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#-catenin mediates anti-adipogenic and anti-cancer effects of arctigenin in preadipocytes and breast cancer cells Jihye Lee, Jee-Young Imm, and Seong-Ho Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00112 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 10, 2017
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Journal of Agricultural and Food Chemistry
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β-catenin mediates anti-adipogenic and anti-cancer effects of arctigenin in
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preadipocytes and breast cancer cells
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Jihye Lee#, Jee-Young Imm^, Seong-Ho Lee#*
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Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742,
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USA ^
Department of Foods and Nutrition, Kookmin University, Seoul 136-702, Korea
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* Corresponding author
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Address: Department of Nutrition and Food Science, 3307 Marie Mount Hall, University of
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Maryland, College Park, MD, 20742, USA.
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Tel.: +1 301 405 4532
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Fax: +1 301 314 3313
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E-mail address:
[email protected] (S-H. Lee)
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ABSTRACT
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Arctigenin is a lignan abundant in Asteraceae plants and has anti-inflammatory, anti-obese and
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anti-cancer activities. Obesity is one of the leading causes of several types of cancers including
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breast cancer. The current study was performed to investigate if arctigenin suppresses
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differentiation of preadipocytes and proliferation of breast cancer cells, and explore potential
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molecular mechanisms. Treatment of arctigenin reduced lipid accumulation in differentiated
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3T3-L1 adipocytes in a dose- and time-dependent manner without toxicity. Arctigenin
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suppressed the expression of peroxisome proliferator-activated receptor-gamma (PPARγ),
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CCAAT/enhancer-binding protein-alpha (C/EBPα), perilipin, and fatty acid binding protein 4
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(FABP4) in a dose-dependent manner in differentiated 3T3-L1 cells. Both total- and
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unphosphorylated (active) β-catenin were increased in whole cell lysates and nuclear fraction of
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differentiated 3T3-L1 cells treated with 25 µM of arctigenin. On the other hand, arctigenin
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decreased proliferation of two human breast cancer cells (MCF-7 and MDA-MB-231).
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Arctigenin induced apoptosis and decreased expression of total and unphosphorylated (active) β-
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catenin and cyclin D1 in MCF-7, but not in MDA-MB-231. This data indicate that arctigenin
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suppresses adipogenesis in preadipocytes and activated apoptosis in estrogen receptor (ER)
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positive breast cancer cells through modulating expression of β-catenin.
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Obesity is a primary risk factor of diverse metabolic diseases including cancer 1. In particular,
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obesity is highly associated with increased incidence of hormone-dependent cancers 2 3. Among
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them, breast cancer is the most prevalent in the occurrence and second leading cause of cancer
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mortality in the US 4. Epidemiological studies demonstrated that the incidence and prognosis of
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breast cancer is affected by high body mass index and obesity
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role in growth and differentiation of breast tissue 7 and most breast cancer patients are diagnosed
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as estrogen receptor (ER) positive 8. Recently, several mechanisms were proposed to explain the
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positive correlation between obesity and breast cancer progression; they include adipokines such
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as leptin 9, adiponectin 10 and pro-inflammatory cytokines 11.
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. Estrogen plays a significant
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Arctigenin is an aglycone form of arctiin which is a major compound of Arctium lappa, and
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bioactivity of arctigenin is higher than that of arctiin in vivo 12. Anti-cancer 13, anti-inflammatory
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previously. However, the mechanisms of these health benefits remain unanswered.
, neuroprotective
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, anti-diabetic
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, and anti-obese
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activities of arctigenin were reported
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β-catenin is a major mediator of the Wnt signaling pathway and inhibits adipogenesis
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Without Wnt ligands, β-catenin undergoes phosphorylation by glycogen synthase kinase-3β
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(GSK-3β) and casein kinase-1 and subsequent proteasomal degradation. In the presence of Wnt
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ligands, β-catenin is unphosphorylated and stabilized, and this active β-catenin translocates into
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the nucleus, binds to T-cell factor (TCF)/Lymphoid enhancer-binding factor (LEF), and stimulate
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expression of their target genes 18.
.
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The present study was designed to investigate anti-adipogenic and anti-cancer effect of
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arctigenin focusing on modulation of the β-catenin. Here, we report that β-catenin might be a 3
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molecular target to suppress the adipogenesis of preadipocytes and proliferation of breast cancer
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cells.
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MATERIALS AND METHODS
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Materials
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Arctigenin was obtained from Tocris Bioscience (Bristol, UK). 3T3-L1 preadipocytes, MCF-7,
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and MDA-MB-231 cells were purchased from ATCC (Manassas, VA, USA). Dulbecco's
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Modified Eagle Medium (DMEM), penicillin-streptomycin, and trypsin were purchased from GE
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healthcare (Logan, UT, USA). Antibodies for PPARγ (CST#2435), C/EBPα (CST#8178), FABP4
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(CST#3544), perilipin (CST#9349), PARP (CST#9542) and unphosphorylated (active) β-catenin
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(CST#19807) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies
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for total β-catenin (SC#1496), cyclin D1 (SC#718) and actin (SC#1616) were purchased from
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Santa Cruz Biotechnology (Dallas, TX, USA). 3-isobutyl-methylxanthine, dexamethasone, and
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insulin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and
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reagents were purchased from Fisher Scientific (Pittsburgh, PA, USA).
