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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*,† †

Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand § Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand ⊗ School of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164, United States ‡

S Supporting Information *

ABSTRACT: Diarylheptanoids from Curcuma comosa, of the Zingiberaceae family, exhibit diverse estrogenic activities. In this study we investigated the estrogenic activity of a major hydroxyl diarylheptanoid, 7-(3,4 -dihydroxyphenyl)-5-hydroxy-1-phenyl(1E)-1-heptene (compound 092) isolated from C. comosa. The compound elicited different transcriptional activities of estrogen agonist at low concentrations (0.1−1 μM) and antagonist at high concentrations (10−50 μM) using luciferase reporter gene assay in HEK-293T cells. In human breast cancer (MCF-7) cells, compound 092 showed an anti-estrogenic activity by downregulating ERα-signaling and suppressing estrogen-responsive genes, whereas it attenuated the uterotrophic effect of estrogen in immature ovariectomized rats. Of note, compound 092 promoted mouse pre-osteoblastic (MC3T3-E1) cell differentiation and the related bone markers, indicating its positive osteogenic effect. Our findings highlight a new, nonsteroidal, estrogen agonist/ antagonist of catechol diarylheptanoid from C. comosa, which is scientific evidence supporting its potential as a dietary supplement to prevent bone loss with low risk of breast and uterine cancers in postmenopausal women. KEYWORDS: estrogen agonist, estrogen antagonist, diarylheptanoid, Curcuma comosa, phytoestrogens, bone, breast cancer, uterus

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INTRODUCTION Estrogens are steroid hormones that play important roles in the growth and development of reproductive and nonreproductive tissues.1 At menopause, the circulating level of estrogen drastically declines and causes a number of unpleasant symptoms including hot flushes, mood swings, urogenital atrophy, and, in particular, bone loss.2 An estrogen replacement therapy is effective in providing a health beneficial effect and reducing osteoporosis in menopausal women.3,4 However, chronic estrogen therapy creates several undesirable effects including the risk of breast cancer.5 Estrogens mainly mediate their actions through nuclear receptors, estrogen receptors alpha (ERα) and beta (ERβ).1 Selective estrogen receptor modulators (SERMs) are naturally occurring as well as synthetic compounds that selectively bind to, and cause conformational changes of, ERs and have received increased investigative attention. They induce responses in various tissues leading to a potential rational therapeutic design. Currently, SERMs are selected as one of the first-line therapeutic approaches for menopause-induced osteoporosis.6 Tamoxifen, one of the SERMs, has been widely used for treatment of breast cancer due to its anti-estrogenic activity. However, it appears that the unanticipated estrogenic action of tamoxifen increased the risk for endometrial cancer.7,8 Therefore, in view of the undesirable effects of SERMs, it is essential to identify new SERM candidates that possess estrogenic and anti© 2017 American Chemical Society

estrogenic actions in a tissue-selective manner for postmenopausal women. Phytoestrogens are naturally occurring plant chemicals that exhibit biological activities similar to those of the endogenous estrogens. 7 Phytoestrogens bind to ERα or ERβ and subsequently activate the transcription of specific genes in the nucleus.7,8 Similar to SERMs, phytoestrogen−ER interaction causes conformational changes that recruit different co-activators or co-repressors in a tissue-specific manner.9 Accordingly, they exhibit both estrogen-agonist and estrogen-antagonist activities in different tissues that have been the targets for hormone replacement therapy6,10 as well as cancer prevention.11 Soybeanderived phytoestrogens such as genistein, diadzein, and glycitein have been shown to prevent bone loss,12,13 but they also induce uterine growth both in vivo and in vitro.9 Additionally, genistein and other flavonoids have also been reported to inhibit cancer cell proliferation.10,14−17 We previously showed that several diarylheptanoids isolated from Curcuma comosa Roxb., family Zingiberaceae, exhibit diverse estrogenic activities.18,19 As dietary supplement products derived from C. comosa have been Received: Revised: Accepted: Published: 3490

