Selective Estrogen Receptor Modulator (SERM)-like Activities of

Subscriber access provided by HACETTEPE UNIVERSITESI KUTUPHANESI

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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

Page 1 of 31

Journal of Agricultural and Food Chemistry

1

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

2

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

3

and Rat Uterine Tissues

4 5

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

6

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

7

and Pawinee Piyachaturawat*,†

8 9

†

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

10

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

11

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

12

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

13

Veterinary Medicine, Washington State University, Washington 99164, USA

14 15

The authors declare no competing financial interest.

16 17

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

18 19

Category: Bioactive Constituents, Metabolites, and Functions

ACS Paragon Plus Environment

1


Page 2 of 31

20

ABSTRACT

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35 36

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

37

phytoestrogens, bone, breast cancer, uterus


2

Page 3 of 31


38

INTRODUCTION

39

Estrogens are steroid hormones that play important roles in growth and

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

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

49

received increased investigative attentions. They induce responses in various tissues

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

Phytoestrogens are the naturally occurring plant chemicals that exhibit biological

58

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

59

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

60

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


3


Page 4 of 31

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

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

69

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

70

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

71

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

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

selective manner.


4

Page 5 of 31


84

MATERIALS AND METHODS

85

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

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

Park, NC, USA).20

100

Cell cultures

101

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

102

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

103

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

104

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

105

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

106

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


5


Page 6 of 31

107

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

108

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

109

Transactivation Assay for ER Activity.

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

transfected with pcDNA3.1 vector alone.

118

Cell proliferation assessment

119

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

120

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

121

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

122

(Invitrogen, MA, USA).

123

Western blot analysis

124

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

125

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

126

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

127

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

128

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


6

Page 7 of 31


129

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

130

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

131

Total RNA isolation and Real-time PCR

132

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

133

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

134

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

135

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

136

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

137

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

138

The primers used are present in Supplementary Table 1.

139

Alkaline phosphatase (ALP) assay

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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


7


Page 8 of 31

152

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

153

de-ionized water and observed under the light microscope.

154

Animals and uterotrophic assay

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

standard histological procedure.

167

Statistical analysis

168

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

169

p

Selective Estrogen Receptor Modulator (SERM)-like Activities of

Recommend Documents