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Selective Estrogen Receptor Modulator (SERM)-like activities of Diarylheptanoid, a Phytoestrogen from Curcuma comosa, in Breast Cancer Cells, Pre-Osteoblast Cells, and Rat Uterine Tissues Natthakan Thongon, Nittaya Boonmuen, Kanoknetr Suksen, Patsorn Wichit, Arthit Chairoungdua, Patoomratana Tuchinda, Apichart Suksamrarn, Wipawee Winuthayanon, and Pawinee - Piyachaturawat J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00769 • Publication Date (Web): 15 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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Journal of Agricultural and Food Chemistry

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Selective Estrogen Receptor Modulator (SERM)-like activities of Diarylheptanoid, a

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Phytoestrogen from Curcuma comosa, in Breast Cancer Cells, Pre-Osteoblast Cells,

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and Rat Uterine Tissues

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Natthakan Thongon†, Nittaya Boonmuen†, Kanoknetr Suksen†, Patsorn Wichit†, Arthit

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Chairoungdua†, Patoomratana Tuchinda‡, Apichart Suksamrarn§, Wipawee Winuthayanon#,

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and Pawinee Piyachaturawat*,†

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Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400,

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Thailand, ‡Department of Chemistry, Faculty of Science, Mahidol University, Bangkok

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10700, Thailand, §Department of Chemistry, Faculty of Science, Ramkhamhaeng

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University, Bangkok 10240, Thailand, and #School of Molecular Biosciences, College of

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Veterinary Medicine, Washington State University, Washington 99164, USA

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The authors declare no competing financial interest.

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*Corresponding Author: Phone: +66 2 201 5620, E-mail: [email protected]

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Category: Bioactive Constituents, Metabolites, and Functions

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ABSTRACT

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Diarylheptanoids from Curcuma comosa (C. comosa), Zingiberaceae family, exhibit

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diverse estrogenic activities. In this study we investigated the estrogenic activity of a major

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hydroxyl diarylheptanoid, 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene

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(compound-092) isolated from C. comosa. The compound elicited different transcriptional

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activities of estrogen agonist at low concentrations (0.1-1 µM) and antagonist at high

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concentrations (10-50 µM) using luciferase reporter gene assay in HEK-293T cells. In

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human breast cancer (MCF-7) cells, compound-092 showed an anti-estrogenic activity by

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down-regulating ERα-signaling and suppressing estrogen-responsive genes whereas it

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attenuated uterotrophic effect of estrogen in immature ovariectomized rats. Of note,

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compound-092 promoted mouse preosteoblastic (MC3T3-E1) cells differentiation, and the

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related bone markers indicating its positive osteogenic effect. Our findings highlight a new,

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nonsteroidal, estrogen agonist/antagonist of catechol diarylheptanoid from C. comosa,

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which is the scientific evidence supporting its potential as a dietary supplement to prevent

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bone loss with low risk of breast and uterine cancers in post-menopausal women.

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Keywords: estrogen agonist, estrogen antagonist, diarylheptanoid, Curcuma comosa,

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phytoestrogens, bone, breast cancer, uterus

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INTRODUCTION

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Estrogens are steroid hormones that play important roles in growth and

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development of reproductive and non-reproductive tissues.1 At menopause, circulating

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level of estrogen has drastically declines and causes a number of unpleasant symptoms

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including hot flushes, mood swings, urogenital atrophy and, in particular, bone loss.2 An

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estrogen replacement therapy is effective in providing health beneficial effect and reducing

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osteoporosis in menopausal women.3,4 However, chronic estrogen therapy creates several

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undesirable effects including the risk of breast cancer.5 Estrogens mainly mediate their

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actions through nuclear receptors, estrogen receptor alpha (ERα) and beta (ERβ).1 Selective

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estrogen receptor modulators (SERMs) by naturally-occurring as well as synthetic

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compounds that selectively bind to, and cause conformational changes of ERs have

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received increased investigative attentions. They induce responses in various tissues

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leading to a potential rational therapeutic design. Currently, SERMs are selected as one of

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the first-line therapeutic approaches for menopause-induced osteoporosis.6 Tamoxifen, one

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of the SERMs, has been widely used for treatment of breast cancer due to its anti-

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estrogenic activity. However, it appears that the unanticipated estrogenic action of

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tamoxifen increased risk for endometrial cancer.7,8 Therefore, in view of undesirable

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effects of SERMs, it is essential to identify new SERM candidates that possess estrogenic

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and anti-estrogenic actions in a tissue-selective manner for post-menopausal women.

