genistein modulates mmp 26 and estrogen receptor expression in endometrial cancer cells

Genistein modulates MMP-26 and estrogen receptor expression in endometrialcancer cells Xiaoli Liu, Xiaoou Xue, Hao Wang, Xiaomei Xu, Dong Zhi DOI: 10.1016/j.jtcms.2016.12.008 Reference:

Genistein modulates MMP-26 and estrogen receptor expression in endometrial

cancer cells

Xiaoli Liu, Xiaoou Xue, Hao Wang, Xiaomei Xu, Dong Zhi

DOI: 10.1016/j.jtcms.2016.12.008

Reference: JTCMS 97

To appear in: Journal of Traditional Chinese Medical Sciences

Received Date: 30 December 2016

Accepted Date: 30 December 2016

Please cite this article as: Liu X, Xue X, Wang H, Xu X, Zhi D, Genistein modulates MMP-26 and

estrogen receptor expression in endometrial cancer cells, Journal of Traditional Chinese Medical Sciences (2017), doi: 10.1016/j.jtcms.2016.12.008.

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Genistein modulates MMP-26 and estrogen receptor

expression in endometrial cancer cells

Xiaoli Liu, XiaoouXue*, Hao Wang, Xiaomei Xu

Dong Zhi Men Hospital of Beijing University of Chinese Medicine, Beijing 100700, China

*

Corresponding Author

Email:xxo123@sina.com (X Xue)

Tel: 8610-84013150

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Abstract

Objective: To clarify the mechanism of the estrogen-like effect of genistein by

observing the expression of Matrix metalloproteinases-26 (MMP-26) and ERα in

human endometrial cancer cells (Ishikawa and HEC-1B) in vitro Methods: The

effect of genistein on MMP-26 and ERα expression was examined by western blot in cultured Ishikawa and HEC-1B cells Additionally, the effects of genistein on ERα-ERE-luc and ERβ-ERE-luc reporter gene expression in HEC-1B cells were

analyzed by luciferase activity assays Results: MMP-26 and ERα protein expression

was down-regulated by genistein treatment in Ishikawa cell induced by high concentration E2, whereas MMP-26 and ERα protein expression was up-regulated by genistein treatment in Ishikawa cells induced by low concentration E2 Expression of the ERα-ERE-luc and ERβ-ERE-luc reporter genes was significantly increased after

E2 induction and was further up-regulated by genistein Expression of ERα-ERE-luc and ERβ-ERE-luc reporter genes decreased significantly following genistein treatment in high E2 concentrations, and increased significantly following genistein treatment in low E2 concentrations Conclusions: Genistein showed estrogen-like

effects in endometrial cancer cells and influenced estrogen receptor signaling by modulating ERα and ERβ expression

Keywords: genistein; endometrial disease; estrogen receptor; MMP-26; reporter gene

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Introduction

Endometrial cancer has become the most common gynecological tumor in developed countries, and its incidence is increasing.1 Nearly 80% of newly diagnosed cases were type 1 endometrial cancer, regulated by estrogen.2-3 Chinese medicine is widely used for the prevention and treatment of menopausal symptoms and endometrial diseases,4 and some clinical effects are related to the estrogen-like activities of chemical components in herbs, which are called phytoestrogens Compared with estrogen, phytoestrogens have lower estrogen receptor affinity and act

as weak hormones, which could allow bidirectional regulation of estrogen signaling in patients Phytoestrogens may stimulate the effect of estrogen when low level estrogen

is present, whereas they may be inhibitory when the level of estrogen is high.5Genistein is an isoflavone and phytoestrogen present in significant quantities in soybean, which is extensively used as a source of dietary protein across the globe.6Genistein has been shown to prevent cancer cell proliferation, and its regulation of the estrogen receptor has attracted significant attention.7-8

Matrix metalloproteinases (MMPs) play an important role in cancer development; MMP-26, known as endometase or matrilysin-2, is a member of the MMP family9that

is expressed in normal endometrial epithelial tissue and endometrial, breast and prostate cancers.10-12In a variety of malignant tumors, such as endometrial, breast and lung, MMP-26 expression is closely related to the infiltration and diffusion tumor cells Some studies have shown that MMP-26 participates in estrogen receptorβ (ERβ) hydrolysis, which can lead to improved prognosis and prolonged survival in breast cancer patients.13-15 Additionally, MMP-26 and ER have similar promoters16; therefore, we hypothesized that different coordinate changes in MMP-26 and ER expression may occur in endometrial cancers of different pathological stage, and studied the effect of genistein by evaluating expression of both genesin cells treated with the drug.17

In this study, MMP-26 and ERα expression in endometrial cancers of different pathological stages were observed In cell systems, the effect of genistein on MMP-26 and ERα protein expression was documented, as well as its effect on the expression of

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ERα and ERβ luciferase reporter genes

