Glycyrrhizin Attenuates the Process of Epithelial-to-Mesenchymal

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Bioactive Constituents, Metabolites, and Functions

Glycyrrhizin attenuates the process of epithelial-tomesenchymal transition by modulating HMGB1 initiated novel signaling pathway in prostate cancer cells Heng-Yu Chang, Sheng-Yi Chen, Chi-Hao Wu, Chi-Cheng Lu, and Gow-Chin Yen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00251 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 6, 2019

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Journal of Agricultural and Food Chemistry

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Glycyrrhizin attenuates the process of epithelial-to-mesenchymal

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transition by modulating HMGB1 initiated novel signaling pathway

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in prostate cancer cells

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Heng-Yu Chang †,, Sheng-Yi Chen †,, Chi-Hao Wu ‡, Chi-Cheng Lu #,

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Gow-Chin Yen †,,§,*

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Department of Food Science and Biotechnology, National Chung Hsing

†

University, 145 Xingda Road, Taichung 40227, Taiwan

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Department of Human Development and Family Studies, National

‡

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Taiwan Normal University, 162, Section 1, Heping E. Road, Taipei City

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106, Taiwan

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# Department

16, Sec. 1, Shuang-Shih Rd., Taichung City 40404, Taiwan

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of Sport Performance, National Taiwan University of Sport

Graduate Institute of Food Safety, National Chung Hsing University,

§

145 Xingda Road, Taichung 40227, Taiwan

16 17

These authors contributed equally to this work.

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

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*Corresponding author:

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Gow-Chin Yen, PhD

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Phone: 886-4-2287-9755. Fax: 886-4-2285-4378.

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E-mail: [email protected]

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Running title: Glycyrrhizin diminishes HMGB1 initiated EMT process

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ABSTRACT:

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High mobility group box 1 (HMGB1) is upregulated in nearly every tumor

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type. Importantly, clinical evidences also prop that HMGB1 is particularly

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increased in metastatic prostate cancer patients. Besides, a growing number

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of studies highlighted that HMGB1 could be a successful therapeutic target

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for prostate cancer patients. Glycyrrhizin is a novel pharmacological

7

inhibitor of HMGB1 that may repress prostate cancer metastasis. This

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research was aimed to investigate the effect of glycyrrhizin on inhibition

9

of HMGB1-induced epithelial-to-mesenchymal transition (EMT), a key

10

step of tumor metastasis, in prostate cancer cells. In this study, HMGB1

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knock-downed DU145 prostate cancer cells were used. Silencing the

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HMGB1 gene expression triggered a change of cell morphology to a more

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epithelial-like shape which was accompanied by a reduction of Cdc42

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/GSK-3β/Snail and induction of E-cadherin levels estimated by

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immunoblotting. Furthermore, HMGB1 facilitated cell migration and

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invasion via downstream signaling whereas HMGB1 targeting by 10 mM

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ethyl pyruvate effectively inhibited EMT characteristics. Interestingly, cell

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migration capacity induced by HMGB1 in DU145 cells was abolished in a

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dose dependent effect of 25-200 M glycyrrhizin treatment. In conclusion,

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glycyrrhizin successfully inhibited HMGB1-induced EMT phenomenon

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suggesting that glycyrrhizin may serves as a therapeutic agent for

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metastatic prostate cancer.

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KEYWORDS: Glycyrrhizin, HMGB1, EMT, Prostate cancer, E-

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cadherin

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INTRODUCTION

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Prostate cancer is the most common type of malignancy cancers and is the

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third leading cause of death in global countries. Approximately 1.3 million

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new patients and 359,000 associated deaths of prostate cancer worldwide

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in 2018 that indicates the highly association of incidence and mortality of

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cancer among men.1, 2 Although prostate cancer is commonly diagnosed

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cancer in the world that 5-year relative survival rate beyond the local or

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localized stage prostate cancer is nearly 100%, while that for matastatic

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prostate cancer is only 29%.3 Besides, metastatic prostate cancer,

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particularly to the lung and liver, may results in high mortality rates in

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patients. Therefore, it is necessary to uncover the underlying mechanisms

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of metastatic prostate cancer to improve the therapeutic capacity.