February April 13, April 15, April 15,

19, 2017 2017 2017 2017 DOI: 10.1021/acs.jafc.7b00769 J. Agric. Food Chem. 2017, 65, 3490−3496

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Journal of Agricultural and Food Chemistry

were obtained from American Type Culture Collection (ATCC) and cultured in phenol red-free DMEM/F12, DMEM, and minimum essential medium alpha modification (αMEM), respectively. Cells were maintained in 10% fetal bovine serum (FBS) and incubated at 37 °C under 5% CO2 incubation. Ten percent SFBS was used in the experiments. MC3T3-E1 cells were cultured in differentiation medium containing hydrocortisone hemisuccinate (200 nM), β-glycerophosphate (10 mM), ascorbic acid (50 μg/mL), and calcium chloride (2 mM) for 7 days before treatments. Transactivation Assay for ER Activity. HEK-293T and MCF-7 cells were seeded in 96-well plates for 24 h. Cells were transiently transfected with 0.1 μg of pcDNA 3.1-hERα, 0.05 μg of 3x-vit-ERETATA-LUC, and 0.05 μg of pRSV-β-gal plasmids by using Lipofectamine 2000 according to the manufacturer’s instruction (Invitrogen). Twenty-four hours after transfection, cells were treated with 1 nM E2, 1 μM ICI, or different concentrations of compound 092 for 24 h. Afterward, luciferase activities were measured using the dual-luciferase reporter assay system. The luciferase activities were expressed as the fold change compared to the cells transfected with pcDNA3.1 vector alone. Cell Proliferation Assessment. Cells were seeded in 96-well plates and then treated with various concentrations of compound 092 (0−100 μM) for 24 h (MCF-7 cells) or for 7 days (MC3T3-E1 cells). Percentage of cell viability was determined using the MTT assay and DAPI staining (Invitrogen). Western Blot Analysis. MCF-7 cells were lysed with modified RIPA lysis buffer.22 An equal amount of protein was resolved by SDS-PAGE and subsequently transferred to a nitrocellulose membrane. The membranes were probed with anti-ERα (sc-7207, 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin (sc-8432, 1:5000; Santa Cruz Biotechnology) antibodies. HRP-conjugated goat antimouse IgG from Jackson Immuno Research (West Grove, PA, USA) was used and incubated (1:5000 dilution) for 1 h. The signal was detected using enhanced SuperSignal West Pico Cheminescent (Thermo Scientific, Rockford, IL, USA). Total RNA Isolation and Real-Time PCR. MCF-7 and MC3T3-E1 cells were treated with tested compounds at indicated concentrations, and total RNAs were extracted using Trizol reagent according to the manufacturer’s recommendations. Equal amounts of total RNA samples were subjected to cDNA synthesis using an iScript select cDNA synthesis kit. Quantitative RT-PCRs of PGR, C-MYC, and TFF1 expression in MCF-7 cells and Runx2, Alp, Osteocalcin, Col1a1, Opg, and Rankl expression in MC3T3-E1 cells were determined using KAPA SYBR FAST qPCR. The primers used are presented in the Supporting Information. Alkaline Phosphatase (ALP) Assay. MC3T3-E1 cells were treated with 10 nM E2 and various concentrations of compound 092 in the differentiation medium for another 7 days. Cells were harvested with lysis buffer containing 0.2% Triton X-100, 1 mM DTT, and 100 mM potassium phosphate buffer (pH 7.8) as previously described.23 ALP activity was determined by mixing the cell extract with 5 mM freshly prepared p-nitrophenyl phosphate (PNPP) substrate in 0.1 M glycine (pH 10.4), 1 mM MgCl2, and 1 mM ZnCl2 at room temperature for 60 min. The enzymatic reaction was stopped by adding 3 M NaOH solution, and absorbance was measured at 405 nm. ALP activity was normalized to total protein concentration by using BCA method. For ALP staining assay, MC3T3-E1 cells were cultured in differentiation medium and treated with 10 nM E2 or various concentrations of compound 092 for 21 days. Cells were washed and fixed with fixative solution (60% citrate and acetone) for 1 min and then incubated in buffer containing 0.4 mg/mL naphthol AS-MX phosphate disodium salt and 1 mg/mL Fast Blue RR salt from Sigma-Aldrich for 15 min and then rinsed with deionized water and observed under the light microscope. Animals and Uterotrophic Assay. The uterotrophic assay in immature ovariectomized rats was done according to a previous study.19 The experimental protocol was approved by the Institutional Animal Care and Use Committee, Faculty of Science, Mahidol University (Proj. #208-2553). Immature female Wistar rats (3 weeks old) were ovariectomized (OVX) and were allowed to recover for 5 days after the operation. The OVX rats were randomly assigned to receive vehicle (olive oil, ip), E2 (2.5 μg/kg body weight, sc), ICI (50 μg/kg body

