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

Accepted Manuscript Genistein modulates MMP-26 and estrogen receptor expression in endometrial cancer cells Xiaoli Liu, Xiaoou Xue, Hao Wang, Xiaomei Xu, Dong Zhi PII: S2095-7548(16)30227-7 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 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Genistein modulates MMP-26 and estrogen receptor expression in endometrial cancer cells RI PT Xiaoli Liu, XiaoouXue*, Hao Wang, Xiaomei Xu Dong Zhi Men Hospital of Beijing University of Chinese Medicine, Beijing 100700, SC China * Email:xxo123@sina.com (X Xue) AC C EP TE D Tel: 8610-84013150 M AN U Corresponding Author ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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 TE D treatment in low E2 concentrations Conclusions: Genistein showed estrogen-like effects in endometrial cancer cells and influenced estrogen receptor signaling by EP modulating ERα and ERβ expression AC C Keywords: genistein; endometrial disease; estrogen receptor; MMP-26; reporter gene ACCEPTED MANUSCRIPT 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 endometrial cancer, regulated by estrogen.2-3 Chinese medicine is RI PT 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 SC as weak hormones, which could allow bidirectional regulation of estrogen signaling in patients Phytoestrogens may stimulate the effect of estrogen when low level estrogen M AN U 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 TE D 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 EP 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) AC C 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 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ERα and ERβ luciferase reporter genes ACCEPTED MANUSCRIPT Materials and methods Materials 1.1 Reagents Mouse monoclonal anti-human MMP-26 antibody was purchased from Abcam RI PT (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 SC (31985-070; Carlsbad, CA), and Lipofectamine 2000 Reagent from Invitrogen (11668-019; Carlsbad, CA).The luciferase positive control (Quantilum Recombinant M AN U 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 TE D 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 EP 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 AC C 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) ACCEPTED MANUSCRIPT 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 RI PT atmosphere at 37°C Cells were washed times with PBS 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 SC Ishikawa cells were selected and cultured in phenol-red-freeOpti-MEM containing 10% CDT-FBS for days, then treated with Opti-MEM containing vehicle, M AN U 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 TE D HEC-1B cells in logarithmic growth were seeded in 24-well plates at 4.0ì108 cells/L (500 àL per well) in antibiotic-and phenol-red-free NEAA medium containing 10% CDT-FBS 24 hours before transfection After 24 hours, cells were transfected EP 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 AC C tube and Lipofectamine 2000 to the other All tubes were incubated for 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 hours, genistein or vehicle alone (0.1% DMSO), was added for 24h ours Cells were lysed for minutes at room temperature in 150 µL 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 ACCEPTED MANUSCRIPT 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 RI PT 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 AC C EP TE D M AN U SC (SPSS, Chicago, IL) Significant difference was defined as P