of cell growth, development and differentiation. In mammals, the cell cycle comprises the G1, S, G2 and M phases. Cell cycle checkpoints ensure the maintenance of genomic integrity by inhibiting damaged or incomplete DNA. In particular, the G2/M checkpoint is an important cell cycle checkpoint in eukaryotic organisms. This checkpoint insures that the cells do not initiate mitosis before repairing damaged DNA after replication. The cell cycle progression depends on a cascade of enzymes by sequential activation and inactivation of cyclin, cyclin-dependent kinases (CDKs) and cyclin-dependent kinase inhibitors (CDKIs) (Nigg,

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1995). The G2/M transition is regulated by the sequential activation and inactivation of the cdc2/cyclin B complex (King, Jackson, & Kirschner, 1994; Krek & Nigg, 1991). Apoptosis (programmed cell death) plays a crucial role for cell homeostasis and differs from necrosis. Among the morphological and biological changes that occur in apoptosis, the main manifestations of cell death are nuclear fragmentation, chromatin condensation, cell shrinkage and DNA fragmentation (Han, Kim, & Kim, 2008). Apoptosis is controlled by the Bcl-2 family of proteins and by caspases, a family of cysteine proteases (Reed et al., 1996; Stennicke & Salvesen, 1998). Apoptosis induced by these molecules can prevent carcinogenesis by eliminating damaged cells or inhibiting abnormal cell development (Hengartner, 2000). Therefore, the induction of cell cycle arrest and programmed cell death play crucial roles in the anticancer properties of many anticancer agents. Although the induction of apoptotic death and cell cycle arrest by flavonoids has already been reported, the mechanisms of the anti-cancer activity of flavonoids isolated from Korean Citrus aurantium L. still remain unclear (Auyeung & Ko, 2010; Kobayashi, Nakata, & Kuzumaki, 2002). We hypothesised that flavonoid isolated from Korean C. aurantium L. could induce cell cycle arrest and apoptosis on A549 lung cancer cells through the regulation of cell cycle arrest and apoptosis-related proteins. Hence, we investigated the anticancer effect and mechanism of flavonoids isolated from Korean C. aurantium L. on A549 human lung cancer cells. 2. Materials & methods 2.1. Reagents and antibodies RPMI 1640 was purchased from Hyclone (Logan, UT). Foetal bovine serum (FBS) and antibiotics (streptomycin/penicillin) were purchased from Gibco (BRL Life Technologies, Grand Island, NY). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), propidium iodide (PI) and RNase A were obtained from Sigma–Aldrich (St. Louis, MO). Hoechst 33342 was purchased from Invitrogen Molecular Probes (Eugene, OR). Antibodies against cyclin B1, cdc25c, cdc 2, p21WAF1/CIP1, cleaved PARP and b-actin were obtained from Millipore (Billerica, MA). Anti-BAX, anti-Bcl-xL and anti-caspase-3, -6, -8 and -9 were purchased from Cell Signalling Technology (Danvers, MA). Horseradish peroxidase-coupled goat anti-mouse IgG and anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescein isothiocyanate (FITC) Annexin-V apoptosis detection kit 1 was purchased from BD Biosciences (San Diego, CA). Materials and chemicals used for electrophoresis were obtained from Bio-Rad Laboratories (Hercules, CA). The enhanced chemiluminescence kit was purchased from Amersham Life Sciences (Amersham, UK). All other chemicals were of the purest grade available. 2.2. Isolation of flavonoids from Korean C. aurantium L. Korean C. aurantium L. fruits were obtained from the Citrus Genetic Resources Bank, Jeju National University. The fruits were washed with water and stored at 70 °C until extraction. The fruit peel was finely ground after lyophilisation in a PVTED50A apparatus (Ilsin BioBase, Yangju, Korea). The powder of the fruit peel (1500 g) was extracted using a PT-MR 2100 apparatus (Kinematica, Lucerne, Switzerland) in 3 L of 70% aqueous methanol for 24 h (n = 3). The extracts were combined and filtered using a Buchner funnel. The filtrate was concentrated to 140 mL using an Eyela NVC-2100 rotary evaporator (Tokyo Rikakikai, Tokyo, Japan). Twenty-five millilitres of water and 3 g of NaOH were added to the concentrate, which was extracted with 200 mL ethyl acetate.

