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Selective Receptor Modulator (SERM)-like Activities of Diarylheptanoid, a from Curcuma comosa, in 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 down- regulating 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, , 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, , bone, breast cancer, uterus

■ INTRODUCTION estrogenic actions in a tissue-selective manner for postmeno- Estrogens are hormones that play important roles in the pausal women. growth and development of reproductive and nonreproductive Phytoestrogens are naturally occurring plant chemicals that 1 exhibit biological activities similar to those of the endogenous tissues. At menopause, the circulating level of estrogen 7 α β drastically declines and causes a number of unpleasant symptoms estrogens. Phytoestrogens bind to ER or ER and fl subsequently activate the transcription of specific genes in the including hot ushes, mood swings, urogenital atrophy, and, in 7,8 − particular, bone loss.2 An estrogen replacement therapy is nucleus. Similar to SERMs, phytoestrogen ER interaction ff fi ff causes conformational changes that recruit different co-activators e ective in providing a health bene cial e ect and reducing fi 9 osteoporosis in menopausal women.3,4 However, chronic or co-repressors in a tissue-speci c manner. Accordingly, they estrogen therapy creates several undesirable effects including exhibit both estrogen-agonist and estrogen-antagonist activities 5 in different tissues that have been the targets for hormone the risk of breast cancer. Estrogens mainly mediate their actions 6,10 11 through nuclear receptors, estrogen receptors alpha (ERα) and replacement therapy as well as cancer prevention. Soybean- beta (ERβ).1 Selective modulators (SERMs) derived phytoestrogens such as , diadzein, and have been shown to prevent bone loss,12,13 but they also induce are naturally occurring as well as synthetic compounds that 9 selectively bind to, and cause conformational changes of, ERs and uterine growth both in vivo and in vitro. Additionally, genistein and other flavonoids have also been reported to inhibit cancer cell have received increased investigative attention. They induce 10,14−17 responses in various tissues leading to a potential rational proliferation. We previously showed that several diary- lheptanoids isolated from Curcuma comosa Roxb., family therapeutic design. Currently, SERMs are selected as one of the 18,19 first-line therapeutic approaches for menopause-induced osteo- Zingiberaceae, exhibit diverse estrogenic activities. As dietary porosis.6 , one of the SERMs, has been widely used for supplement products derived from C. comosa have been treatment of breast cancer due to its anti-estrogenic activity. However, it appears that the unanticipated estrogenic action of Received: February 19, 2017 tamoxifen increased the risk for endometrial cancer.7,8 Therefore, Revised: April 13, 2017 in view of the undesirable effects of SERMs, it is essential to Accepted: April 15, 2017 identify new SERM candidates that possess estrogenic and anti- Published: April 15, 2017