extensively used, it is essential to evaluate the activity of these major compounds. Of these seven isolated diarylheptanoids, nonphenolic compounds (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (compound 001) and (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien3-ol (compound 049) exhibit high estrogenic activities in both an in vitro system using a recombinant yeast system and a uterotrophic assay in vivo.19,20 However, the naturally occurring diarylheptanoid with two hydroxyl substituents on the benzene ring, (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3ol (compound 092), lacks an estrogenic activity in both in vitro and in vivo systems.19 The presence of the OH substituent in the aromatic ring has been reported to be associated with the antagonistic properties of estrogenic compounds.21 Therefore, the present study aimed to investigate the estrogenic and antiestrogenic effects of compound 092. Specifically, we characterized whether compound 092 possesses a SERM-like activity in breast cancer, bone, and uterine epithelial cells using in vitro and in vivo models. Herein, we show that compound 092 exhibited estrogenic and anti-estrogenic properties in a tissue-selective manner.

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MATERIALS AND METHODS

Reagents, Antibodies, Plasmids, and Compound 092 from C. comosa. Diarylheptanoid 7-(3,4-dihydroxyphenyl)-5-hydroxy-1phenyl-(1E)-1-heptene (compound 092, Figure 1) was isolated and

Figure 1. Effect of diarylheptanoid, 7-(3,4 dihydroxyphenyl)-5-hydroxy1-phenyl-(1E)-1-heptene (compound 092) on ERα/ERE-mediated transactivation in HEK-293T cells. HEK-293T cells were transiently transfected with hERα, 3x-ERE-TATA-Luc, and pRSV-β-galactosidase. After 24 h, transfected cells were treated with tested compounds or combinations of tested compounds. Data (from three independent experiments with technical triplicates) are presented as relative luciferase activity and compared to vehicle control (DMSO) in the presence of hERα/ERE alone (dotted line). (∗) p < 0.05 compared to vehicle control (hERα/ERE alone); (#) p < 0.01 compared to E2 (hERα/ERE). purified as previously described.18 The purity of the compound was >98% by HPLC. The following reagents were used: 17β-estradiol (E2), Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham (DMEM/F12), minimum essential medium Eagle (MEM), and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) from Sigma-Aldrich (St. Louis, MO, USA); ICI 182,780 (ICI) from Tocris Bioscience (Ellisville, MS, USA); charcoal-stripped fetal bovine serum (SFBS) and Trizol reagent from Invitrogen (Carlsbad, CA, USA); bicinchoninic acid (BCA) from Thermo Scientific (Rockford, IL, USA); dual-luciferase reporter assay from Promega (Madison, WI, USA); iScript select cDNA-synthesis kit from Bio-Rad (Hercules, CA, USA); and SYBR master mix from Applied Biosystem (Carlsbad, CA, USA). Plasmid of hER (WTERα) and 3x-vit-ERE-TATA-LUC were kindly provided by Dr. Kenneth Korach (National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA).20 Cell Cultures. Human breast cancer (MCF-7), human embryonic kidney (HEK-293T), and mouse pre-osteoblastic (MC3T3-E1) cells 3491