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After three extraction cycles, the organic layer was neutralised with brine and dried over anhydrous MgSO4 to yield 0.6 g of flavonoid components. HPLC analysis was carried out using an Agilent 1100 series LC system (Agilent Technologies, Santa Clara, CA). Chromatographic separation was performed on a Zorbax StableBond Analytical SB-C18 column (4.6  250 mm, 5 lm, Agilent Technologies). The binary solvent system consisted of 0.1% aqueous formic acid (A) and methanol/acetonitrile (1:1, v/v) (B). The elution was performed by a linear gradient from 0% to 25% B over 10 min and from 40% to 70% B over 10 min followed by 30 min of isocratic elution, decreased from 40% to 25% B over 5 min and followed by 10 min of isocratic elution. The flow rate was 0.5 mL/min with a column temperature of 35 °C. Chromatographic data were collected and manipulated using ChemStation, Rev.B.0301. Spectral data were collected (200–400 nm, 2-nm resolution) for the entire run, and the flavonoids were quantified by extracting the chromatograms at 280 nm. All of the flavanones and flavones were quantified using the external calibration curves of hesperetin and nobiletin, respectively. MS/MS experiments were conducted using a 3200 QTRAP LC–MS/MS system (Applied Biosystems, Foster City, CA) with a Turbo V™ source and a Turbo Ion Spray probe. The mass spectrometer was operated in positive ion mode with selected ion monitoring (SIM). BioAnalyst™, Version 1.4.2, and The Analyst software, Version 1.4.2, were used for instrumental control and data acquisition, respectively. Nitrogen at a pressure of 45 psi was used as a nebulising and drying gas. The electron spray voltage was set at 5.2 kV and the source temperature at 500 °C. The mass spectra were recorded between m/z 100 and m/z 1000 with a step size of 0.06 amu. 2.3. Cell culture and treatment A549 human lung carcinoma cells and WI-38 (Human embryonic fibroblast, lung-derived cell line) obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin (P–S) in a 5% CO2 atmosphere at 37 °C. Cells grown to 70–80% confluence were untreated (control) or treated with flavonoids for 24 h in complete growth medium. 2.4. Evaluation of growth inhibitory potential The cytotoxicity of flavonoids on A549 and WI-38 cells were measured by a standard MTT assay. A549 and WI-38 cells were cultured in wells of a 12-well plate at a density of 5  105 per well and incubated for 24 h at 37 °C. The flavonoids isolated from Korean C. aurantium L. were dissolved in DMSO. The cells were treated with flavonoids at various concentration (50, 100, 150, 200 and 250 lg/mL) for 24 h at 37 °C, and then 100 lL of a solution of 5 mg/mL MTT in phosphate-buffered saline (PBS) were added to each well, followed by incubation for 3 h at 37 °C. After the fluid was discarded, 500 lL of DMSO were added to each well to dissolve the crystalline deposits that had formed. The optical density (OD) of the cells at 540 nm was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader. All experiments were done in triplicate. 2.5. Morphological studies Variations in cell morphology were analysed by light and fluorescence microscopy. Treated A549 cells were centrifuged at 1500 rpm for 5 min, fixed for 15 min in PBS containing 4% paraformaldehyde, washed with PBS, and stained with Hoechst 33342 (20 lg/mL). Nuclear morphology was observed with a LEICA DM

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6000 B fluorescence microscope with excitation wavelength of 350 nm (blue fluorescence).

for 2–4 h with the reaction buffer. Samples were measured as optical density at 405 nm with a plate reader. The data were represented as mean ± SD of three independent experiments.

2.6. DNA cell cycle analysis 2.9. Western blot When the A549 cell grows to 70–80% confluent, they were treated with 10, 50, 150 and 250 lg/mL flavonoids for 24 h in complete medium. The floating and adherent cells were collected, washed twice with cold PBS and centrifuged. The pellet was fixed in 70% (v/v) ethanol for 24 h at 4 °C. The cells were washed once with PBS and resuspended in cold propidium iodide (PI; 50 lg/mL) containing RNase A (0.1 mg/mL) in PBS (pH 7.4) for 30 min in the dark. Cellular DNA content was analysed by flow cytometry using a FACSCalibur apparatus (Becton Dickinson, San Jose, CA). Forward light scatter characteristics were used to exclude cell debris from the analysis. At least 10,000 cells were used for each analysis. Cell cycle distribution was analysed using the ModFit LT programme. (Verily Software House, Topsham, ME). 2.7. Apoptosis analysis A549 cells were harvested 24 h after flavonoids treatment (10, 50, 150 and 250 lg/mL), and the magnitude of apoptosis was determined using a FITC Annexin-V apoptosis detection kit 1 (BD Pharmingen, San Diego, CA). In brief, the cells were washed with ice-cold PBS and resuspended in 100 lL of Annexin-V binding buffer containing 10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2 according to the manufacturer’s protocol. Aliquots of the cells were incubated in 5 lL of an Annexin V-FITC (BD Biosciences) solution and 5 lL of PI at room temperature for 15 min in the dark. Then, 400 lL of binding buffer were added. The apoptotic cells were measured by a fluorescence-activated cell sorter analysis in a flow cytometer (Becton Dickinson, San Jose, CA).