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Materials and methods

1 Materials

1.1 Reagents

Mouse monoclonal anti-human MMP-26 antibody was purchased from Abcam (ab39146; Cambridge, UK), and rabbit monoclonal anti-human ERα antibody from Santa Cruz Biotechnology (SC8002; Santa Cruz, CA).Fetal bovine serum(FBS) and estrogen-free FBS (CDT)were purchased from Hyclone (SH30070.03 and SH30068.01, respectively; South Logan, UT) Opti-MEM was purchased from Gibco (31985-070; Carlsbad, CA), and Lipofectamine 2000 Reagent from Invitrogen (11668-019; Carlsbad, CA).The luciferase positive control (Quantilum Recombinant Luciferase), Steady-Glo Luciferase Assay System and β-gal assay kit were purchased from Promega (E1701, E2510 and E2000, respectively; Madison, WI)

Genistein was provided by the Cellular and Biochemical Lab, Beijing University

of Chinese Medicine The purity was 99.8%, and the molecular weight was 270.2 Genistein was dissolved in ethanol, and the final ethanol concentration was between 0.1% and 1% in all experiments According to the results of previous experiments, 40×10-6mol/L genistein was chosen for all experiments, and the treatment duration was 48 hours

1.2 Plasmids

The β-gal-control plasmid (pβ-gal-control) was purchased from Clontech (Mountain View, CA) Five ERE elements were inserted into pTAL-lucto construct the recombinant vector, which was synthesized by Beijing Di’an Biological Ltd (Beijing, China) pCXN2-hERα and pCXN2-hERβ were generously provided by Doctor Satoshi Inoue, from the Department of Geriatric Medicine, University of Tokyo (Japan)

1.3Cells

The human endometrial cancer cell lines Ishikawa (expressing estrogen and progesterone receptors) and HEC-1B (negative for estrogen and progesterone receptor expression) were purchased from the Cell Experiment Center, School of Basic Medicine, Peking Union Medical University (Beijing, China)

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2 Methods

2.1 Cell culture

Ishikawa cells were cultured with high glucose-DMEMcontaining10% FBS, and HEC-1B cells were cultured with NEAA medium containing 10% FBS, in a 5% CO2

atmosphere at 37°C Cells were washed 2 times with PBS 4 days before experiments, and then cultured in phenol-red-free Opti-MEM containing 10% CDT-FBS to consume residual estrogen in the cell

2.2 Western blotting

Ishikawa cells were selected and cultured in phenol-red-freeOpti-MEM containing 10% CDT-FBS for 4 days, then treated with Opti-MEM containing vehicle,

10-6 mol/L E2, 10-8 mol/L E2, 80×10-6 mol/Ldaidzein+10-6 mol/L E2, 80×10-6 mol/L daidzein+10-8 mol/L E2, 40×10-6 mol/Lgenistein +10-6 mol/L E2, or40×10-6 mol/L genistein+ 10-8 mol/L E2 After 48 hours, cells were collected, and ERα and MMP-26 protein expression was measured by western blot

2.3 ER reporter luciferase activity assays

HEC-1B cells in logarithmic growth were seeded in 24-well plates at 4.0×108 cells/L (500 µ L per well) in antibiotic-and phenol-red-free NEAA medium containing 10% CDT-FBS 24 hours before transfection After 24 hours, cells were transfected with the indicated plasmids Briefly, 50 µL serum- and antibiotic-free Opti-MEM was added to two different tubes per treatment Then, 0.8 µg pERE-TAL-luc, 0.1 µg β-gal-control, 0.1 µg pCXN2-hERα or 0.1µg pCXN2-hERβ DNA was added to one tube and Lipofectamine 2000 to the other All tubes were incubated for 5 min and combined (DNAs to liposomes, respectively); the ratio of liposomes to medium was 2.8:100 After 20 minutes incubation at room temperature, DNA-liposome mixtures were added to HEC-1B cells, mixed well and placed at 37°C After 6 hours, genistein

or vehicle alone (0.1% DMSO), was added for 24h ours Cells were lysed for 5 minutes at room temperature in 150 µ L lysis buffer (supplied with kit) Luciferase activity was measured according to the manufacturer’s instructions using 100 µL of cell lysate β-gal activity was measured according to the instructions supplied with the kit using 50 µL of cell lysate Standard luciferase activities from different samples

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were calculated according to A420 values which were read by ELISA; reported luciferase activity values are the intensity values/A420 values For each experiment, treatments were performed in triplicate, and all experiments were independently repeated three times

2.4 Statistical analysis

All results are presented as mean (SEM), and all data were analyzed by one-way analysis of variance (ANOVA) Multiple comparisons were made with the least significant difference post-hoc test All analyses were conducted using SPSS v17.0

(SPSS, Chicago, IL) Significant difference was defined as P<.05

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Results

1 Synchronous changes in ERαand MMP-26 expression in Ishikawa cells

Compared with the control group, ERα and MMP-26 expression decreased after 48

h treatment with 40×10-6 mol/L genistein and high concentration E2(10-6 mol/L; E2-H)