DOI: 10.1021/acs.jafc.7b00769 J. Agric. Food Chem. 2017, 65, 3490−3496

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Figure 2. Anti-estrogenic effect of compound 092 in breast cancer cells. (A) Viability of MCF-7 cells determined using the MTT assay after 24 h of treatment with vehicle control (DMSO), E2, or various doses of compound 092. (∗) p < 0.05 compared to DMSO. (B) Representative images from three independent experiments of DAPI staining of MCF-7 cells after 48 h of treatment with DMSO, E2, ICI, or compound 092 (100× magnification). (C) Transactivation induction by the tested compounds in MCF-7 cells in the presence of hERα and 3x-vit-ERE-TATA-Luc plasmids. Data (from three independent experiments with technical triplicates) are presented as relative luciferase activity and compared to vehicle control in the presence of hERα/ ERE alone. (∗) p < 0.05 compared to vehicle control (hERα/ERE alone); (#) p < 0.01 compared to E2 (hERα/ERE). (D) MCF-7 cells treated with DMSO, E2 (10 nM), ICI (1 μM), or compound 092 (1, 10, and 50 μM) for 24 h. Endogenous ESR1 mRNA levels of MCF-7 cells were analyzed by quantitative RT-PCR. (∗) p < 0.05 compared to DMSO. (E) Endogenous ERα protein expression after 24 h treatment of DMSO, E2, ICI, or compound 092 in comparison to β-actin (a loading control). A representative blot from three biological experiments is shown. Densitometric analysis was performed and represented as relative values compared to DMSO. (∗) p < 0.05. (F) Endogenous expression of E2-target genes (PGR, C-MYC, and TFF1) after 24 h of treatment with DMSO, E2 (10 nM), ICI (1 μM), or compound 092 (10 μM) in MCF-7 cells. (∗) p < 0.05 compared to DMSO; (#) p < 0.01 compared to E2. weight, ip), compound 092 (100 mg/kg body weight, ip), or combinations of E2 and ICI or E2 and compound 092. Animals were treated once a day for 3 consecutive days, and 24 h after the last treatment, they were then sacrificed with an overdose of pentobarbital sodium injection (ip). Uteri were removed, blotted, and weighed. A portion of each uterus was fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) using a standard histological procedure. Statistical Analysis. Data were expressed as the mean ± SEM. Statistical significance was considered at p < 0.05 and determined by one-

way analysis of variance (ANOVA) followed by the post hoc (Bonferroni) multiple comparisons using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA).

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RESULTS Transactivation Activity of Compound 092 Is Mediated via the ERα/ERE-Dependent Pathway. A phenolic diarylheptanoid, (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6hepten-3-ol, from C. comosa (compound 092) consists of two 3492


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Figure 3. Osteogenic effect of compound 092 on pre-osteoblast differentiation. (A) MC3T3-E1 cell proliferation determined by MTT assay after 7 days of treatment with DMSO (dotted line), E2, or compound 092. (∗) p < 0.05 compared to DMSO on designated days. (B) Expression of alkaline phosphatase (Alp) in MC3T3-E1 cells after 7 days of treatment with DMSO, E2 (10 nM), or compound 092. (C) ALP activity at day 7 after treatment with DMSO, E2, or compound 092. (D) Expression of ALP in MC3T3-E1 cells determined by naphthol AS-MX phosphate staining after 21 days of treatment with DMSO, E2, or compound 092. Representative images from three independent experiments are shown (50× and 200× magnifications).