Western blot analysis was performed. Briefly, A549 cells were cultured in wells of 6-well plates and incubated in DMSO as the solvent control or in the presence of 10, 50, 150 and 250 lg/mL flavonoids for 24 h at 37 °C. After washing with ice-cold PBS, the cells were lysed and protein concentration was determined using a Bradford assay (Bio-Rad, Hercules, CA). An equal amount of protein was subjected to 12% sodium dodecyl sulphate– polyacrylamide gels (SDS–PAGE) and the resolved proteins transferred to a 0.45-mm Immunobilon polyvinyldene fluoride membrane (Millipore). Blots were probed with a 1:1000 dilution of the desired primary antibody overnight at 4 °C, incubated with a 1:2000 diluted enzyme-linked secondary antibody, visualised by enhanced chemiluminescence and Western Blotting Detection Reagents (GE Healthcare Life Sciences, Piscataway, NJ) and exposed to X-ray film (Fuji, Tokyo, Japan). Each band was quantitatively determined using the Image J program (http://rsb.info.nih.gov). The densitometry readings of the bands were normalised to b-actin expression. 2.10. Statistical analysis Data are expressed as the mean ± standard deviation of at least three independent experiments and statistical analysis was done using a Student’s t-test, using SPSS Version 10.0 for Windows (SPSS, Chicago, IL). The level of statistical significance was set at p < 0.05. 3. Results

2.8. Caspase-3 activity assay 3.1. Characterisation and quantification of flavonoids Caspase-3 activity was evaluated by the detection of the cleavage of a colorimetric caspase-3 substrate, N-acetyl-AspGlu-Val-Asp (DEVD)-p-nitroaniline, using an assay kit (Millipore, Billerica, MA). In brief, A549 cells were treated with 10, 50, 150 and 250 lg/mL flavonoids for 24 h. The floating and adherent cells were collected, and lysed in ice-cold lysis buffer for 30 min in an ice bath. The supernatants were collected and incubated at 37 °C

The structures of 14 different flavonoids were characterised by retention times, molecular ion masses, and comparison with literature data. The HPLC chromatogram is depicted in Fig. 1A. The mass spectral and quantification data are compiled in Table 1. The 14 flavonoids were quantified from the peak areas of the HPLC chromatogram recorded at 280 nm. Quantification of those flavonoids

Fig. 1. Characterization of flavonoids isolated from Korean Citrus aurantium L. in A549 cells. (A) HPLC chromatogram of Korean Citrus aurantium L. at 280 nm. (1) naringin, (2) hesperidin, (3) poncirin, (4) isosinesetin, (5) hexamethoxyflavone, (6) sinesetin, (7) hexamethoxyflavone, (8) tetramethyl-O-isoscutellarein, (9) nobiletin, (10) heptamethoxyflavone, (11) 3-hydoxynobiletin, (12) tangeretin, (13) hydroxypentamethoxyflavone, (14) hexamethoxyflavone. (B) Growth inhibition of A549 and WI-38 cells after treatment with various concentrations (0–250 lg/mL) of flavonoids for 24 h.

