Article pubs.acs.org/JAFC
Daidzein Suppresses Tumor Necrosis Factor‑α Induced Migration and Invasion by Inhibiting Hedgehog/Gli1 Signaling in Human Breast Cancer Cells Cheng Bao,†,§ Hyeju Namgung,†,§ Jaehoo Lee,† Hyun-Chang Park,† Jiwon Ko,† Heejung Moon,‡ Hyuk Wan Ko,‡ and Hong Jin Lee*,† †
Department of Food Science and Technology, Chung-Ang University, 4726 Seodongdaero, Anseong 456-756, South Korea College of Pharmacy, Dongguk University, Goyang 410-820, South Korea
‡
ABSTRACT: In breast cancer, the cytokine tumor necrosis factor-α (TNF-α) induces cell invasion, although the molecular basis of it has not been clearly elucidated. In this study, we investigated the role of daidzein in regulating TNF-α induced cell invasion and the underlying molecular mechanisms. Daidzein inhibited TNF-α induced cellular migration and invasion in estrogen receptor (ER) negative MCF10DCIS.com human breast cancer cells. TNF-α activated Hedgehog (Hh) signaling by enhancing Gli1 nuclear translocation and transcriptional activity, which resulted in increased invasiveness; these effects were blocked by daidzein and the Hh signaling inhibitors, cyclopamine and vismodegib. Moreover, these compounds suppressed TNF-α induced matrix metalloproteinase (MMP)-9 mRNA expression and activity. Taken together, mammary tumor cell invasiveness was stimulated by TNF-α induced activation of Hh signaling; these effects were abrogated by daidzein, which suppressed Gli1 activation, thereby inhibiting migration and invasion. KEYWORDS: breast cancer, daidzein, Hedgehog signaling, invasion, TNF-α
■
INTRODUCTION Consumption of soy-based foods has been associated with a lower risk of breast cancer, the most frequently diagnosed cancer in women worldwide.1,2 Isoflavones, especially genistein, are the main ingredients responsible for the cancer chemopreventive activity of soy foods, which include modulation of estrogen signaling, inhibition of tyrosine kinase activity, and suppression of Akt and nuclear factor-κB (NF-κB) signaling pathways.3 Daidzein, another isoflavone present in soy food, has not been as extensively studied as genistein due to its lower biological efficacy in terms of estrogen receptor (ER) binding and inhibiting proliferation in MCF-7 or other breast cancer cell types.4,5 It was recently reported that daidzein suppressed MDA-MB-231 breast cancer cell invasion by reducing matrix metalloproteinase (MMP) 2 activity,6 suggesting an important role of daidzein in breast carcinogenesis. Tumor invasion is the rate-limiting process for metastasis during which fibroblasts and macrophages in the microenvironment actively communicate with tumor cells.7 Many lines of evidence indicate that inflammation and cytokines regulation are involved in cancer progression.8 Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that plays a critical role in tumor malignancy, including tumor cell motility, invasion, and metastasis.9 In breast cancer, the expression of TNF-α ligand and receptors in infiltrating macrophages or stromal cells were reportedly higher than in benign breast tissue,10,11 highlighting the importance of the tumor microenvironment in the interaction between epithelial and stromal cells in mammary carcinogenesis.12 The induction of mammary tumor cell migration and invasion by TNF-α has been attributed to the activation of NF-κB signaling.13,14 However, a recent study showed that TNF-α activated the Hh signaling © 2014 American Chemical Society
pathway in esophageal adenocarcinoma cells via regulation of mechanistic target of rapamycin (mTOR)/S6K1, which led to an increase in Gli1 phosphorylation independently of Smoothened (Smo) function.15 Dysfunction of Hh signaling is implicated in breast cancer development and poor overall survival rate,16,17 although the precise mechanisms are not well understood.18 Kubo et al. reported that all of human breast carcinomas tested had higher Gli1 immunoreactivity than adjacent normal tissues, indicating a key role for the Hh/Gli signaling pathway in mammary tumorigenesis.19 Interestingly, recent accumulating evidence has elucidated that Gli1 enhanced the progression to invasive phenotype from noninvasive ductal carcinoma in situ (DCIS)20 and promoted cellular migration and invasion in estrogen receptor (ER) negative breast cancer cells,21,22 underscoring a key role in more aggressive, ER-independent breast tumors including basal-like and triple negative subtypes.17,23,24 These data suggest that the Hh/Gli signaling pathway can be potentially targeted to prevent progression of breast cancer. However, the role of the natural isoflavone, daidzein, on TNF-α associated breast cancer and molecular mechanisms have not been thoroughly studied. This study investigated the potential role for daidzein in modulating TNF-α induced cell migration and invasion and examined whether Hh/Gli signaling is linked to this process in ER negative, basal-like MCF10DCIS.com human breast cancer cells. Received: Revised: Accepted: Published: 3759
January 13, 2014 March 26, 2014 April 11, 2014 April 11, 2014 dx.doi.org/10.1021/jf500231t | J. Agric. Food Chem. 2014, 62, 3759−3767
Journal of Agricultural and Food Chemistry
Article
Figure 1. Effect of daidzein on TNF-α induced migration and invasion in MCF10DCIS.com human breast cancer cells. (A) Cells (5 × 105 cells/well in a 6-well plate) were treated with TNF-α (5 ng/mL) alone or together with daidzein (30 μM) for 24 h. Cell migration was assessed by light microscopy at 100× magnification and the number of cells at the injured area at 0 and 24 h was compared. (B) Cells (5 × 104 cells/well in a 24-well invasion chamber) were treated with TNF-α alone or together with daidzein for 24 h. Invasiveness was quantified by counting the number of cells that migrated to the lower side of the filter within randomly selected microscope fields. (C) Cells (5 × 103cells/well in a 96-well plate) were treated with TNF-α or daidzein alone at the indicated concentrations or cotreated TNF-α (5 ng/mL) with daidzein (30 μM), cyclopamine (10 μM), or vismodegib (10 μM) for 24 h. Cell viability was assessed with MTT assay. Values represent means ± SD **p < 0.01, ***p < 0.001 compared to TNF-α treated cells.