© 2017 American Chemical Society 3490 DOI: 10.1021/acs.jafc.7b00769 J. Agric. Food Chem. 2017, 65, 3490−3496 Journal of Agricultural and Food Chemistry Article extensively used, it is essential to evaluate the activity of these were obtained from American Type Culture Collection (ATCC) and major compounds. Of these seven isolated diarylheptanoids, cultured in phenol red-free DMEM/F12, DMEM, and minimum essential medium alpha modification (αMEM), respectively. Cells were nonphenolic compounds (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol ° (compound 001) and (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien- maintained in 10% fetal bovine serum (FBS) and incubated at 37 C 049 under 5% CO2 incubation. Ten percent SFBS was used in the 3-ol (compound ) exhibit high estrogenic activities in both an experiments. MC3T3-E1 cells were cultured in differentiation medium in vitro system using a recombinant yeast system and a β 19,20 containing hydrocortisone hemisuccinate (200 nM), -glycerophos- uterotrophic assay in vivo. However, the naturally occurring phate (10 mM), ascorbic acid (50 μg/mL), and calcium chloride (2 mM) diarylheptanoid with two hydroxyl substituents on the benzene for 7 days before treatments. ring, (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3- Transactivation Assay for ER Activity. HEK-293T and MCF-7 ol (compound 092), lacks an estrogenic activity in both in vitro cells were seeded in 96-well plates for 24 h. Cells were transiently and in vivo systems.19 The presence of the OH substituent in the transfected with 0.1 μg of pcDNA 3.1-hERα, 0.05 μg of 3x-vit-ERE- aromatic ring has been reported to be associated with the TATA-LUC, and 0.05 μg of pRSV-β-gal plasmids by using Lipofect- ’ antagonistic properties of estrogenic compounds.21 Therefore, amine 2000 according to the manufacturer s instruction (Invitrogen). Twenty-four hours after transfection, cells were treated with 1 nM E2, 1 the present study aimed to investigate the estrogenic and anti- μ ff ff fi M ICI, or di erent concentrations of compound 092 for 24 h. estrogenic e ects of compound 092. Speci cally, we charac- Afterward, luciferase activities were measured using the dual-luciferase terized whether compound 092 possesses a SERM-like activity in reporter assay system. The luciferase activities were expressed as the fold breast cancer, bone, and uterine epithelial cells using in vitro and change compared to the cells transfected with pcDNA3.1 vector alone. in vivo models. Herein, we show that compound 092 exhibited Cell Proliferation Assessment. Cells were seeded in 96-well plates estrogenic and anti-estrogenic properties in a tissue-selective and then treated with various concentrations of compound 092 (0−100 manner. 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 ■ MATERIALS AND METHODS (Invitrogen). Western Blot Analysis. MCF-7 cells were lysed with modified RIPA Reagents, Antibodies, Plasmids, and Compound 092 from ff 22 C. comosa. Diarylheptanoid 7-(3,4-dihydroxyphenyl)-5-hydroxy-1- lysis bu er. An equal amount of protein was resolved by SDS-PAGE phenyl-(1E)-1-heptene (compound 092, Figure 1) was isolated and 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 anti- mouse 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 Figure 1. Effect of diarylheptanoid, 7-(3,4 dihydroxyphenyl)-5-hydroxy- Rankl expression in MC3T3-E1 cells were determined using KAPA 1-phenyl-(1E)-1-heptene (compound 092)onERα/ERE-mediated SYBR FAST qPCR. The primers used are presented in the Supporting transactivation in HEK-293T cells. HEK-293T cells were transiently Information. transfected with hERα, 3x-ERE-TATA-Luc, and pRSV-β-galactosidase. Alkaline Phosphatase (ALP) Assay. MC3T3-E1 cells were treated After 24 h, transfected cells were treated with tested compounds or with 10 nM E2 and various concentrations of compound 092 in the combinations of tested compounds. Data (from three independent differentiation medium for another 7 days. Cells were harvested with experiments with technical triplicates) are presented as relative luciferase lysis buffer containing 0.2% Triton X-100, 1 mM DTT, and 100 mM activity and compared to vehicle control (DMSO) in the presence of potassium phosphate buffer (pH 7.8) as previously described.23 ALP hERα/ERE alone (dotted line). (∗) p < 0.05 compared to vehicle control activity was determined by mixing the cell extract with 5 mM freshly (hERα/ERE alone); (#) p < 0.01 compared to E2 (hERα/ERE). 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 purified as previously described.18 The purity of the compound was solution, and absorbance was measured at 405 nm. ALP activity was >98% by HPLC. The following reagents were used: 17β- (E2), normalized to total protein concentration by using BCA method. For Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham ALP staining assay, MC3T3-E1 cells were cultured in differentiation (DMEM/F12), minimum essential medium Eagle (MEM), and 3- medium and treated with 10 nM E2 or various concentrations of (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) compound 092 for 21 days. Cells were washed and fixed with fixative from Sigma-Aldrich (St. Louis, MO, USA); ICI 182,780 (ICI) from solution (60% citrate and acetone) for 1 min and then incubated in buffer Tocris Bioscience (Ellisville, MS, USA); charcoal-stripped fetal bovine containing 0.4 mg/mL naphthol AS-MX phosphate disodium salt and 1 serum (SFBS) and Trizol reagent from Invitrogen (Carlsbad, CA, USA); mg/mL Fast Blue RR salt from Sigma-Aldrich for 15 min and then rinsed bicinchoninic acid (BCA) from Thermo Scientific (Rockford, IL, USA); with deionized water and observed under the light microscope. dual-luciferase reporter assay from Promega (Madison, WI, USA); Animals and Uterotrophic Assay. The uterotrophic assay in iScript select cDNA-synthesis kit from Bio-Rad (Hercules, CA, USA); immature ovariectomized rats was done according to a previous study.19 and SYBR master mix from Applied Biosystem (Carlsbad, CA, USA). The experimental protocol was approved by the Institutional Animal Plasmid of hER (WTERα) and 3x-vit-ERE-TATA-LUC were kindly Care and Use Committee, Faculty of Science, Mahidol University (Proj. provided by Dr. Kenneth Korach (National Institute of Environmental #208-2553). Immature female Wistar rats (3 weeks old) were Health Sciences, Research Triangle Park, NC, USA).20 ovariectomized (OVX) and were allowed to recover for 5 days after Cell Cultures. Human breast cancer (MCF-7), human embryonic the operation. The OVX rats were randomly assigned to receive vehicle kidney (HEK-293T), and mouse pre-osteoblastic (MC3T3-E1) cells (olive oil, ip), E2 (2.5 μg/kg body weight, sc), ICI (50 μg/kg body