K.I. Park et al. / Food Chemistry 135 (2012) 2728–2735 Table 1 The quantitative value and the retention time of identified flavonids isolated from Korean Citrus aurantium L.. No. Retention time (min)

MS Compound [M + H]+

Quantity ± SD (mg/kg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

581 611 595 373 403 373 403 343 403 433 419 373 389 403

299 ± 0.5 210 ± 1.3 109 ± 1.1 30.4 ± 0.1 15.5 ± 0.05 19.7 ± 0.1 23.6 ± 0.07 35.6 ± 0.1 201 ± 0.1 169 ± 1.3 28.7 ± 0.1 81.5 ± 0.09 12.8 ± 0.04 7.0 ± 0.04

16.46 18.02 29.56 41.67 42.88 43.40 44.60 45.91 46.52 47.50 48.91 50.78 52.06 52.72

Naringin Hesperidin Poncirin Isosinesetin Hexamethoxyflavone Sinesetin Hexamethoxyflavone Tetramethyl-O-isoscutellarein Nobiletin Heptamethoxyflavone 3-Hydroxynobiletin Tangeretin Hydroxypentamethoxyflavone Hexamethoxyflavone

was validated on the basis of a representative flavonoid standard from the same group. Thus, the flavanones (naringin, hesperidin and poncirin) and flavones (isosinensetin, hexamethoxyflavone, sinesetin, hexamethoxyflavone, tetramethyl-O-isoscutellarein, nobiletin, heptamethoxyflavone, 3-hydroxynobiletin, tangeretin, hydroxypentamethoxyflavone and hexamethoxyflavone) were quantified using the calibration curves of hesperetin and nobiletin,

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respectively. The calibration curves were constructed by plotting the concentration of the standard against the peak area. Plant flavonoids for which no standards were available have been routinely quantified using the calibration curve of structurally-related compounds. 3.2. Growth inhibition by flavonoids To evaluate the effects of inhibition of growth and survival in A549 and WI-38 cells, cells were treated with various concentrations (0–250 lg/mL) of flavonoids. Compared to the control (DMSO only), after 24 h treatment with flavonoids, as shown in Fig. 1B, flavonoids had a strong inhibitory effect on cell proliferation of A549 in a dose-dependent manner. Especially, flavonoids at 200 and 250 lg/mL decreased the cell growth by approximately 52% and 46%, respectively. The IC50 (50% inhibitory concentration) of flavonoids was approximately 230 lg/mL. Moreover, the results showed that flavonoids had a weak anti-proliferative effect on normal cell line, human embryonic fibroblast, lung-derived cell line WI-38. 3.3. Induction of G2/M phase cell cycle arrest by flavonoids To determine the effect of flavonoids on the cell cycle progression of A549 cells, flow cytometry was performed on cells treated

Fig. 2. Effect of flavonoids on cell cycle distribution in A549 cells. Cells were incubated with various concentrations (0–250 lg/mL) of flavonoids for 24 h, and the distribution of the cell cycle was ascertained by FACS analysis. (A) Flow cytometry of cell cycle phase distribution. (B) After incubation with flavonoids for 24 h, the cells were examined by Olympus CKX41 microscope (400). (C) Statistical analysis of cell cycle phase distribution. The data are representative examples for triplicate independent tests. ⁄p < 0.05 vs control group.

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Fig. 3. Induction of apoptosis of A549 cells by flavonoids. Apoptosis ratio was detected by Annexin V-FITC/PI double staining. (A) Flow cytometric analysis. (B) Statistical analysis for apoptosis. (C) Cells were incubated with flavonoids for 24 h and fixed, stained with Hoechst stain. Microscopic observations were conducted by LEICA DM 6000 B microscope (400). Arrows indicate chromatin condensation and nuclear fragmentation. The data are representative examples from triplicate independent tests. ⁄p < 0.05 vs control group.

with 0, 10, 50, 150 and 250 lg/mL of flavonoids for 24 h. As shown in Figs. 2A and C, untreated control cells had 4.21% of G2/M phase, whereas the cells treated with 150 and 250 lg/mL flavonoids were significantly increased in the G2/M phase (18.88% and 32.3%, respectively). This was accompanied by a significant decrease in the G1 phase when compared with the untreated control cells at 250 lg/mL. However, flavonoids did not influence the percentage of A549 cells in the S phase. Furthermore, the exposure of flavonoids was associated with cellular phenotypic changes, such as cell shrinkage and density (Fig. 2B). These results were consistent with the suggestion that the growth inhibitory effect of flavonoids in A549 cells was caused by G2/M phase arrest. 3.4. Flavonoid-induced apoptosis in A549 cells As shown in Fig. 3A, untreated cells did not show any significant apoptosis, whereas flavonoid-treated cells increased apoptosis in a dose-dependent manner. We analysed the apoptosis rate of A549 cells treated with various concentrations of flavonoids for 24 h by Annexin V-FITC/PI double-labelled flow cytometry. Flavonoids-treated A549 cells showed that early apoptotic cell proportions (lower right quadrant) were increased 19.7% and 21.8% at 150 and 250 lg/mL, respectively, compared with 3.9% for the control. The late apoptotic cell proportions (upper right quadrant) were also increased 11.2% and 12.1% at 150 and 250 lg/ml, respectively, compared with 7.4% for the control. The total apoptotic cell proportion was increased 30.9% and 34% at 150 and 250 lg/mL, respectively, compared with 11.3% for the control (Fig. 3A and B). Also, A549 cells were stained with Hoechest 33342. Cells treated with flavonoids at 250 lg/mL, showed apoptotic changes, such as nuclear fragmentation and chromatin condensation (Fig. 3C). These data were consistent with induction of apoptosis by the flavonoids. 3.5. Flavonoids inhibit the expression of cell cycle proteins in A549 cells A549 cells were untreated or were treated with flavonoids (10, 50, 150 and 250 lg/mL) for 24 h, and the expression of cyclin B1,

