Lin et al. BMC Cancer (2015) 15:958 DOI 10.1186/s12885-015-1965-7 RESEARCH ARTICLE Open Access Flavones inhibit breast cancer proliferation through the Akt/FOXO3a signaling pathway Chia-Hung Lin1, Ching-Yao Chang2, Kuan-Rong Lee1*, Hui-Ju Lin3,4, Ter-Hsin Chen5 and Lei Wan2,4,6* Abstract Background: Flavones found in plants display various biological activities, including anti-allergic, anti-viral, anti-inflammatory, anti-oxidation, and anti-tumor effects. In this study, we investigated the anti-tumor effects of flavone, apigenin and luteolin on human breast cancer cells. Methods: The anti-cancer activity of flavone, apigenin and luteolin was investigated using the MTS assay. Apoptosis was analyzed by Hoechst 33342 staining, flow cytometry and western blot. Cell migration was determined using the culture inserts and xCELLigence real-time cell analyzer instrument equipped with a CIM-plate 16. Real-time quantitative PCR and western blot were used to determine the signaling pathway elicited by flavone, apigenin and luteolin. Results: Flavone, apigenin and luteolin showed potent inhibitory effects on the proliferation of Hs578T, MDA-MB-231 and MCF-7 breast cancer cells in a concentration and time-dependent manner. The ability of flavone, apigenin and luteolin to inhibit the growth of breast cancer cells through apoptosis was confirmed by Hoechst33342 staining and the induction of sub-G1 phase of the cell cycle. Flavone, apigenin and luteolin induced forkhead box O3 (FOXO3a) expression by inhibiting Phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB)/Akt. This subsequently elevated the expression of FOXO3a target genes, including the Cyclin-dependent kinase inhibitors p21Cip1 (p21) and p27kip1 (p27), which increased the levels of activated poly(ADP) polymerase (PARP) and cytochrome c. Conclusion: Taken together, these data demonstrated that flavone, apigenin and luteolin induced cell cycle arrest and apoptosis in breast cancer cells through inhibiting PI3K/Akt activation and increasing FOXO3a activation, which suggest that flavone, apigenin and luteolin will be the potential leads for the preventing and treating of breast cancer. Keywords: Breast cancer, Flavones, FOXO3a, Akt, p27, Apoptosis Background metastasis, decrease the rate of apoptosis, or both. These Breast cancer is one of the most common types of can- changes often involve dysregulation of key signal trans- cer affecting women in western countries, and in recent duction pathways within the cell that transmit extra- years, the number of deaths caused by breast cancer has cellular signals to transcription factors, resulting in been increasing in Taiwan. Despite the new promising changes in gene expression. Previous studies have breakthrough in therapeutics, the annual breast cancer shown that increased protein kinase B (PKB)/Akt activity mortality rate continues to increase, and one million can promote breast cancer cell survival and therapeutic new cases are diagnosed every year [1]. Numerous risk resistance [6, 7]. Forkhead box O3 (FOXO3a), a down- factors for breast cancer etiology have been identified, stream target of the phosphatidylinositol-3-kinase including genetic, gender, age, alcohol consumption, (PI3K)/Akt pathway, belongs to a family of transcrip- smoking and obesity [2–5]. tion factors that are characterized by a distinct fork- Breast cancer develops as a consequence of cellular head DNA-binding domain [8]. Activation of PI3K/Akt changes that increase the rate of cell division and signaling causes phosphorylation of FOXO3a, thereby inhibiting its activity and translocating it out of the nu- * Correspondence: [email protected]; [email protected] cleus, where it becomes subject to proteasomal degrad- 1 Institute of Molecular Medicine, National Tsing Hua University, No. 101, ation in the cytosol [9]. The cytoplasmic expression Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan 2Department of Biotechnology, Asia University, Taichung, Taiwan level of FOXO3a is correlated with Akt phosphorylation Full list of author information is available at the end of the article © 2015 Lin et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lin et al. BMC Cancer (2015) 15:958 Page 2 of 12 and is associated with poor prognosis in breast cancer Collection. Cells were cultured in Dulbecco’s modified [10]. Nuclear localization of FOXO3a promotes the ex- Eagle medium (Invitrogen), supplemented with 10 % fetal pression of multiple target genes such as p21Cip1 (p21), bovine serum (Invitrogen) and 1 % penicillin-streptomycin kip1 p27 (p27), and cyclin D, which results in cell cycle solution (Invitrogen) at 37 °C in a 5 % CO2 incubator. No arrest to inhibit the growth of cancer cells [9, 11, 12]. approvals were required for this study, which complied with Cell cycle arrest in the G1, G2-M, and