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High mobility group box 1 (HMGB1) protein was disclosed in calf

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thymus in 1973, which is a non-histone fraction of ubiquitous chromatin

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presented in nucleated mammalian cells that regulates gene expression and

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nucleosome stability.4 Overexpression of HMGB1 is related with each

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hallmarks of cancer including cell proliferation, sustained angiogenesis,

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evasion of apoptosis, energy metabolism, inflammation environment, and

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tissue invasion and metastasis.5, 6 Furthermore, co-expression of receptor

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for advanced glycation end products (RAGE), receptor engage with

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HMGB1, and HMGB1 is associated with poor prognosis of prostate cancer

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that is believed HMGB1 may play a critical role of clinical impact on

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prostate cancer.7 Indeed, abundant expression of HMGB1 has been

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invsetegated not only in prostate cancer cell lines (LNCaP, PC3 and

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DU145) but also increased in prostate tumor compared with normal

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prostate tissue that exhibited HMGB1 extremely associated with tumor

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progression and poor patient prognosis of prostate cancer.7-10

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Tumor progression from normal cell to malignant invasive carcinoma 3 ACS Paragon Plus Environment


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goes through several stages. Epithelial-to-mesenchymal transition (EMT),

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the key feature of tumor metastasis, is a dynamic process that regulates

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epithelial cells losing their polarity and detaching from the basement

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membrane to establish another tumor colonies at distant sites. The dynamic

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process of EMT to undergo multiple biological changes that authorize it to

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assume a mesenchymal cell phenotype, enhanced apoptosis resistance, cell

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differentiation, cell migratory capacity, invasiveness, and promoted

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production of extracellular matrix (ECM) components.11 The switch of

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EMT in cell morphology and behavior alter is mediated by the signaling

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networks, including the receptor tyrosine kinases (RTKs), transforming

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growth factor-beta (TGF-, Notch, Wnt, tumor necrosis factor-alpha

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(TNF-, and bone morphogenetic proteins (BMPs) pathways and

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downstream key transcription factor of Snail. Snail interacts with

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epigenetic remodeling complexes and several corepressors to control EMT

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process by repressing specific target genes, such as the E-cadherin gene.12,

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13

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in FaDu hypopharyngeal carcinoma cells through decreased Snail

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expression and upregulation the level of epithelial cadherin (E-cadherin).14

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Moreover, epithelial cells losing their polarity appertain to EMT process is

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regulated by Ras homolog gene family, member A (RhoA)/ cell division

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control protein 42 (cdc42) and downstream glycogen synthase kinase-3

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(GSK-3) kinase.15 Additionally, GSK-3 has been demonstrated to

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regulate Snail stabilization during EMT process in gastrointestinal and

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colorectal cancer.16, 17 However, there is a knowledge gap of HMGB1 in

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regulating EMT process through GSK-3/Snail/E-cadherin axis in prostate

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cancer. Therefore, to reveal the underlying mechanisms of HMGB1

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initiated signaling in regulating EMT process of prostate cancer may

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advance the therapeutic capacity.

Previously study showed that HMGB1 attenuates TGF--induced EMT


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Currently, several ways for prostate cancer treatment such as

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radiotherapy,

chemotherapy,

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deprivation therapy are used to manage this disease effectively.18-21 Herein,

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we focus on future challenges in the handle for metastatic prostate cancer,

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which continues to be connected with a high mortality despite multiple

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methods have been used. The root or extract from licorice plant or

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glycyrrhizin (GR) are spacious used to be a flavor enhancer and surface-

8

active agent in foods including bread, cakes, drinks, non-alcoholic

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beverages, chewing gum, candies, seasonings, herbs, vegetarian protein

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products, vitamin or dietary amino acid supplements, and other foods.22

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Licorice is also frequently used in Traditional Chinese medicine (TCM)

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and alternative medicine in enhancing health benefits.23 Besides,

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antioxidant constituent from the roots and stems of licorice are found to

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prevent colon and lung tumors.24 GR, a natural compound extracted from

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the roots of Glycyrrhiza glabra (Licorice), has anti-inflammatory and

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antiviral activities as well as a novel pharmacological inhibitor of

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HMGB1.25-27 Increasing evidence displayed GR suppresses cell growth by

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downregulating HMGB1 level in both human lung cancer and mouse

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melanoma.25,

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migration and invasion abilities of human gastrinoma cells via the

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ROS/PKC-/ERK pathway.29 Noteworthily, our previously results

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demonstrated that glycyrrhizic acid or 18β-glycyrrhetinic acid significantly

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prohibited the amassment of HMGB1 in cisplatin-induced nephrotoxicity

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of BALB/c mice.30 Nevertheless, it remains unknown the role of GR in

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HMGB1-mediated EMT process in prostate cancer. On this basis, we aim

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to investigate whether GR abolish HMGB1-mediated EMT process

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through GSK-3/Snail/E-cadherin signaling pathway.

