Tumor Biol. DOI 10.1007/s13277-013-1542-0

RESEARCH ARTICLE

Genistein exhibits anti-cancer effects via down-regulating FoxM1 in H446 small-cell lung cancer cells Tiantian Tian & Jisheng Li & Bei Li & Yawei Wang & Meiying Li & Daoxin Ma & Xiuwen Wang

Received: 13 October 2013 / Accepted: 11 December 2013 # International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Genistein, a major isoflavone constituent in soybeans, has been reported to exhibit multiple anti-tumor effects, such as inducing cell cycle arrest, triggering apoptosis, and inactivating critical signaling pathways in a few human cancer cells. Here, we investigated the anti-tumor effects of genistein on the small-cell lung cancer (SCLC) cell line H446 and the underlying molecular mechanisms. H446 cells were treated with various concentrations of genistein, and experiments including CCK-8 assay, colony formation assay, flow cytometry analysis, wound healing assay, real-time polymerase chain reaction (PCR), western blot analysis, and plasmid transfection were used to investigate the influence of genistein on cell proliferation, migration ability, apoptosis, cell cycle progression, as well as the mRNA and protein alterations of FoxM1 pathway molecules. We found that genistein significantly inhibited the proliferation and migration ability of H446 cell, accompanied by apoptosis and G2/M phase cell cycle arrest. In addition, genistein enhanced the antiproliferative effect of cisplatin on H446 cells. Importantly, genistein led to attenuation of the FoxM1 protein and down-regulated a series of FoxM1 target genes regulating cell cycle and apoptosis including Cdc25B, cyclin B1, and survivin. In addition, up-regulation of FoxM1 by cDNA transfection prior to genistein treatment could reduce genistein-induced H446 proliferation inhibition. Thus, for the first time, we demonstrated that genistein exerted multiple

Tiantian Tian and Jisheng Li contributed equally to this work. T. Tian : J. Li : B. Li : Y. Wang : M. Li : X. Wang (*) Department of Medical Oncology, Cancer center, Qilu Hospital of Shandong University, Jinan 250012, China e-mail: [email protected] D. Ma Department of Hematology, Cancer Center, Qilu Hospital of Shandong University, Jinan 250012, China

anti-tumor effects in H446 SCLC cell line at least partly mediated by the down-regulation of FoxM1. FoxM1 has the potential as a novel therapeutic agent in SCLC and is worthy of further study. Keywords SCLC . Genistein . FoxM1 . Cell cycle arrest . Apoptosis

Introduction Lung cancer is one of the leading causes of cancer deaths worldwide [1]. Small-cell lung cancer (SCLC) is the deadliest type and comprises about 15–20 % of all lung cancers. Although the remission rate of SCLC was significantly improved by combined chemotherapy and radiotherapy, it is prone to drug resistance and relapse with a quite poor 5-year survival rate [2]. Above facts underscored the importance of exploring new therapeutic approaches for the prevention and treatment for this deadly malignancy. To date, many natural compounds from some kinds of food and traditional Chinese medicine have been demonstrated to exhibit anti-tumor effects on human cancers. Among them, soy isoflavones mainly derived from soybeans have received lots of attention because of its protective effect on multiple common cancers [3–6]. Genistein, a major isoflavone constituent (about 50 %) in soybeans, is a well known phytoestrogen and is shown to be the main contributing member among soybean isoflavones for the chemoprevention effect. Genistein not only could reduce the risk of both hormone-dependent and hormoneindependent cancers including breast, prostate, colon, gastric, non-small-cell lung cancers, as well as leukemia [3–6]. Studies on the inhibition effects of genistein on cancer cell growth, invasion, and metastasis were also reported [7–10]. In addition, multiple studies proved that

Tumor Biol.

genistein could enhance the anti-tumor activities of chemotherapy and radiotherapy in multiple human cancers [11–13]. These studies clearly demonstrated that genistein could be a potent agent for both cancer prevention and treatment either alone or in combination with conventional therapies. Notably, epidemiologic studies shown that genistein is associated with a decreased risk of lung cancer [14]. However, the anti-tumor role of genistein in SCLC has not been fully investigated by now. Genistein was shown to exert its anti-tumor activity via its multi-targeted biological abilities. It can modulate several genes related to cell cycle and apoptosis regulation and eventually inhibit carcinogenesis and cancer cell growth [15–18]. Importantly, a recent report showed that genistein inhibited pancreatic cancer cell growth by inactivating a proliferation-specific transcription factor Forkhead box protein M1 (FoxM1) [19]. FoxM1 is a member of the forkhead family of transcription factors. By regulating the expression of Plk1, Aurora B, cyclin B1, Cdc25B, p21, p27, and other critical genes, all of which are essential regulators of G1-S and G2-M transition, FoxM1 plays an essential role in modulating cell cycle progression [20]. Moreover, FoxM1 also regulates diverse biological processes including apoptosis, cell differentiation, DNA damage response, senescence, and tissue homeostasis. Importantly, FoxM1 is one of the most commonly over-expressed oncogenes in solid tumors including SCLC [21, 22]. Furthermore, it has been shown that FoxM1 can also promote angiogenesis, invasion, metastasis, drug resistance, and so on [23–25]. These results suggest a principal role for FoxM1 in the development and progression of human cancers. Therefore, targeting FoxM1 could be a novel therapeutic strategy for human cancers. In this study, we used SCLC cell line H446 as the experimental model and demonstrated that genistein could inhibit the proliferation and migration of H446 cell via inducing both apoptosis and G2/M phase cell cycle arrest. In addition, genistein also enhanced the chemosensitivity of H446 cells to the treatment of cisplatin. Further study suggested that the anti-tumor effects of genistein in SCLC cell at least partly depended on its ability of suppressing FoxM1 expression.