S phases can lead to all relevant regulations. apoptosis [13, 14]. FOXO transcription factors are also in- volved in the cellular stress response, and they regulate cell Cell viability assay cycle progression and apoptosis [15]. In particular, FOXO3a Cell viability was determined using the MTS/PMS ((3- plays a vital role in the initiation of cell cycle arrest, in (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2 addition to its involvement in DNA damage-mediated (4-sulfophenyl)-2H-tetrazolium, inner salt)/phenazine apoptosis [16]. Moreover, FOXO3a is an important tumor methosulfate) assay. Hs578T (2 × 103 cells/well), MDA- suppressor that is underexpressed in many breast cancers. MB-231 (2 × 103 cells/well) and MCF-7 (4 × 103 cells/well) Several anti-cancer drugs have been shown to increase the cells were seeded in 96-well plates. Media containing dif- expression of FOXO3a, which suggest it is a tangible thera- ferent concentrations (0–100 μM) of the flavone (HPLC > peutic target for breast cancer therapy [17]. 98 %, Sigma), apigenin (HPLC > 95 %, Sigma) and luteolin Surgical resection, radiation therapy, and chemotherapy (TLC > 98 %, Sigma) were added and incubated for 72 h. are among the main treatment options for breast cancer Subsequently, 20 μl of MTS was added from a stock patient. In addition, there is growing need to discover new solution (2 mg/mL) and incubated for an additional chemopreventive agents that are effective in preventing/ 2 h. The absorbance was read at 490 nm in the micro- treating breast cancer. Polyphenols are compounds found plate reader 550 model (Bio-rad). in food plants and Chinese herbs, and they can be divided into various classes on the basis of their molecular struc- Hoechst 33342 staining for detection of cell apoptosis ture. Flavonoids constitute a class of polyphenols that can Apoptosis was analyzed by Hoechst 33342 staining. The be further divided into the following six subclasses: fla- MCF-7, Hs578T, and MDA-MB-231 cells were seeded in 5 vones, flavanones, flavanols, flavonols, flavonols, isofla- 6-well plates (2 × 10 cells/well) and treated with IC50 con- vones, and anthocyanidins [18, 19]. A recent study centrations (Table 1) of flavone, apigenin and luteolin for showed that flavonoids exhibit various beneficial biological 24 h. Cells were stained with 40 mg/mL Hoechst 33342 properties such as anti-inflammatory [20, 21], anti-viral (Sigma). Nuclear morphology was assessed using the cell [22, 23], anti-allergic [24], anti-oxidant, [25, 26] and anti- membrane penetration DNA dye Hoechst 33342. Then, tumor activities [27–31]. Researchers have also discovered the cells were visualized under a fluorescence microscope that flavones inhibit tumor growth by promoting apop- with a blue filter. tosis in cancer cells [27, 28, 32]. Compounds in the fla- vone subclass, including flavone, apigenin, and luteolin, Cell cycle analysis are present in fruits and vegetables and are considered to For cell cycle analysis, cells were seeded in a 10-cm dish be potent dietary phytochemicals that are effective in can- (5 × 105 cells) and allowed to adhere overnight. Cells cer chemoprevention [33, 34]. were then treated with IC50 concentrations (Table 1) of fla- Although different mechanisms and signaling pathways vone, apigenin and luteolin for 24 h. Cells were harvested have been proposed as targets of flavone, apigenin, and and fixed in ice-cold 70 % ethanol overnight at −20 °C. luteolin, these compounds were studied individually and Cells were washed with ice-cold PBS and subsequently occasionally by using different model cancer cells. How- suspended in staining buffer (20 μg/mL propidium ever, whether these structurally similar compounds could iodide; 0.1 % Tween 20; 0.2 mg/mL RNase A in PBS), induce common pathways among different types of cancer and incubated at room temperature for 30 min. Using cells to potentiate their chemoprevention activity remains flow cytometry (FACSort instrument and analysis soft- to be elucidated. The objectives of the present study were ware; ModFit LT), the cells were analyzed with regard to investigate the anti-proliferative role of flavone, api- to cell cycle distribution and apoptosis. genin, and luteolin in MCF-7, Hs578T, and MDA-MB-231 human breast cancer cells, and to elucidate the common Colony formation assay molecular pathways in these cells. Cells were plated in a 25 T flask at a density of 2 × 103 cells and treated with IC50 concentrations (Table 1) of Methods flavone, apigenin and luteolin for 21 d. Thereafter, the Cell culture cells were then fixed with methanol for 15 min, followed The human Breast cancer cell lines Hs578T, MDA-MB-231 by incubation with 0.5 % crystal violet for 30 min and and MCF-7 were purchased from American Type Culture rinsing with deionized distilled water.