activity in the presence of hERα/ERE in MCF-7 cells, suggesting that it exhibits no estrogenic activity in these cells. However, cotreatment of compound 092 at a dose of 10 μM with E2 caused a significant reduction of relative luciferase activity when compared to E2 treatment with hERα/ERE (Figure 2C). These findings indicate that compound 092 exerts its antiestrogenic activity in MCF-7 cells through a hERα/ERE dependent pathway. The effects of compound 092 on the transcription and translation of ERα in MCF-7 cells were further determined. We found that E2 treatment slightly decreased ESR1 transcript as well as ERα protein expression (p < 0.05), whereas ICI strongly inhibited it (Figure 2D,E). Compound 092 decreased endogenous ESR1 transcript and ERα protein levels in a doserelated manner, but showed no statistical significance at mRNA levels (1 and 10 μM). A significant decrease (p < 0.05) was seen only at a high dose (50 μM). E2 significantly increased mRNA levels of known estrogen targets, PGR, C-MYC, and TFF1 in MCF-7 cells, which were significantly attenuated by cotreatment with compound 092 (Figure 2F). However, the inhibitory effect of compound 092 on E2-induced endogenous gene expression was less than ICI activity (Figure 2F). Taken together, compound 092 profoundly exhibits anti-estrogenic action and requires ERα for its inhibition of transcriptional activity in the breast cancer cell line. Estrogenic Activity of Compound 092 on Pre-osteoblast Differentiation. Osteoblast differentiation can be characterized into three stages, which are discriminated by expression of specific osteoblast differentiation markers including

aromatic rings linked by a linear seven-carbon aliphatic chain with two hydroxyl substituents on the benzene ring, as shown in Figure 1. To determine whether compound 092 mediates its estrogenic/anti-estrogenic activity through the activation of human ERα (hERα) and estrogen responsive element (ERE), a luciferase reporter assay was conducted in HEK-293T cells. E2 significantly increased the luciferase activity in the presence of hERα/ERE, which was completely inhibited by an ER antagonist, 1 μM ICI 182,780 (Figure 1). However, E2 failed to induce EREreporter activity in the absence of ERα, suggesting an important role of ERα in transactivation regulation. Compound 092 at low concentrations of 0.1 and 1 μM induced ERE-mediated transcriptional activity and enhanced E2-induced activities. However, the estrogenic activity of compound 092 was decreased at higher concentrations of 10 and 50 μM. The compound at a concentration of 50 μM did not affect the viability of HEK-293T cells; its IC50 at 24 h was 72.12 ± 1.27 μM. Thus, the inhibition by compound 092 was not related to its cytotoxicity. Anti-estrogenic Activity of Compound 092 in Breast Cancer Cells. To evaluate the effect of compound 092 in breast cancer cells, first, we assessed the cell viability of MCF-7 cells using the MTT assay. E2 (10 nM) treatment for 24 h significantly increased MCF-7 cell viability, whereas compound 092 at 1−10 μM had virtually no effect (Figure 2A). On the other hand, high doses of compound 092 at 50 and 100 μM significantly decreased cell viability (Figure 2A). This effect was also illustrated by DAPI staining (Figure 2B). Then, the involvement of the antiestrogenic effect of compound 092 in the hERα/ERE-driven pathway was assessed. Compound 092 did not induce luciferase 3493


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compound 092 did not affect the expression of Col1a1 or Rankl (Figure 4D,E). Lastly, we evaluated the Rankl/Opg ratio using the increased ratio as a predictive index of bone resorption.27 We found that E2 and compound 092 significantly decreased the Rankl/Opg ratio (Figure 4F). The results suggest that compound 092 has an osteogenic effect in mouse pre-osteoblast cell differentiation comparable to that of E2. Inhibitory Effect of Compound 092 on E2-Induced Uterotrophic Activity in the Rat Model. To determine whether compound 092 exerts a uterotrophic effect, we assessed its activity in the immature OVX rat model. After 3 consecutive days of treatment, E2 significantly increased uterine weight compared with that in the OVX control, whereas treatment with compound 092 alone did not alter the uterine weight (Figure 5A). However, cotreatment of compound 092 with E2 significantly (p < 0.05) attenuated the E2-induced increased uterine weight (Figure 5A). Histological examination of the uterine mucosa in the E2-treated group showed the elongation of uterine columnar epithelial cells (Figure 5B). This uterotrophic effect of E2 was markedly suppressed by ICI and compound 092. The finding that compound 092 is devoid of uterotrophic activation suggests that the compound has selective activity without undesirable effects on reproductive target tissues.