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radical

prostatectomy,

or

androgen

Also, glycyrrhetinic acid has been showed that repress

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MATERIALS AND METHODS

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Materials. RPMI 1640 medium, trypsin-EDTA and penicillin-

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streptomycin antibiotic were obtained from Invitrogen (Carlsbad, CA,

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USA). Polyethylene glycol sorbitan monolaurate (tween 20), bovin serum

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albumin

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trishydroxymethylaminomethane (Tris), sodium dodecyl sulfate (SDS), -

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actin antibody, GR and ethyl pyruvate (EP) were obtained from Sigma-

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Aldrich Co. (St. Louis, MO, USA). GSK3 antibody, phospho-

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GSK3 antibody, Snail antibody, PI3K antibody, phospho-PI3K antibody,

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AKT antibody, phospho-AKT antibody, Rac1/cdc42 antibody, phospho-

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Rac1/cdc42 antibody, ERK1/2 antibody, phospho-ERK1/2 antibody, JUN

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N-terminal kinase (JNK) antibody, phospho-JNK antibody, P38 antibody,

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and phospho-P38 antibody were purchased from Cell signaling technology

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(Beverly, MA, USA). E-cadherin antibody, phospho-P65 antibody, and

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Vimentin antibody were obtained from Santa Cruz Biotechnology (Dallas,

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Texas, USA). HMGB1 antibody was obtained from Novus Biologicals

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(Littleton, Colorado, USA). Grams crystal violet solution was purchased

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from Merck Co. (Kenilworth, NJ, USA). RIPA lysis buffer, ECL Detection

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System, and Polyvinylidene difluoride (PVDF) membrane were purchased

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from Millipore (Billerica, MA, USA). Culture insert was purchased from

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ibidi Applied BioPhysics (Troy, NY, USA). Transwell® Permeable was

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purchased from Corning (Lowell, MA, USA). Human IL-6, IL-8 and TGF-

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 ELISA kit were purchased from R&D system (Minneapolis, MN, USA).

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MG132 proteasome inhibitor was purchased from Apex Biotechnology Co.

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(Hsinchu, Taiwan)

(BSA),

Tetramethylethylenediamine

(TEMED),

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Cell Lines and Maintenance. The human prostate cancer cell line

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(DU145) was purchased from the Bioresource Collection and Research

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Center (Hsinchu, Taiwan). HMGB1 stable knockdown DU145 cells were 6 ACS Paragon Plus Environment

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kindly provided by Dr. Chi-Hao Wu (National Taiwan Normal University,

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Taiwan). Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad,

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CA, USA) supplemented with 10% v/v fetal bovine serum (FBS) and 1%

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penicillin-streptomycin in a 5% CO2 incubator at 37°C.

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Cell Viability Assay. 2 × 104 cells/well were plated in 96-well

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microtiter plates incubated at 37°C overnight. DU145 parental and

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HMGB1-L cells were treated with 50, 100, and 200 M GR for 24 h. After

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treatment, the cell viability was measured according our previously

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article.31

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Cell Migration Assay. To assess cell motility, a wound-healing assay

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was performed. Culture inserts were put in twelve-well plates and then 4 ×

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104 cells were seeded into the chambers. After cell attachment overnight at

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37°C, culture-inserts were gently removed to form the cell-free gap. Cell

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was washed with PBS for debris removing, and the cells were exposed to

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various concentrations of GR or EP, respectively. Cell migration distance

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was recorded using a microscope (PowerIX71, OLYMPUS, Osaka, Japan)

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at various time points.