■
amplified using 5× HOT FIREPol EvaGreen qPCR Mix Plus (Solis Biodyne, Tartu, Estonia) and the following primer sequences for Gli1, 5′-CCTTCTTCGC GAACGGTTT-3′ (forward) and 5 ′GGGTGAAGGCTGCTCACGTA-3′ (reverse), and for GAPDH, 5′CAATGCCAAGTATGACAT-3′ (forward) and 5′-CCTGTTATTATGGGGGTCTG-3′ (reverse). To detect MMP-2 and -9 mRNA expression levels, labeled TaqMan primers and TaqMan Fast Advanced Master Mix from Applied Biosystems (Foster City, CA, USA) were used, along with the ABI PRISM 7900 sequence detection system (Applied Biosystems). Results were normalized to GAPDH expression level, and relative quantitation was performed using the comparative 2−ΔΔCT method. Protein Extraction. After treatment with a compound, cells were collected and washed with PBS. Total protein was extracted with radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.1 mM Na3VO4, 1% phenylmethylsulfonyl fluoride, 1% protease inhibitor). Cytosolic and nuclear fractions were extracted using NE-PERTM Nuclear and cytoplasmic extraction reagents kit (Thermo Scientific) according to the manufacturer’s instructions. Western Blot Analysis. An equivalent amount of protein from cell lysates were resolved by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF, Millipore, Billerica, MA, USA) membranes which were probed with primary and secondary antibodies followed by chemiluminescence detection using EZ capture MG (ATTO, Tokyo, Japan). Primary antibodies used in this study were against Gli1, p-NFκB p65 (Ser536) (Cell Signaling Technology,
MATERIALS AND METHODS
Reagents and Cell Culture. Daidzein and equol were purchased from Sigma (St. Louis, MO, USA), TNF-α from R&D Systems (Minneapolis, MN, USA), 6,7,4′-trihydroxyisoflavone from Indofine Chemical Company, Inc. (Hillsborough, NJ, USA), and vismodegib and cyclopamine from LC Laboratories (Woburn, MA, USA). MCF10DCIS.com human breast cancer cells were purchased from Asterand (Detroit, MI, USA) and cultured in DMEM/F12 (Life Technologies, Grand Island, NY, USA) supplemented with 5% horse serum, 1% HEPES, and 1% penicillin/streptomycin (Life Technologies) at 37 °C, 5% CO2. Patched1 mutant (Ptch1(−/−)) embryonic fibroblasts (MEFs) were kindly provided by K. Anderson at Sloan Kettering Institute (New York, USA) and cultured in DMEM (Life Technologies, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies). Cell Viability Assay. Cells were incubated with compounds at different concentrations for 24 h, and 200 μL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added 4 h before harvest. Dark-blue formazan crystals were dissolved with DMSO, and absorbance was measured with an ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm. Cell growth was defined as the percentage of viable cells in comparison with the control. RNA Extraction and Quantitative Real-Time PCR. Total cellular RNA was extracted using Trizol reagent (Life Technologies) and was reverse-transcribed to obtain cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Lafayette, CO, USA) in an Atlas Thermal Cycler (Astec, Fukuoka, Japan). The cDNA was 3760
dx.doi.org/10.1021/jf500231t | J. Agric. Food Chem. 2014, 62, 3759−3767
Journal of Agricultural and Food Chemistry
Article
Figure 2. Effect of TNF-α on Hh signaling in MCF10DCIS.com human breast cancer cells. (A) Cells (7 × 105 cells in a 60 mm dish) were treated with TNF-α (5 ng/mL) alone or together with daidzein (30 μM) for 24 h. IκBα expression and NFκB p65 phosphorylation level at Ser 536 were determined by Western blotting. (B) Cytosolic and nuclear fractions of TNF-α treated cells were separated, and Gli1 nuclear translocation was determined by Western blotting. (C) Cells (2 × 105 cells/well in a glass bottom dish) treated with TNF-α were probed with anti-Gli1 antibody, with nuclei stained with DAPI, and cells were visualized by confocal microscopy. (D) Cells were transfected with 8X-Gli-BS-Luc and pCMV-β-gal vectors and treated with TNF-α. Relative luciferase activity from the Gli1 promoter was determined using a luminometer and normalized to β-galactosidase activity. **p < 0.001 compared to the control. Boston, MA, USA), IκBα, α-tubulin, lamin B, (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), β-actin (Sigma, St. Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology Inc. The blots were quantified using EZ West Lumi Plus (ATTO) Confocal Microscopy. Cells were cultured in a glass bottom dish (MatTex, Ashland, MA, USA) and fixed with 4% paraformaldehyde in PBS 24 h after treatment with a compound. Cells were then treated with Gli1 antibody (1:100, Santa Cruz) and Alexa Fluor 488conjugated secondary antibody (1:200, Life Technologies), and DAPI (Vector, Burlingame, CA, USA) was used to stain nuclei. Cells were visualized using a Nikon A1 confocal microscope (Nikon, Tokyo, Japan). Transfection. Cells were transfected with 200 ng of Gli1 vector (pCMV-FLAG-mGli1 from Dr. Chi Chung Hui, University of Toronto) or Gli1 siRNA (200 nM, Bioneer, Daejeon, Korea) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) in serum free medium. After 6 h, the treatment compound was added to the culture medium for 24 h. The expression of Gli1 protein was confirmed by Western blotting. Gli1 vector-transfected cells were seeded for the invasion assay. Luciferase Assay. 8XGli-BS-Luc and pCMV-β-gal vectors were provided by Dr. Hiroshi Sasaki (RIKEN, Wako, Japan) and Dr. David Mangelsdorf (University of Texas Southwestern Medical School, Dallas, Texas), respectively. Cells were transfected with 200 ng of 8XGli1-BS-Luc and 50 ng of pCMV-β-gal using FuGENE HD transfection reagent in serum free medium. After 24 h, the culture medium was replaced and cells were treated with TNF-α (5 ng/mL) for 24 h. Cells were dissolved in 100 μL of reporter lysis buffer (Promega), and luciferase activity was determined using GLOMAX luminometer (Promega). Transcriptional activity was normalized to βgalactosidase activity. Wound-Healing Cell Migration Assay. Cells were grown to confluence in six-well plates and then scratched with a pipet tip to
create a gap. Wells were washed with PBS before adding fresh medium containing TNF-α (5 ng/mL) alone or with daidzein (30 μM). Cells were visualized under a light microscope (100× magnification) at 0 and 24 h. after treatment, and the relative wound area at these time points was calculated using Image Pro-plus 6.0 software (MediaCybernetics, Rockville, MD, USA). Invasion Assay. The invasion assay was performed using Matrigelcoated invasion chambers (BD Biosciences, Bedford, MA, USA). The lower section was filled with 750 μL of culture medium containing 0.1% bovine serum albumin. Cells were placed in the upper section of the transwell plate in 500 μL of the same medium. After treatment with the compounds, cells in the lower and upper chambers were incubated for 24 h, fixed with methanol, and stained with hematoxylin and eosin for 10 and 5 min, respectively. After cells on the upper surface of the transwell plate were removed with a cotton swab, invading cells were observed under a light microscope, and cells in five randomly selected fields were counted. Zymography Assay. The enzymatic activity of MMP-2 and -9 was determined by gelatin zymography. Conditioned medium was collected 24 h after treatment with a compound and was resolved by electrophoresis using a polyacrylamide gel containing 0.1% (w/v) gelatin. The gel was washed with 2.5% Triton X-100 to remove the SDS, rinsed with 50 mM Tris-HCl buffer (pH 7.6) containing 5 mM CaCl2, 200 mM NaCl, and 0.02% Brij-35 (Merck, Darmstadt, Germany), and then incubated overnight at 37 °C. The following day, the gel was stained with 0.5% Coomassie Brilliant Blue R-250 (AMRESCO, Solon, OH, USA) solution containing 10% acetic acid and 20% methanol for 30 min, then destained with 7.5% acetic acid solution containing 10% methanol. The gelatinase activity was determined using EZ West Lumi Plus (ATTO). Statistical Analysis. Data are presented as mean ± standard deviation (SD). Mean differences were assessed using the Student’s ttest, and a p-value