3491 DOI: 10.1021/acs.jafc.7b00769 J. Agric. Food Chem. 2017, 65, 3490−3496 Journal of Agricultural and Food Chemistry Article

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 way analysis of variance (ANOVA) followed by the post hoc combinations of E2 and ICI or E2 and compound 092. Animals were (Bonferroni) multiple comparisons using GraphPad Prism version 5.0 treated once a day for 3 consecutive days, and 24 h after the last for Windows (GraphPad Software, San Diego, CA, USA). treatment, they were then sacrificed with an overdose of pentobarbital sodium injection (ip). Uteri were removed, blotted, and weighed. A ■ RESULTS portion of each uterus was fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) using a Transactivation Activity of Compound 092 Is Mediated standard histological procedure. via the ERα/ERE-Dependent Pathway. A phenolic diary- Statistical Analysis. Data were expressed as the mean ± SEM. lheptanoid, (3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6- Statistical significance was considered at p < 0.05 and determined by one- hepten-3-ol, from C. comosa (compound 092) consists of two

3492 DOI: 10.1021/acs.jafc.7b00769 J. Agric. Food Chem. 2017, 65, 3490−3496 Journal of Agricultural and Food Chemistry Article

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). aromatic rings linked by a linear seven-carbon aliphatic chain with activity in the presence of hERα/ERE in MCF-7 cells, suggesting two hydroxyl substituents on the benzene ring, as shown in that it exhibits no estrogenic activity in these cells. However, Figure 1. To determine whether compound 092 mediates its cotreatment of compound 092 at a dose of 10 μM with E2 caused estrogenic/anti-estrogenic activity through the activation of a significant reduction of relative luciferase activity when human ERα (hERα) and estrogen responsive element (ERE), a compared to E2 treatment with hERα/ERE (Figure 2C). luciferase reporter assay was conducted in HEK-293T cells. E2 These findings indicate that compound 092 exerts its anti- significantly increased the luciferase activity in the presence of estrogenic activity in MCF-7 cells through a hERα/ERE hERα/ERE, which was completely inhibited by an ER antagonist, dependent pathway. 1 μM ICI 182,780 (Figure 1). However, E2 failed to induce ERE- The effects of compound 092 on the transcription and reporter activity in the absence of ERα, suggesting an important translation of ERα in MCF-7 cells were further determined. We role of ERα in transactivation regulation. Compound 092 at low found that E2 treatment slightly decreased ESR1 transcript as well concentrations of 0.1 and 1 μM induced ERE-mediated as ERα protein expression (p < 0.05), whereas ICI strongly transcriptional activity and enhanced E2-induced activities. inhibited it (Figure 2D,E). Compound 092 decreased However, the estrogenic activity of compound 092 was decreased endogenous ESR1 transcript and ERα protein levels in a dose- at higher concentrations of 10 and 50 μM. The compound at a related manner, but showed no statistical significance at mRNA concentration of 50 μM did not affect the viability of HEK-293T levels (1 and 10 μM). A significant decrease (p < 0.05) was seen ± μ μ fi cells; its IC50 at 24 h was 72.12 1.27 M. Thus, the inhibition by only at a high dose (50 M). E2 signi cantly increased mRNA compound 092 was not related to its cytotoxicity. levels of known estrogen targets, PGR, C-MYC, and TFF1 in Anti-estrogenic Activity of Compound 092 in Breast MCF-7 cells, which were significantly attenuated by cotreatment Cancer