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Cell Invasion Assay. Cell invasion ability was measured by using

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Corning transwell inserts with 8.0 m pore polycarbonate membrane. The

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matrigel was coated on the upper chambers of 8.0 m pore polycarbonate

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membrane. Cell loading number and the measure method were following

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our previously report.31

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Reverse Transcription-Polymerase Chain Reaction (RT-PCR).

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Total RNA was prepared using TRIzol (Invitrogen, Carlsbad, CA, USA)

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according to the manufacturer's instructions. Then, cDNA products were

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generated and amplified by RT-PCR. The following sense and antisense

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primer sequences were used for RT-PCR analysis: E-cadherin: 5’-

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ACAGGATGGCTGAAGGTGACAG-3’ (forward) and5’7 ACS Paragon Plus Environment


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CTCAGGATCTTGGCTGAGGATG-3’ (reverse); -catenin: 5’-

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GGTTCACCTGTGACTCCTGTTG-3’ (forward) and5’-

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GTGCCTCAGCAATCCCTTTCTC-3’ (reverse); 18S: 5’-

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GTAACCCGTTGAACCCCATT-3’ (forward) and5’-

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CCATCCAATCGGTAGTAGCG-3’ (reverse). The PCR products were

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analyzed by electrophoresis on a 1.8% agarose gel and observable via

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SYBR® Safe DNA gel staining (Invitrogen, Carlsbad, CA, USA).

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Western Blot Analysis. Western blotting was performed according

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previously reported.31 Briefly, cells were lysed by ice-cold RIPA lysis

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buffer containing 1X Protease inhibitor. The protein concentration was

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calculated with the Bio-Rad DC protein assay kit. Total proteins (50 g)

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were separated by 8-12% SDS-PAGE and then transferred onto a PVDF

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membrane. After antibody hybridization, the protein band was visualized

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using the ECL Detection System (Millipore, Billerica, MA, USA).

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Immunofluorescence (IFC) Analysis. IFC staining of HMGB1, E-

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cadherin, and vimentin was performed according to a previous method.32

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Briefly, cells were fixed by 2% paraformaldehyde and permeabilized with

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0.1% Triton X-100. Then, cells were washed, blocked, and subsequently

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incubated with antibody (1:500) at room temperature for 2 h. The cells

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were then washed in PBS and stained with DAPI to counterstain the DNA.

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The cells were captured by Olympus fluorescence microscope (Osaka,

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Japan).

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Statistical Analysis. The results are expressed as the mean ± SD.

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Each experiment was performed in triplicate. The statistical analysis was

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performed using SAS software, and the variances were assessed according

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to the ANOVA procedure. Significant differences (p < 0.05) between the

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means were identified by Duncan’s multiple range tests.


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RESULTS

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Knocking Down HMGB1 Gene. To examine the knock-down

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efficiency of HMGB1 gene in DU145 prostate cells, protein expression

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was measured by western blot analysis and immunofluorescence staining,

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respectively (Fig. 1). Both cytoplasmic and nuclear HMGB1 protein

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expression was decreased in HMGB1 knock-downed DU145 prostate

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cancer cells (denoted as L cells) compared with DU145 parental cells

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(denoted as P cells) (Fig. 1A, B). HMGB1 protein may translocate from

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the nucleus to the cytoplasm and secrete into the medium. Further to test

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the secreted HMGB1 protein level from culture medium, about 3-folds

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reduction of HMGB1 protein production from L cells conditioned medium

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compared that with P cells (Fig. 1C). Previously study manifested that

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serum integrants bind to HMGB1 may meddle with its detection.33 To rule

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out this possibility, we treated cell with serum starvation culture condition,

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and conformed the HMGB1 protein expression was still abolished in L

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cells (Fig. 1D-F). On the other hand, constitutive knockdown of gene

17

expression is not durable by cells for a long time.34, 35 The HMGB1 protein

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expression was analyzed at different times, included 3rd, 6th, and 12th cell

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generation (Fig. 1G, H). HMGB1 protein stable knockdown cell lines, cell

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generation from 3rd to 12th, were be used in following experiments to

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eliminate the need for multiple times of transfection and steadily increasing

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reproducibility of results. In present study, we knocked down the HMGB1

23

gene of DU145 cells and found the cell shape changed to a more epithelial-

24

like morphology (Fig. 1I).