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Cell culture and differentiation of preadipocytes
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Two human breast cancer cells (MCF-7 and MDA-MB-231) were maintained at 37℃ with 5%
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CO2 in media supplemented with serum (10% fetal bovine serum) and antibiotics (penicillin-
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streptomycin). The cells were plated onto 96-well (for MTT assay), 24-well (for luciferase
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activity) or 60-mm culture dishes (for immunoblot) and then treated with different concentrations
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of arctigenin for indicated times as indicated detail in figure legends. 4
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3T3-L1 cells were cultured in media containing 10% bovine calf serum and antibiotics. The cells
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were plated onto 96-well (for MTT assay), 24-well (for Oil Red O staining) or 60-mm culture
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dishes (for immunoblot). After 2 days of post-confluence, the cells were incubated with
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differentiation media (containing 1 µM of dexamethasone, 0.5 mM of 3-isobutyl-1-
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methylxanthine, and 1 µg/mL of insulin) for 48 hours and then incubated with maintenance
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media (containing 1 µg/mL of insulin) in the presence of different concentrations of arctigenin.
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The fresh maintenance media containing different concentrations of arctigenin were changed
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every other day until the cells were harvested as indicated in detail (Fig. 1A). DMSO was used as
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a vehicle in 0.1% concentration (v/v).
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Measurement of triglyceride accumulation
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The cells were washed using PBS and fixed with 500 µL of 10% formalin at room temperature
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for 10 minutes. Then, cells were washed with distilled water twice, and stained with 500 µL of
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Oil Red O (2 mg/mL) for 15 minutes. After washing with distilled water four times, 500 µL of
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isopropanol was added to extract Oil Red O dye incorporated into triglycerides. The absorbance
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was measured at 500 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA).
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Cell proliferation
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Cell proliferation was measured as we described previously 19. Briefly, the cells were grown onto
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96-well culture dishes with media containing different concentrations of arctigenin. After
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removal of all media, 1 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
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(MTT) was added and incubated for 3 hours at 37℃ CO2 incubator. Then, MTT dye was 5
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extracted using 200 µL of DMSO and the absorbance was measured at 540 nm using the same
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microplate reader described above.
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Transient transfection and luciferase activity assay
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Transient transfection was carried out using PolyJet reagent (SignaGen Laboratories, Ijamsville,
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MD, USA). The cells (6×105cells/well) were plated in 24-well culture dishes. On next day,
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master mixture of plasmid (0.5 µg of TOP Flash or FOP Flash plasmids and 0.05 µg of pRL-null
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plasmids) were prepared per each well and incubated with cells for 24 hours. The transfected
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cells were treated with different doses of arctigenin for 24 hours. The cellular extracts were
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harvested using lysis buffer. Luciferase activity (a ratio of firefly/renilla) was measured
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according to protocol of a dual-luciferase assay kit (Promega, Madison, WI, USA).
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Apoptosis assay
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The amount of cleaved DNA/histone complexes (nucleosomes) was analyzed using ELISAPLUS
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Kit (Roche Diagnostics, Indianapolis, IN, USA). The extract was obtained from MCF-7 and
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MDA-MB-231 treated with vehicle or arctigenin for 24 hours and mixed with immune reagent
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for 2 hours. After washing, the extract was incubated with 100 µL of ABTS solution for 10
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minutes. The absorbance was recorded at 405 nm and 490 nm in the same microplate reader
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described above.
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Immnoblot analysis
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Protein expression was measured as we described previously 19. 6
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Fractionation of nuclear and cytoplasmic extracts
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The nuclear and cytoplasmic fraction was isolated separately using Active Motif Nuclear Extract
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kit (Active Motif, Carlsbad, CA, USA) as we described previously 40.
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Statistical analysis
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All experiments were conducted in triplicates and the results are presented as mean values ±
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standard deviations. Significant differences (P < 0.05) were analyzed using a student t-test.
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RESULTS
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Arctigenin inhibits adipogenesis in differentiated 3T3-L1 cells.
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To examine if arctigenin leads to toxicity in adipocytes, cell viability was measured in fully
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differentiated 3T3-L1 cells treated with different doses (0, 1, 5, 10, 25, and 50 µM) of arctigenin.
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As shown in Fig. 1B, cell proliferation was not affected by treatment of arctigenin (0-25 µM) in
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the differentiated adipocytes while treatment of 50 µM arctigenin decreased cell viability. Next,
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fat accumulation was measured in differentiated 3T3-L1 cells treated with 0, 6, 12 and 25 µM of
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arctigenin for different days (Days 6, 8 and 10). Time-dependent increase of fat accumulation
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was observed in the vehicle-treated cells (Fig. 1C). However, in the presence of arctigenin, lipid
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accumulation was significantly decreased in a dose-dependent manner [Day 6: 0.655±0.013,
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0.537±0.012, 0.384±0.020, 0.211±0.010 (p