cdc25c, cdc 2 and p21WAF1/CIP1 proteins was examined by Western blot. All protein levels decreased in a dose-dependent manner, with significant inhibition occurring at 50, 150 and 250 lg/mL (Fig. 4). These data indicates that flavonoids can induce G2/M arrest by blocking the expression levels of cyclin B1, cdc25c, cdc2 and p21WAF1/CIP1. 3.6. Flavonoids induce Bcl-2 family proteins and caspase activation in A549 cells After treatment with flavonoids in A549 cells, Bcl-xL expression was decreased dose-dependently, but significant changes of Bax level were not found. Also, flavonoids decreased the expression level of pro-caspases-3, -6, -8 and -9 and increased the caspase-3 activity and the expression level of cleaved PARP in a dose-dependent manner (Fig. 5A). Furthermore, a densitometric analysis of the bands revealed that flavonoids increased the Bax/Bcl-xL ratio in a dose-dependent manner (Fig. 5B). These results suggest that flavonoids induced apoptosis through the regulation of the ratio of Bax/Bcl-xL and the activation of caspases. 4. Discussion Plant-derived herbal medicines have been used as a traditional medicine in Asian countries including Korea and China. More than half of the known anti-cancer drugs originated from herbal plants. Natural herb products such as flavonoids are gaining attention as therapeutic agents for cancer. The common dietary flavonoids in plant foods have anti-cancer activity against human cancer cell lines (Middleton, Kandaswami, & Theoharides, 2000). In particular, naringin, nobiletin and hesperetin exhibit an anti-cancer effect by cell cycle arrest and apoptosis pathway in human cell lines (Kim et al., 2008; Luo, Guan, & Zhou, 2008). Cell cycle control is an important process that involves a complex cascade of events on cell growth. The controlled function of cell cycle regulatory proteins such as cyclins and cdks is an important means of inhibition of cancer cell growth and division (Chen

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Fig. 4. Effect of flavonoids on cell cycle-related proteins (cyclin B1, cdc2, cdc25c and p21WAF1/CIP1) expression level in A549 cells. Cells were treated with flavonoids (0, 10, 50, 150 and 250 lg/mL) for 24 h. Cell lysates were subjected to SDS–PAGE and analysed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of flavonoids on expression of cell cycle-related proteins level were represented. The data are representative examples for triplicate independent tests. ⁄p < 0.05 vs control group.

et al., 2002). The flavonoid silibinin inhibits proliferation of human colon cancer cells by arresting the cell cycle at G2/M (Hogan, Krishnegowda, Mikhailova, & Kahlenberg, 2007). Flavones such as luteolin, apigenin and chrysin, and flavonols such as quercetin, kaempferol and myricetin induce G2/M arrest in a human oesophageal adenocarcinoma cell line (Zhang, Zhao, & Wang, 2008). Cdc2 interacts with cyclin B1 and forms a cdc2-cyclin complex. The cdc2-cyclin B1 complex plays a critical role in regulation of the G2/M phase. Inactivation of the cyclin B1/cdc2 complex inhibits the transition from the G2 to M phase (Grana & Reddy, 1995). Cdc25c plays an important role in the dephosphorylation of cdc2 on Thr14/Tyr15. The activation of cdc25c induces the subsequent activation of the cyclin B1/cdc2 complex (Jin, Gu, & Morgan, 1996). p21WAF1/CIP1, cyclin-dependent kinase inhibitor, regulated the cell cycle progression. Especially, the level of expression p21WAF1/CIP1 increased at the G2/M transitions in a variety of cancer cells (Cho et al., 2011; Dash & El-Deiry, 2005, Niculescu et al., 1998, Zhao, Xiang, Domann, & Zhong, 2009). In the present study, flavonoids inhibited cell proliferation in A549 human lung cancer cells. The anti-cancer effect was associated with the capacity of flavonoids to trigger growth inhibition at the G2/M phase (Fig. 2). Flavonoids induced G2/M arrest through the down-regulation of cdc2, cdc25c and cyclin B1 and the up-regulation of p21WAF1/CIP1 (Fig. 4). To maintain cell homeostasis, regulation of apoptosis is pivotal. The apoptosis signal is regulated mainly by the caspases family,