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Modulation of EMT Progression by HMGB1 Downstream

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Signaling through Cdc42, GSK-3β, Snail, and E-cadherin Kinases. The

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varied cell morphology of L cells elicited us to investigate the impact of

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HMGB1 on the EMT-signaling cascade transduction. The PI3K, AKT, 9 ACS Paragon Plus Environment


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ERK, MAPK, p38 and JNK signaling pathways have been showed that

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involved in EMT progression.13 Besides, ethyl pyruvate (EP), a aliphatic

3

ester from pyruvic acid, has already showed to be an effectual HMGB1

4

inhibitor in suppressing tumor growth.36,

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HMGB1-related epithelial-like morphology is mediated by PI3K, AKT,

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ERK, p38 and JNK kinases, we explored kinase activities in L cells and

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pharmacological inhibition of parental cells (denoted as EP cells),

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respectively. As shown in Fig. 2A, both L cells and EP treated cells had

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alike phosphorylation activities of PI3K, AKT, ERK, p38 and JNK kinases

10

compared with that of the corresponding controls, suggesting these kinases

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could not be involved in HMGB1 initiated EMT-relative signaling pathway.

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As mentioned earlier, epithelial cells losing their polarity is regulated

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by RhoA/cdc42 and downstream GSK-3 kinase in EMT process.15

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Further investigation to uncover whether HMGB1-initiated EMT

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progression involves the Cdc42/GSK-3 signaling pathways. Under

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standard culture conditions, the phosphorylation of Cdc42 and GSK-

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3 kinases were significantly repressed by knocking-down HMGB1 gene

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expression or EP treatment compared with that of parental DU145 cells

19

(Fig. 2B). Moreover, knocking down of HMGB1 gene drastically

20

decreased the phosphorylation of GSK-3 kinase by about 4-fold

21

compared with the corresponding controls in serum-free condition (Fig.

22

2C).

37

To examine whether the

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Snail, a transcription factor, directly enhances the repression of the

24

adhesion molecule E-cadherin to regulate EMT process. On top of that,

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Snail stabilization during EMT process is regulated by GSK-3 in

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gastrointestinal and colorectal cancer.16, 17 To confirm the involvement of

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HMGB1 initiated signaling through Snail/E-cadherin axis in the

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modulation of EMT process, we estimated Snail gene expression by using 10 ACS Paragon Plus Environment

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western blot analysis. Knocking-down HMGB1 gene expression reduced

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nuclear Snail activity to 50% that of the parental cells in both complete

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medium and serum-free condition (Fig. 3A, B). Indeed, GSK-3

4

phosphorylates Snail that allows its export from the nucleus and resulting

5

in ubiquitination and degradation of Snail.38, 39 We hypothesized that Snail

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might be tightly controlled by the ubiquitin-proteasome pathway (UPP).

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Surprisingly, the level of Snail was markedly enhanced in cells which were

8

treated with 10 M MG132 proteasome inhibitor (Fig. 3C). To extend

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these observations, we found that Snail expression effectively reduced in

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L cells compared with P cells despite treatment with MG132 (Fig. 3C).

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There was a slight decrease of E-cadherin protein expression in L cells

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treated with 10 M MG132 compared with untreated L cells, revealing that

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HMGB1 dominated in EMT process through regulating Snail/E-cadherin

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signaling (Fig. 3C). Similarly, we observed abundantly heighten E-

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cadherin protein expression in HMGB1 gene knockdown cells (Fig. 4A,

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C). Furthermore, the pharmacological inhibition of HMGB1 gene

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expression by EP (10 mM) significantly increased E-cadherin protein

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expression by around 6.7-fold, indicating that E-cadherin directly regulated

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by HMGB1 (Fig. 4B).

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-catenin is an essential regulator of cadherin stability and cell-cell

21

adhesion.40, 41 Under serum free condition for a short time period (3 h, 6 h,

22

and 12 h) significantly elevated of -catenin and E-cadherin mRNA

23

expression at 6 h in HMGB1 gene knockdown cells compared that with

24

control cells (Fig. 4D). E-cadherin greatly increased in lower HMGB1

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gene expression prostate cancer cells (Fig. 4E, F). These data suggested

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that HMGB1 directly involved in EMT process by downstream

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cdc42/GSK-3/Snail/E-cadherin signaling pathway.