Cells. To evaluate the effect of compound 092 in breast with compound 092 (Figure 2F). However, the inhibitory effect cancer cells, first, we assessed the cell viability of MCF-7 cells of compound 092 on E2-induced endogenous gene expression using the MTT assay. E2 (10 nM) treatment for 24 h significantly was less than ICI activity (Figure 2F). Taken together, compound increased MCF-7 cell viability, whereas compound 092 at 1−10 092 profoundly exhibits anti-estrogenic action and requires ERα μM had virtually no effect (Figure 2A). On the other hand, high for its inhibition of transcriptional activity in the breast cancer cell doses of compound 092 at 50 and 100 μM significantly decreased line. cell viability (Figure 2A). This effect was also illustrated by DAPI Estrogenic Activity of Compound 092 on Pre-osteo- staining (Figure 2B). Then, the involvement of the anti- blast Differentiation. Osteoblast differentiation can be estrogenic effect of compound 092 in the hERα/ERE-driven characterized into three stages, which are discriminated by pathway was assessed. Compound 092 did not induce luciferase expression of specific osteoblast differentiation markers including

3493 DOI: 10.1021/acs.jafc.7b00769 J. Agric. Food Chem. 2017, 65, 3490−3496 Journal of Agricultural and Food Chemistry Article cell differentiation (Runt-related transcription factor 2 or Runx2 compound 092 did not affect the expression of Col1a1 or Rankl and collagen type I alpha 1 chain or Col1a1),23,24 matrix (Figure 4D,E). Lastly, we evaluated the Rankl/Opg ratio using the maturation (alkaline phosphatase or Alp),25 and matrix increased ratio as a predictive index of bone resorption.27 We mineralization (Alp, Osteocalcin, and Osteoprogerin or Opg).25,26 found that E2 and compound 092 significantly decreased the Therefore, to investigate the SERM-like activity of compound Rankl/Opg ratio (Figure 4F). The results suggest that compound 092 on bone tissues, we evaluated these parameters in mouse pre- 092 has an osteogenic effect in mouse pre-osteoblast cell osteoblast (MC3T3-E1) cells in the presence of compound 092 differentiation comparable to that of E2. compared to E2. Compound 092 significantly increased cell Inhibitory Effect of Compound 092 on E2-Induced viability of MC3T3-E1 cells after 7 days of culture comparable to Uterotrophic Activity in the Rat Model. To determine that of E2, especially at doses of 0.1 and 1 μM(Figure 3A). Both whether compound 092 exerts a uterotrophic effect, we assessed E2 and compound 092 significantly increased bone differ- its activity in the immature OVX rat model. After 3 consecutive entiation marker, alkaline phosphatase (Alp) transcript (Figure days of treatment, E2 significantly increased uterine weight 3B), ALP activity after 7 days (Figure 3C) and 21 days after the compared with that in the OVX control, whereas treatment with treatment (Figure 3D). Because the osteogenic activity of compound 092 alone did not alter the uterine weight (Figure compound 092 was effectively observed at a dose of 1 μM, this 5A). However, cotreatment of compound 092 with E2 dose was selected for further evaluation on the expression of significantly (p < 0.05) attenuated the E2-induced increased endogenous bone differentiation markers. Both E2 and uterine weight (Figure 5A). Histological examination of the compound 092 significantly increased Osteocalcin, Runx2, and uterine mucosa in the E2-treated group showed the elongation of Opg mRNA levels (Figure 4A−C). However, treatment with 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.