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Phytoestrogens are the naturally occurring plant chemicals that exhibit biological

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activities similar to the endogenous estrogens.7 Phytoestrogens bind to ERα or ERβ and

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subsequently activate the transcription of specific genes in the nucleus.7,8 Similar to

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SERMs, phytoestrogen-ER interaction causes conformational changes that recruit different

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co-activators or co-repressors in a tissue specific manner.9 Accordingly, they exhibit both

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estrogen-agonist and estrogen-antagonist activities in different tissues which have been the

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targets for hormone replacement therapy 6,10 as well as cancer prevention.11 Soybean-

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derived phytoestrogens such as genistein, diadzein, and glycitein have been shown to

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prevent bone loss12,13 but they also induce the uterine growth both in vivo and in vitro.9

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Additionally, genistein and other flavonoids have also been reported to inhibit cancer cell

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proliferation.10,14–17 We previously showed that several diarylheptanoids isolated from

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Curcuma comosa Roxb. (C. comosa), family Zingiberaceae, exhibit diverse estrogenic

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activities.18,19 As dietary supplement products derived from C. comosa have been

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extensively used, it is essential to evaluate the activity of these major compounds. Of these

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seven isolated diarylheptanoids, nonphenolic compounds (3S)-1,7-diphenyl-(6E)-6-hepten-

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3-ol (compound-001) and (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (compound-049)

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exhibit high estrogenic activities both in vitro system using a recombinant yeast system,

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and uterotrophic assay in vivo.19,20 However, the naturally occurring diarylheptanoid with

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two hydroxyl substituents on the benzene ring, (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-

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(6E)-6-hepten-3-ol (compound-092), lacks an estrogenic activity both in vitro and in vivo

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system.19 The presence of OH substituent to the aromatic ring has been reported to

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associate with antagonistic properties of estrogenic compounds.21 Therefore, the present

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study aimed to investigate the estrogenic and anti-estrogenic effects of the compound-092.

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Specifically, we characterized whether compound-092 possesses a SERM-like activity in

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breast cancer, bone, and uterine epithelial cells using in vitro and in vivo models. Herein,

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we show that compound-092 exhibited estrogenic and anti-estrogenic properties in a tissue-

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selective manner.

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MATERIALS AND METHODS

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Reagents, antibodies, plasmids and Compound-092 from C. comosa

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Diarylheptanoid 7-(3,4 dihydroxyphenyl)-5-hydroxy-1-phenyl-(1E)-1-heptene (compound-

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092) (Figure 1), was isolated and purified as previously described.18 Purity of compound

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was more than 98% purified by HPLC. The following reagents were used: 17β-estradiol

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(E2), Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham (DMEM/F12),

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Minimum Essential Medium Eagle (MEM), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

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tetrazolium bromide (MTT) from Sigma-Aldrich (St. Louis, MO, USA); ICI 182,780 (ICI)

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from Tocris Bioscience (Ellisville, MS, USA); Charcoal Stripped Fetal Bovine Serum

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(SFBS), and Trizol reagent from Invitrogen (Carlsbad, CA, USA); biscinchoninic acid

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(BCA) from Thermo Scientific (Rockford, IL, USA); dual-luciferase reporter assay from

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Promega (Madison, WI, USA); iScriptTM select cDNA-synthesis kit from BioRad

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(Hercules, CA, USA); SYBR master mix from Applied Biosystem (Carlsbad, CA, USA).

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Plasmid of hER (WTERα) and 3x-vit-ERE-TATA-LUC were kindly obtained from Dr.

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Kenneth Korach (National Institute of Environmental Health Sciences, Research Triangle

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Park, NC, USA).20

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Cell cultures

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Human breast cancer (MCF-7), human embryonic kidney (HEK-293T), and mouse

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preosteoblastic (MC3T3-E1) cells were obtained from American Type Culture Collection

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(ATCC) and cultured in phenol red-free DMEM/F12, DMEM, and Minimum essential

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medium alpha modification (αMEM), respectively. Cells were maintained in 10% fetal

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bovine serum (FBS) and incubated at 37°C under 5% CO2 incubator. 10% SFBS was used

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in the experiments. MC3T3-E1 cells were cultured in differentiation medium containing

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hydrocortisone hemisuccinate (200 nM), β-glycerophosphate (10 mM), ascorbic acid (50

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µg/ml), and calcium chloride (2 mM) for 7 days before treatments.

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Transactivation Assay for ER Activity.

