β-Catenin Mediates Anti-adipogenic and Anticancer Effects of

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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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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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14

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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