(P<.01) However, ERα and MMP-26 expression increased after 48 h treatment with

40×10-6 mol/L genistein and low concentration E2 (10-8 mol/L; E2-L), compared with the E2-L group (P 01) Finally, compared with the E2-H group, ERα and MMP-26 expression decreased after 48 hours treatment with 40×10-6 mol/L genistein and E2-H

(P 01) (Fig.1, Fig 2 and Table 1)

Control E2-H E2-L D+E2-H D+E2-L G+E2-H G+E2-L

ERα

β-Actin

A B C D E F G

Figure 1ERα expression in Ishikawa cells treated withE 2 and daidzein(D)orgenistein (G).Control:

E2(−), genistein (−),daidzein(−); E2-H: 10-6 mol/L E2; E2-L: 10-8 mol/L E2; D+E2-H: 80×10-6

mol/Ldaidzein+ 10-6 mol/L E2; D+E2-L: 80×10-6 mol/Ldaidzein+ 10-8 mol/L E2; G+E2-H: 40×10-6 mol/L genistein+10-6 mol/L E2; G+E2-L: 40×10-6 mol/L genistein + 10-8 mol/L E2.

Control E2-H E2-L D+E2-H D+E2-L G+E2-H G+E2-L

MMP-26

β-Actin

A B C D E F G

Figure 2 MMP-26 expression in Ishikawa cells treated with E2 and daidzein (D), or genistein (G).Control: E2 (−), genistein (−),daidzein (−); E2-H: 10-6mol/L E2; E2-L: 10-8mol/L E2; D+E2-H: 80×10-6 mol/Ldaidzein + 10-6 mol/L E ; D+E -L: 80×10-6 mol/Ldaidzein + 10-8mol/L E ; G+E -H:

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40×10-6 mol/L genistein + 10-6 mol/L E2; G+E2-L: 40×10-6 mol/L genistein + 10-8 mol/L E2.

Table 1ERα and MMP-26 protein expression in Ishikawa cells after genistein and E 2 treatment

Genistein + E 2 -H 40×10-6+10-6 0.1152(0.0155)

*

0.2608(0.0217)* Genistein + E 2 -L 40×10-6+10-8 0.9417(0.0528)

#

0.9116(0.0413)#

Note: All data are presented as mean (SEM), and n=3

For statistical analysis of ER-α/β-actin and MMP-26/β-actin: Genistein+E2-H vsE2-H

*P<.01 and Genistein +E2-L vsE2-L, #P<.01.

2 Two-way-competition regulation of endometrial estrogen receptor expression

HEC-1B cells were transfected with pβgal-control, pTAL-ERE-luc, pCXN2-hERα orpCXN2-hERβ, and then induced with high (10-6mol/L) or low (10-8 mol/L) concentration E2, and 40×10-6 mol/L genistein for 48 hours The luciferase activity results showed that E2and genistein increased luciferase reporter gene activity; however, the induction capacity of genistein was lower than estrogen (Table2)

Table 2 The effect of estrogen on ERE-mediated luciferase activity

Group

Concentration (mol/L)

Luciferase activity (Intensity/A 420 )

Control 0 4110 (83.90) 3066(46.21)

E2-H 10-6 12367(817.8)* 10472(43.32)*

E2-L 10-8 22562(469.4)* 15119(236.6)*

Genistein 40×10-6 9970.9(552.8)* 13565.5(516.6)*

Note: All data are presented as mean (SEM) For statistical analysis all values are compared with Control, *P<0.01

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To observe differences in luciferase reporter activity attributable to ERα and ERβ that were affected by different treatments, the mean luciferase activity values were calculated for ERα and ERβ respectively and then the values of the control group from corresponding drug concentrations were subtracted to obtain values of the change in luciferase reporter activity for ERα and ERβ, respectively Comparing the changed luciferase values with the different effect of drug concentration, we calculated the difference in activity for E2 and genistein with regard to ERα and ERβ After estrogen treatment at a concentration of 10-6and 10-8mol/L, the change in

ERα luciferase activity was significantly higher than ERβ (P<.01), which indicated

that the reporter activity mediated by ERα was stronger than by ERβ Compared with the E2-H and E2-L groups, luciferase activities in the ERα genistein group were

significantly decreased (P<.01) Furthermore, the change in ERβ luciferase activity was remarkably higher than in ERα (P<.01), which indicated that in the genistein

group ERβ-mediated reporter activity was stronger than ERα-mediated activity (Table 3)

Table 3The effect of genistein on ER-α and ER-βERE-medicated luciferase activity

Group

Concentration (mol/L)

Change in luciferase activity

E2-H 10-6 8780(816.8)* 6894(13.44)*#

E2-L 10-8 18965(469.4)* 11530(235.6)*#

Genistein 40×10-6 6387.5(752.9)*▲ 9982.3(1580.4)*▲#

Note: All data are presented as mean (SEM) For statistical analysis values compared with Control,

*P<.01; compared with E2-L, ▲P<.01; intra-group comparison of ERα and ERβ, #P<.01.

Together with high concentration E2 treatment, ERα and ERβ luciferase activities

were significantly decreased by genistein treatment (P<.01), and with low