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HEK-293T and MCF-7 cells were seeded in 96-well plates for 24 h. Cells were transiently

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transfected with 0.1 µg pcDNA 3.1-hERα, 0.05 µg 3x-vit-ERE-TATA-LUC, and 0.05 µg

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pRSV-β-gal plasmids by using Lipofectamine 2000 according to the manufacturer’s

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instruction (Invitrogen, Carlsbad, CA, USA). Twenty-four hours after transfection, cells

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were treated with 1 nM E2, 1 µM ICI, or different concentrations of compound-092 for 24

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h. Afterward, luciferase activities were measured using the Dual-luciferase Reporter Assay

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System. The luciferase activities were expressed as the fold change compared to the cells

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transfected with pcDNA3.1 vector alone.

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Cell proliferation assessment

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Cells were seeded in 96 well-plates and then treated with various concentrations of

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compound-092 (0-100 µM) for 24 h (MCF-7 cells) or for 7 days (MC3T3-E1 cells).

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Percentage of cell viability was determined using MTT assay and DAPI staining

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(Invitrogen, MA, USA).

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Western blot analysis

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MCF-7 cells were lysed with modified RIPA lysis buffer.22 An equal amount of protein

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was resolved by SDS-PAGE, subsequently transferred to a nitrocellulose membrane. The

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membranes were probed with anti-ERα (sc-7207, 1:1000, Santa Cruz Biotechnology,

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Dallas, TX) and anti-β-actin (sc-8432, 1:5000, Santa Cruz Biotechnology) antibodies.

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HRP-conjugated goat anti-mouse IgG from Jackson Immuno Research (West Grove, PA,

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USA) was used and incubated (1:5000 dilution) for 1 h. The signal was detected using the

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enhanced SuperSignal West Pico Cheminescent Thermo Scientific (Rockford, IL, USA).

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Total RNA isolation and Real-time PCR

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MCF-7 and MC3T3-E1 cells were treated with tested compounds at indicated

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concentrations and total RNAs were extracted using Trizol reagent according to the

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manufacturer’s recommendations. Equal amount of total RNA samples was subjected for

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cDNA synthesis using iScriptTM select cDNA synthesis kit. Quantitative RT-PCRs of PGR,

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C-MYC and PS2 expression in MCF-7 cells and Runx2, Alp, Osteocalcin, Col1a1, Opg, and

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Rankl expression in MC3T3-E1 cells were determined using KAPA SYBR FAST qPCR.

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The primers used are present in Supplementary Table 1.

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Alkaline phosphatase (ALP) assay

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MC3T3-E1 cells were treated with 10 nM E2, and various concentrations of compound-

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092 in the differentiation medium for another 7 days. Cells were harvested with lysis buffer

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containing 0.2% Triton X-100, 1 mM DTT, and 100 mM potassium phosphate buffer (pH

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7.8) as previously described.23 ALP activity was determined by mixing the cell extract with

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5 mM freshly prepared p-nitrophenyl phosphate (PNPP) substrate in 0.1 M glycine (pH

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10.4), 1 mM MgCl2, and 1 mM ZnCl2 at room temperature for 60 min. The enzymatic

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reaction was stopped by adding 3 M NaOH solution and absorbance was measured at 405

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nm. ALP activity was normalized to total protein concentration by using BCA method. For

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ALP staining assay, MC3T3-E1 cells were cultured in differentiation medium and treated

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with 10 nM E2 or various concentrations of compound-092 for 21 days. Cells were washed

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and fixed with fixative solution (60% citrate and acetone) for 1 min, and then incubated in

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buffer containing 0.4 mg/ml Naphthol AS-MX phosphate disodium salt and 1 mg/ml Fast

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Blue RR salt from Sigma-Aldrich (St. Louis, MO, USA) for 15 min and then rinsed with

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de-ionized water and observed under the light microscope.

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Animals and uterotrophic assay

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The uterotrophic assay in immature ovariectomized rats was done according to the previous

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report.19 The experimental protocol was approved by the Institutional Animal Care and Use

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Committee, Faculty of Science, Mahidol University (Proj.#208-2553). Immature female

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Wistar rats (3-week-old) were ovariectomized (OVX) and were allowed to recover for 5

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days after the operation. The OVX rats were randomly assigned to receive vehicle (olive

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oil, ip.), E2 (2.5 µg/kg body weight, sc.), ICI (50 µg/kg body weight, ip.), compound-092

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(100 mg/kg body weight, ip.), combinations of E2 and ICI or E2 and compound-092.

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Animals were treated once a day for 3 consecutive days and 24 h after the last treatment,

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they were then sacrificed with overdose of pentobarbital sodium injection (ip). Uteri were

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removed, blotted, and weighed. A portion of each uterus was fixed in 10% formalin,

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embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) using a

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standard histological procedure.

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Statistical analysis

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Data were expressed as Mean ± S.E.M. Statistical significance was considered when

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p