■ 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 estro- genic responses through ERα via a classical ERE-dependent pathway. The anti-estrogenic activity of compound 092 in the Figure 4. Effect of compound 092 on pre-osteoblast differentiation MCF-7 cells was associated with the down-regulation of ERα and markers. MC3T3-E1 cells were treated with DMSO, E2 (10 nM), or E2-target genes as well as growth inhibition. To our knowledge, compound 092 (1 μM) for 7 days. The mRNA expression of osteoblast this is the first study reporting the selective estrogenic activities of transcription factors: (A) Osteocalcin, (B) Runx2, (C) Opg, (D) Col1a1, a dihydroxy diarylheptanoid compound 092 that displays growth and (E) Rankl and (F) the ratio of Rankl/Opg were evaluated by qRT- inhibitory effect in breast cancer cells while exhibiting a growth PCR relative to Gapdh (a housekeeping gene). (∗) p < 0.05 compared to ff DMSO. stimulatory e ect in the osteoblasts. Therefore, compound 092 may have potential implications and be further developed as a SERM.

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 DOI: 10.1021/acs.jafc.7b00769 J. Agric. Food Chem. 2017, 65, 3490−3496 Journal of Agricultural and Food Chemistry Article

Compound 092 is a diarylheptanoid containing two hydroxyl molecular mechanism of compound 092 is still unclear. The substituents on the benzene ring, which is closely related to binding of different compounds on ER causes differential compounds 001 and 049, nonphenolic diarylheptanoid phytoes- conformational changes and affects coactivator recruitment in trogens.18,28 The addition of OH groups to the aromatic ring has specific cell types.35 Overexpression of the coactivator SRC-1 been reported to result in a great reduction of estrogenic enhances the ER agonistic response to tamoxifen.36 In contrast, activity.21 In some cases, additional OH groups give rise to ER reduced nuclear receptor corepressor N expression is associated antagonistic properties such as shikonin, which decreases both with the development of tamoxifen resistance in breast cancer transcriptional and translational levels of ERα in MCF-7 cells.11 xenografts.37 Therefore, we reason that estrogenic and anti- In addition to the OH group, a long bulky side chain can also estrogenic effects of compound 092 in different target cell models interfere with the binding of compounds to the ERα binding might be due to the recruitment of different coactivators and/or pocket and reduce the estrogenic activity, such as an alkylamide corepressors of each cell type. side chain at the 7α position of ICI.29 Thus, ICI interferes with ER In summary, our study reveals the first evidence demonstrating function and causes the degradation of ERα,29 an action that is the selective effects of a catechol diarylheptanoid, compound different from that of other SERMs including tamoxifen.30 In this 092, from C. comosa. We highlighted the estrogenic or anti- regard, it is likely that the two hydroxyl groups at the side chain of estrogenic activities of compound 092 in a tissue-specific manner. compound 092 are the most important structural feature Compound 092 exhibited osteogenic activity by inducing pre- responsible for the anti-estrogenic action in contrast to the osteoblast differentiation while possessing an anti-estrogenic estrogenic activity of diarylheptanoid compound 049. Of note, effect in female reproductive tissues, specifically breast and compound 092 exhibits biphasic activities of both estrogenic and uterine cells. Therefore, compound 092 from C. comosa may have anti-estrogenic effects in the transactivation assay in HEK-293T potential for development as an alternative treatment to prevent cells (Figure 1). These interesting properties of the compound bone loss with low risk of cancer development. A better prompted us to investigate its SERM activities in different understanding of the molecular mechanism of its biological estrogen responsive tissues. Indeed, compound 