Annexin V-FITC/PI apoptosis detection kit, and cell cycle kit were obtained from Bestbio (Shanghai, China). Real-time polymerase chain reaction (RT-PCR) assay kits were purchased from TOYOBO Biotech (Jiangsu, China). Genistein (Santa Cruz, CA) was dissolved in 100 % dimethyl sulfoxide (DMSO) to make a 20-mM stock solution and was added directly to the media at different concentrations. Cell culture Human small-cell lung cancer cell line H446 was obtained from American Type Culture Collection (ATCC) and cultured in 1640 and 10 % fetal bovine serum (FBS, Hyclone, Utah, USA). Cells were incubated at 37 °C under 5 % CO2 and 100 % humidity. CCK-8 assay Cells were plated in 96-well plates at the concentration of 4×103 per well. The medium was replaced with fresh medium containing genistein (0–100 μM) next day. Cell viability was measured on days 1, 2, and 3 by CCK-8 assay following the manufacturer's instruction. Colony formation assay After 48 h of exposure to 0, 25, 50, and 75 μM of genistein, the cells were collected and plated in 6-well plates at a density of 1000 cells/well. The cells were then incubated at 37 °C under 5 % CO2 for 10 days. Colonies were stained with 2 % crystal violet, washed with water, and counted. Wound healing assay Wound healing assay was used to examine the effect of genistein on the migration ability of H446 cells. Briefly, cells were grown on 6-well plates for 24 h. Then media was removed, and equal size wounds were created with a pipette tip. Cells were washed twice with PBS, and serum free media was added. Each group was incubated with DMSO or 50 μM of genistein for 48 h. Images were captured and analyzed. Apoptosis analyses

Materials and methods Reagents and antibodies Anti-FoxM1 antibody was purchased from Cell Signaling Technology (CST, MA, USA). Primary antibodies for cyclin B1 and survivin were obtained from Abcam Inc (MA, USA). Lipofectamine 2000 was purchased from Invitrogen (CA, USA). The FoxM1 cDNA plasmid was purchased from Genechem (Shanghai, China). Cell counting kit 8 (CCK-8),

H446 cells were plated (1×105 per well) in a 6-well plate and exposed to various concentrations of genistein. After 72 h, apoptosis was detected using the Annexin V-FITC/PI apoptosis detection kit. Briefly, H446 cells were washed twice with ice-cold PBS and re-suspended in binding buffer. Cell suspensions were stained with 5 μL of annexin V-FITC conjugate and 10 μL of PI. Samples were analyzed by FACS Calibur (BD FACS Calibur TM, San Jose, CA) and FlowJo (Tree Star, SanCarlos, CA) analysis software.

Tumor Biol.

Cell cycle analysis H446 cells were treated with various concentrations of genistein for 72 h before harvest. The cells (2×105) were washed with PBS and fixed in 70 % cold alcohol at −20 °C for 1 h. After being washed with cold PBS, 100 μL of RNase A was added and incubated at 37 °C for 30 min. Then the cells were stained with 400 μL of propidium iodide for 1 h at 4 °C. The distribution of cell cycle phase was assayed by flow cytometry (BD FACSCalibur TM, San Jose, CA).

peroxidase (HRP)-conjugated goat-anti-rabbit IgG (Beyotime Institute of Biotechnology, A0208, 1:5000). The protein levels were normalized by β-actin. Plasmids and transfection H446 cells were transfected with FoxM1 cDNA plasmid using Lipofectamine 2000 (Invitrogen, CA, USA) as described earlier [26]. The transfection efficiency was monitored by Western blot.

Real-time reverse transcription-PCR analysis

Statistical analysis

The total RNA from treatment cells was isolated by Trizol (Invitrogen, CA, USA), and cDNA was synthesized by reverse transcription of 2 mg of total RNA. Real-time PCR was conducted using an ABI Prism 7500 sequence detection system (Applied Biosystems, CA, USA) and performed with SYBR Green PCR Master Mix (TOYOBO, Jiangsu, China) in triplicate according to the manufacturer's protocols. The samples were loaded in quadruple, and the results of each sample were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Sequences of PCR primers were shown in Table 1.

Data were expressed as mean±SD. ANOVA and Student t test were used to assess the significant differences between the treatment groups. Statistical analysis was performed with SARS 9.1 software. The difference was considered statistically significant at P

Genistein exhibits anti-cancer effects via down-regulating FoxM1 in H446 small-cell lung cancer cells.

Genistein, a major isoflavone constituent in soybeans, has been reported to exhibit multiple anti-tumor effects, such as inducing cell cycle arrest, t...
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