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Association of HMGB1 Gene with Cell Migration and Invasion. It 11 ACS Paragon Plus Environment


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has been observed that once increased E-cadherin expression during EMT

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inhibits cell migration and invasion.13, 42, 43 We thus considered whether the

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cell migration and invasion ability are associated with HMGB1 expression.

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Besides, we also confirmed cell motility by using HMGB1 inhibitor. Two

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complementary approaches were used, namely wound healing assay and

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cell invasion assay, to estimate the impacts of tumor HMGB1 on cell

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motility. In the cell migration assay, it took about 24 h to heal the scrape

8

made on DU145 parental cells. Under the same condition, lower HMGB1

9

gene expression cells showed about 70% heal at 24 h as well as EP

10

treatment (Fig. 5A, B). Likewise, the apparent results of reduced migration

11

ability by decreasing HMGB1 gene expression compared with

12

corresponding control cells in starvation condition (Fig. 5C, D). In addition,

13

genetic knockdown and pharmacological inhibition of HMGB1 gene

14

expression uniformly reduced cell invasion to about 50% versus parental

15

cells indicating that HMGB1is required for cell movement during EMT

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progression (Fig. 5E, F).

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Impaired EMT Progression by Glycyrrhizin Treatment.

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Previously evidence presented that GR is an inhibitor of HMGB1 protein

19

in anti-cancer therapy.25 Under GR treatment, there was no cytotoxic effect

20

observed between HMGB1 gene silencing cells (HMGB1-L cell group)

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and that corresponding control cells (Parental group) (Fig. 6A and B). As

22

mentioned above, E-cadherin directly regulated by HMGB1 (Fig. 4B). We

23

thus contemplated whether GR directly induce E-cadherin level and

24

suppress EMT progression in prostate cancer cells. GR treatment

25

significantly increased protein expression of E-cadherin in DU145 parental

26

cells but not in HMGB1 knockdown cells (Fig. 6C). Further to examine

27

cell migration ability, parental cells treated with GR showed in a dose

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dependent manner in inhibition of cell migration (Fig. 6D and E). Our 12 ACS Paragon Plus Environment

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results implied that EMT progression may restrain by GR treatment in

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prostate cancer.

3 4

DISCUSSION

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The evidence of HMGB1 facilitates tumor progression is now rapidly

6

accumulating. Overexpression of HMGB1 is allied with the hallmarks of

7

cancer including multiply indefinitely growth potential, angiogenesis

8

ability, escape from cell death, self-sufficiency of replication, resist

9

inhibitory signals of growth, immune manipulation, tissue invasion, and

10

metastasis.5, 6 The most common lead to cancer patient death are tumor

11

invasion and metastasis rather than the primary tumor itself. In clinical,

12

raising the level of HMGB1 is closely related to the invasive and metastatic

13

activity of cancers such as colon cancer and lung cancer indicates HMGB1

14

levels are highly associated with tumor size, clinical stage of cancer, lymph

15

node metastasis, and distant metastasis.44, 45 Clinical evidences support that

16

overexpression of HMGB1 is in prostate cancer patients and may serve as

17

a novel prognostic marker for biochemical recurrence-free survival after

18

radical prostatectomy.10 Up to the present, the underlying mechanisms of

19

HMGB1 initiated signaling in regulating EMT process is still lacking. In

20

the present study, we identified a HMGB1-associated signaling mechanism

21

for EMT which is a major process for successful metastasis. When

22

HMGB1 protein in DU145 prostate cancer cells was knocked down, it

23

shaped cell to a more epithelial-like morphology, resulting in suppressed

24

cdc42/GSK-3β/Snail cascades and drastically raised E-cadherin expression

25

which leading to hamper cell migration and invasion capacities.

26

Recent reports suggest inhibition of the HMGB1 suppresses

27

activation of PI3K, AKT, ERK, MAPK, p38 and JNK kinases which are

28

effector molecules linked to tumor proliferation, invasion, matrix 13 ACS Paragon Plus Environment


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metalloproteinases (MMPs) expression and metastases in human lung,

2

glioma, colon, and cervical carcinoma cell line.46,

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somewhat surprising that the phosphorylation of PI3K, AKT, ERK, MAPK,

4

p38 and JNK kinases were not significantly changed by knocking-down

5

HMGB1 gene or EP treatment in DU145 prostate cancer cell line implying

6

cell type-specific properties were existed (Fig. 2A).