092 showed action would facilitate further development as a therapeutic SERM-like activities giving differential tissue selectivities on agent. breast, uterine, and bone cells. C. comosa contains several diarylheptanoids with diverse ■ ASSOCIATED CONTENT 18 estrogenic activities. Of these diarylheptanoids, compound *S Supporting Information 049 exhibits the highest estrogenic activity in in vivo, in vitro, and 18,20,28 The Supporting Information is available free of charge on the in silico models. In addition, our previous studies showed ACS Publications website at DOI: 10.1021/acs.jafc.7b00769. that compound 049 exerts an estrogenic effect of increased bone formation markers in human osteoblast cells23 and protects Description of primers and probes (PDF) against the loss of bone and deterioration of bone micro- architecture in OVX-induced osteopenia by suppression of bone ■ AUTHOR INFORMATION 31 turnover rate. In addition, compound 049 possesses non- Corresponding Author genomic estrogenic action by inducing osteoblastic cell differ- * β β (P.P.) Phone: +66 2 201 5620. E-mail: pawinee.pia@mahidol. entiation through the ER-Akt-GSK -dependent -catenin signal- ac.th. ing pathway22 and enhancing the endothelium-dependent relaxation of aortic rings through the ER−Akt−eNOS pathway.32 ORCID In the present study, we observed a marked increase of Runx2, Pawinee Piyachaturawat: 0000-0002-1677-0777 Alp, and Osteocalcin expressions as well as ALP activity after Funding treatment with compound 092 compared to those of E2. These This work is supported by Mahidol University, the Development findings suggest that compound 092 may also accelerate and Promotion of Science and Technology Talents project osteoblast differentiation by promoting matrix maturation and (DPST, to N.T.), and the Thailand Research Fund IRN- mineralization phases in mouse pre-osteoblastic cells. However, it 58W0004 (to P.P.), and DBG5980003 (to A.S.). is unclear at present whether compound 092 elicits a nongenomic Notes estrogenic activity similar to that of compound 049.Itis The authors declare no competing financial interest. important to note that a high concentration of compound 092 μ ff (10 M) decreased MCF-7 cell viability but did not a ect pre- ■ ACKNOWLEDGMENTS osteoblast cell viability at a similar dose. Several SERMs and phytoestrogens increase uterine wet We are grateful to Prof. Chumpol Pholpramool for his critical weight in animal models.33,34 Treatment with tamoxifen in reading of the manuscript. women has been associated with an increased risk of endometrial cancer.30 To determine whether compound 092 could ■ REFERENCES potentially increase uterine growth, we evaluated uterine weight (1) Hall, J. M.; Couse, J. F.; Korach, K. S. The multifaceted mechanisms and morphology using an OVX rat model. It was found that of estradiol and estrogen receptor signaling. J. Biol. Chem. 2001, 276 compound 092 did not stimulate uterine weight increase. In (40), 36869−36872. ff (2) Dalal, P. K.; Agarwal, M. Postmenopausal syndrome. Indian J. contrast, it showed an inhibitory e ect on the increase in uterine − weight induced by E2, in a similar manner as the ICI cotreatment. Psychiatry 2015, 57 (Suppl.2), S222 S232. (3) Diamanti-Kandarakis, E.; Sykiotis, G. P.; Papavassiliou, A. G. Taken together, these results highlight the potential SERM Selective modulation of postmenopausal women. Cancer 2003, 97 (1), property of compound 092 and a safety therapeutic implication 12−20. ff with low incidence of the adverse e ects on uterine growth. (4) Gruber, C. J.; Tschugguel, W.; Schneeberger, C.; Huber, J. C. Despite the wide range of estrogenic and anti-estrogenic Production and actions of estrogens. N. Engl. J. Med. 2002, 346 (5), 340− activities of compound 092 in different types of tissue, the 352.

3495 DOI: 10.1021/acs.jafc.7b00769 J. Agric. Food Chem. 2017, 65, 3490−3496 Journal of Agricultural and Food Chemistry Article

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