which performs as inactive zymogens in cells and produces a cascade of catalytic effects at the initiation of apoptosis (Shi, 2004). Many studies have sought to elucidate the cell apoptosis mechanism through the caspase signalling pathway. Caspase-3 activation after stimulation by killer T-cell triggers the cleavage of many important proteins such as full term. The cascade reaction acts to stimulate mitochondria to release cytochrome c. Excessive cytochrome c recruits caspase-9 for the activation of caspase-3. Hesperidin induced apoptosis in in vitro tumour cells, such as colon cancer SNU-C4 cells and leukaemia Thp-1 cells, through caspase-3 signal blocking (Park, Kim, Ha, & Chung, 2008). In this experiment, the total apoptotic cell proportion was increased and the levels of pro-caspase -3, -6, -8 and -9 were significantly down-regulated at 150 and 250 lg/mL. Moreover, apoptotic changes appeared by staining with Hoechst 33342 in flavonoids-treated cells and the up-regulation of caspase-3 activity indicates that flavonoids induce apoptosis in a caspase-3-dependent manner (Figs. 3 and 5). In terms of cell apoptosis, the Bcl family plays an important role. The Bcl family proteins are apoptotic regulatory proteins that control the mitochondrial apoptotic process. The Bcl family in the cell determines whether a cell lives or dies (Wong & Puthalakath, 2008). Bcl-xL interacts with mitochondrial plasma membrane and protects from other apoptotic factors, such as Bax and Bak. This prevents induced cytochrome c from the plasma membrane. Nobiletin induces apoptosis in various tumour cell lines via the inhibited overexpression of Bcl family of proteins (Luo et al.,

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Fig. 5. Effect of flavonoids on apoptotic-related proteins (caspases, PARP, Bax and Bcl-xL) expression level and caspase-3 activity. Cells were treated with flavonoids (0, 10, 50, 150 and 250 lg/mL) for 24 h. Cell lysates were subjected to SDS–PAGE and analysed by Western blotting. Representative blots are shown. Densitometric analyses of the effect of flavonoids on expression of apoptotic related proteins level and caspase-3 activity were represented. The data are representative examples for triplicate independent tests. ⁄ p < 0.05 vs control group.

2008). The ratio of Bax/Bcl-xL seems to be a key point of apoptosis. Presently, Bcl-xL was significantly down-regulated, whereas Bax protein was not significantly changed. However, the data demonstrate that the ratio of Bax/Bcl-xL was up-regulated in flavonoidtreated A549 cells (Fig. 5). In conclusion, our results demonstrated that flavonoids isolated from Korean C. aurantium L. play a pivotal role in the anti-cancer properties in A549 cells. Also, flavonoids induce G2/M arrest by regulating proteins of cell cycle, such as cyclin B1, cdc2, cdc25c and p21WAF1/CIP1. Flavonoids induce apoptosis through the up-regulation of the ratio of Bax/Bcl-xL, caspase 3 activity and cleaved PARP, and the down-regulation of pro-caspases (caspase-3, -6, -8 and -9) proteins. Flavonoids cause G2/M arrest and apoptosis, through the regulation cell cycle dependent and pro-apoptotic proteins. Although detailed studies are needed to examine the effect of flavonoids, this study provides scientific support for the use of Korean C. aurantium L. for the treatment of human lung cancer. Acknowledgments This work was supported by the National Research Foundation (NRF) of Korea Grant funded by the Korean government (MEST) (No. 2009-0084454) and the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family affairs, Republic of Korea (No. 0820050).

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