47

By contrast, it is

7

During EMT, the crucial transcription factor of Snail which switches

8

cell behavior by perturbing of E-cadherin–mediated cell adhesion appear

9

and up-regulating expression of vimentin.48, 49 Although co-expression of

10

E-cadherin and vimentin has been revealed to promote tumor invasion and

11

metastasis of breast cancer, head-neck squamous cell carcinoma, non-

12

small-cell lung cancer, and oral squamous cell carcinomas.50-53

13

Nevertheless, western blot analysis and immunofluorescence staining

14

results showed that E-cadherin excessively increased but not vimentin in

15

HMGB1 knocked-down DU145 cells compared with control cells,

16

respectively (Fig. 4A, C). No changes of vimentin protein expression was

17

observed neither E-cadherin surface-negative prostate cancer cells (S-

18

DU145 and R-DU145) nor E-cadherin-positive expression prostate cancer

19

cells (T-DU145) that hinted E-cadherin is not interrelated to vimentin in

20

DU145 cells.54 Noticeably, loss of E-cadherin sustained prostate cancer

21

metastasis that considered to be the key step to initiate EMT.55, 56

22

GR is widely used in the food as a sugar substitute and as a sweetener

23

in tobacco industries.23,

24

inhibitor of HMGB1 that directly induced E-cadherin level and suppress

25

cell migration competency in prostate cancer cells. However, protein

26

expression of E-cadherin and migration ability were not able to influence

27

by glycyrrhizin treatment in HMGB1 knockdown cells (Fig. 6). Our

28

previously results exhibited that glycyrrhizic acid

57

Furthermore, GR is a novel pharmacological


or 18β-glycyrrhetinic

Page 15 of 35


1

acid significantly blocked the accumulation of HMGB1 in cisplatin-

2

induced nephrotoxicity of BALB/c mice.30 Here, GR treatment showed no

3

effect in HMGB1 knocked-down cells inferred that HMGB1 protein levels

4

fall below a certain threshold resulting in E-cadherin level and migration

5

capacity were not affected after GR treatment (Fig. 1 and Fig. 6).

6

In conclusion, this aberrant signal triggered EMT process through the

7

activation of HMGB1 initiated Rac1/Cdc42 /GSK-3β/Snail/E-cadherin

8

signaling cascade. Knockdown of gene expression, pharmacological

9

inhibition or GR treatment may result in suppression of HMGB1 and

10

subsequent deteriorated GSK-3β activation induced Snail degradation and

11

reduced. Snail degradation enhanced E-cadherin expression and alleviated

12

cell migration ability during EMT process (Fig. 7). Our results illustrated

13

that GR may block the process of EMT by modulating HMGB1 initiated

14

novel signaling pathway in prostate cancer cells.

15 16 17 18

Abbreviations

19

BSA, bovin serum albumin; BMPs, bone morphogenetic proteins; cdc42,

20

cell division control protein 42; DAPI, 4’,6-diamidino-2-phenylindole; E-

21

cadherin, epithelial cadherin; EMT, epithelial-to-mesenchymal transition;

22

EP, ethyl pyruvate; ECM, extracellular matrix; GSK-3 glycogen

23

synthase kinase-3 GR, glycyrrhizin; HMGB1, high mobility group box

24

1; IFC, immunofluorescence; RAGE, receptor for advanced glycation end

25

products; RhoA, Ras homolog gene family, member A; RTKs, receptor

26

tyrosine kinases; RT-PCR, Reverse transcription-polymerase chain

27

reaction; TCM, Traditional Chinese medicine; TGF- transforming

28

growth factor-beta; TNF- tumor necrosis factor-alpha. 15 ACS Paragon Plus Environment


1 2

Conflict of interests

3

The authors declare no conflict of interests.

4 5


Page 16 of 35

Page 17 of 35


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Figure legends

2 3

Figure 1. Alteration of cell morphology by knocking-down HMGB1

4

gene. The cellular HMGB1 protein levels under complete culture condition

5

were detected using (A) western blot analysis and (B) immunofluorescence

6

staining, respectively. Complete condition medium was concentrated with

7

Millipore centrifugal filter devices for HMGB1 protein detection (C). The

8

intracellular or extracellular HMGB1 protein levels under serum free

9

culture condition were detected (D-F). Cells were collected from 3rd, 6th

10

and 12th generation for HMGB1 protein analysis (G) and quantification (H).