cell differentiation (Runt-related transcription factor 2 or Runx2 and collagen type I alpha 1 chain or Col1a1),23,24 matrix maturation (alkaline phosphatase or Alp),25 and matrix mineralization (Alp, Osteocalcin, and Osteoprogerin or Opg).25,26 Therefore, to investigate the SERM-like activity of compound 092 on bone tissues, we evaluated these parameters in mouse preosteoblast (MC3T3-E1) cells in the presence of compound 092 compared to E2. Compound 092 significantly increased cell viability of MC3T3-E1 cells after 7 days of culture comparable to that of E2, especially at doses of 0.1 and 1 μM (Figure 3A). Both E2 and compound 092 significantly increased bone differentiation marker, alkaline phosphatase (Alp) transcript (Figure 3B), ALP activity after 7 days (Figure 3C) and 21 days after the treatment (Figure 3D). Because the osteogenic activity of compound 092 was effectively observed at a dose of 1 μM, this dose was selected for further evaluation on the expression of endogenous bone differentiation markers. Both E2 and compound 092 significantly increased Osteocalcin, Runx2, and Opg mRNA levels (Figure 4A−C). However, treatment with

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DISCUSSION The present study demonstrated the selective estrogenic activities of a dihydroxy diarylheptanoid, compound 092, a naturally occurring compound derived from C. comosa.18,19 The compound displayed estrogen antagonist activity both in hormone-dependent human breast cancer (MCF-7) cells and in immature OVX rat uterine epithelial cells. In contrast, it showed a positive osteogenic effect like estrogen in the mouse osteoblast (MC3T3-E1) cells. Compound 092 mediated estrogenic responses through ERα via a classical ERE-dependent pathway. The anti-estrogenic activity of compound 092 in the MCF-7 cells was associated with the down-regulation of ERα and E2-target genes as well as growth inhibition. To our knowledge, this is the first study reporting the selective estrogenic activities of a dihydroxy diarylheptanoid compound 092 that displays growth inhibitory effect in breast cancer cells while exhibiting a growth stimulatory effect in the osteoblasts. Therefore, compound 092 may have potential implications and be further developed as a SERM.

Figure 4. Effect of compound 092 on pre-osteoblast differentiation markers. MC3T3-E1 cells were treated with DMSO, E2 (10 nM), or compound 092 (1 μM) for 7 days. The mRNA expression of osteoblast transcription factors: (A) Osteocalcin, (B) Runx2, (C) Opg, (D) Col1a1, and (E) Rankl and (F) the ratio of Rankl/Opg were evaluated by qRTPCR relative to Gapdh (a housekeeping gene). (∗) p < 0.05 compared to DMSO.

Figure 5. Effect of compound 092 on uterine weight in immature OVX Wistar rats. OVX rats were treated with E2 (2.5 μg/kg body weight/day), ICI (50 μg/kg body weight/day), compound 092 (100 mg/kg body weight/day), ICI + E2, or compound 092 + E2 for 3 consecutive days. (A) Uterine wet weight data are presented as the mean ± SEM of four to eight animals. (∗) p < 0.05 compared to OVX + oil; (#) p < 0.05 compared to OVX + E2. (B) H&E staining of rat uterine cross sections in sham or OVX treatment groups. Representative images are shown at 200× magnification. Epi, luminal epithelium. 3494


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molecular mechanism of compound 092 is still unclear. The binding of different compounds on ER causes differential conformational changes and affects coactivator recruitment in specific cell types.35 Overexpression of the coactivator SRC-1 enhances the ER agonistic response to tamoxifen.36 In contrast, reduced nuclear receptor corepressor N expression is associated with the development of tamoxifen resistance in breast cancer xenografts.37 Therefore, we reason that estrogenic and antiestrogenic effects of compound 092 in different target cell models might be due to the recruitment of different coactivators and/or corepressors of each cell type. In summary, our study reveals the first evidence demonstrating the selective effects of a catechol diarylheptanoid, compound 092, from C. comosa. We highlighted the estrogenic or antiestrogenic activities of compound 092 in a tissue-specific manner. Compound 092 exhibited osteogenic activity by inducing preosteoblast differentiation while possessing an anti-estrogenic effect in female reproductive tissues, specifically breast and uterine cells. Therefore, compound 092 from C. comosa may have potential for development as an alternative treatment to prevent bone loss with low risk of cancer development. A better understanding of the molecular mechanism of its biological action would facilitate further development as a therapeutic agent.

