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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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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] 18 19
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