Compound 092 is a diarylheptanoid containing two hydroxyl substituents on the benzene ring, which is closely related to compounds 001 and 049, nonphenolic diarylheptanoid phytoestrogens.18,28 The addition of OH groups to the aromatic ring has been reported to result in a great reduction of estrogenic activity.21 In some cases, additional OH groups give rise to ER antagonistic properties such as shikonin, which decreases both transcriptional and translational levels of ERα in MCF-7 cells.11 In addition to the OH group, a long bulky side chain can also interfere with the binding of compounds to the ERα binding pocket and reduce the estrogenic activity, such as an alkylamide side chain at the 7α position of ICI.29 Thus, ICI interferes with ER function and causes the degradation of ERα,29 an action that is different from that of other SERMs including tamoxifen.30 In this regard, it is likely that the two hydroxyl groups at the side chain of compound 092 are the most important structural feature responsible for the anti-estrogenic action in contrast to the estrogenic activity of diarylheptanoid compound 049. Of note, compound 092 exhibits biphasic activities of both estrogenic and anti-estrogenic effects in the transactivation assay in HEK-293T cells (Figure 1). These interesting properties of the compound prompted us to investigate its SERM activities in different estrogen responsive tissues. Indeed, compound 092 showed SERM-like activities giving differential tissue selectivities on breast, uterine, and bone cells. C. comosa contains several diarylheptanoids with diverse estrogenic activities.18 Of these diarylheptanoids, compound 049 exhibits the highest estrogenic activity in in vivo, in vitro, and in silico models.18,20,28 In addition, our previous studies showed that compound 049 exerts an estrogenic effect of increased bone formation markers in human osteoblast cells23 and protects against the loss of bone and deterioration of bone microarchitecture in OVX-induced osteopenia by suppression of bone turnover rate.31 In addition, compound 049 possesses nongenomic estrogenic action by inducing osteoblastic cell differentiation through the ER-Akt-GSKβ-dependent β-catenin signaling pathway22 and enhancing the endothelium-dependent relaxation of aortic rings through the ER−Akt−eNOS pathway.32 In the present study, we observed a marked increase of Runx2, Alp, and Osteocalcin expressions as well as ALP activity after treatment with compound 092 compared to those of E2. These findings suggest that compound 092 may also accelerate osteoblast differentiation by promoting matrix maturation and mineralization phases in mouse pre-osteoblastic cells. However, it is unclear at present whether compound 092 elicits a nongenomic estrogenic activity similar to that of compound 049. It is important to note that a high concentration of compound 092 (10 μM) decreased MCF-7 cell viability but did not affect preosteoblast cell viability at a similar dose. Several SERMs and phytoestrogens increase uterine wet weight in animal models.33,34 Treatment with tamoxifen in women has been associated with an increased risk of endometrial cancer. 30 To determine whether compound 092 could potentially increase uterine growth, we evaluated uterine weight and morphology using an OVX rat model. It was found that compound 092 did not stimulate uterine weight increase. In contrast, it showed an inhibitory effect on the increase in uterine weight induced by E2, in a similar manner as the ICI cotreatment. Taken together, these results highlight the potential SERM property of compound 092 and a safety therapeutic implication with low incidence of the adverse effects on uterine growth. Despite the wide range of estrogenic and anti-estrogenic activities of compound 092 in different types of tissue, the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00769. Description of primers and probes (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(P.P.) Phone: +66 2 201 5620. E-mail: pawinee.pia@mahidol. ac.th. ORCID

Pawinee Piyachaturawat: 0000-0002-1677-0777 Funding

This work is supported by Mahidol University, the Development and Promotion of Science and Technology Talents project (DPST, to N.T.), and the Thailand Research Fund IRN58W0004 (to P.P.), and DBG5980003 (to A.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Chumpol Pholpramool for his critical reading of the manuscript.

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REFERENCES

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