11

Morphology of cells was recorded by inverted microscope (I).

12

Immunofluorescence staining for HMGB1 (red). Cell nuclei (blue) were

13

visualized by DAPI stain. Shown are the averages of three independent

14

experiments. *: p < 0.05 compared with control cells. (P, parental; L,

15

HMGB1-L)

16 17

Figure 2. Suppression of Rac1/cdc42 and GSK-3 phosphorylation by

18

knocking-down HMGB1 gene. Protein levels of PI3K, p-PI3K, AKT, p-

19

AKT, ERK, p-ERK, JNK, p-JNK, p38 and p-p38 were determined by

20

Western blot analysis (A). Rac1/cdc42 and GSK-3 protein levels were

21

detected under complete culture condition treatment with or without 10

22

mM ethyl pyruvate for 6 h by using western blot analysis (B). GSK-

23

3 protein level was detected under serum free culture condition treatment

24

with or without 10 mM ethyl pyruvate for 6 h by using western blot

25

analysis (C). Shown are the averages of three independent experiments. *:

26

p < 0.05 compared with control cells. (P, parental; L, HMGB1-L; EP, ethyl

27

pyruvate)



1

Figure 3. HMGB1 directly regulated EMT-relative Snail/E-cadherin

2

signaling pathway. Both cytoplasmic and nuclear Snail protein levels were

3

detected under complete culture condition treatment with or without 10

4

mM ethyl pyruvate for 6 h by using western blot analysis (A). Both

5

cytoplasmic and nuclear Snail protein levels were detected under serum

6

free culture condition (B). Cells were treated with or without 10 μM

7

MG132 (a protease inhibitor) for 6 h or 12 h to detect protein levels of snail

8

or E-cadherin, respectively (C and D). Shown are the averages of three

9

independent experiments. *: p < 0.05 compared with control cells. (P,

10

parental; L, HMGB1-L; EP, ethyl pyruvate)

11 12

Figure 4. E-cadherin gene expression directly regulated by HMGB1 in

13

DU145 cells. E-cadherin and vimentin protein expression measured by (A)

14

western blot analysis and (C) immunofluorescence staining, respectively.

15

E-cadherin protein levels were detected under complete culture condition

16

treatment with or without 10 mM ethyl pyruvate for 24 h (B). mRNA levels

17

of -catenin and E-cadherin were measured in serum free condition for 3,

18

6, and 12 h. 18S mRNA served as the internal control (D). E-cadherin

19

protein levels were detected under serum free condition treatment with or

20

without 10 mM ethyl pyruvate for 24 h (E). Under serum free condition,

21

E-cadherin (red) and vimentin (green) protein expressions were assessed

22

by immunofluorescence staining. Cell nuclei (blue) were visualized by

23

DAPI stain (F). Shown are the averages of three independent experiments.

24

*: p < 0.05 compared with control cells. (P, parental; L, HMGB1-L; EP,

25

ethyl pyruvate)

26 27

Figure 5. Reducing HMGB1 gene expression decreased cell migration

28

and invasion abilities. Polarized migration was measured by a wound 26 ACS Paragon Plus Environment

Page 26 of 35

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1

healing assay in complete culture condition with or without ethyl pyruvate

2

for 24 h (A and B). Polarized migration was measured in serum free

3

condition (C and D). Cell invasion assay was performed by transwell

4

culture system. Complete condition medium with or without ethyl pyruvate

5

were used as an attractant and the invasive cells were fixed, stained with

6

crystal violet, and counted (E and F). Shown are the averages of three

7

independent experiments. *: p < 0.05 compared with control cells. (P,

8

parental; L, HMGB1-L; EP, ethyl pyruvate)

9 10

Figure 6. Induction of EMT-associated E-cadherin expression

11

resulting in impaired cell migration capacity. Cells with or without

12

knocked-down of HMGB1 gene were treated with 50, 100, and 200 M

13

GR for 24 h. Cell viability of DU145 parental (A) and DU145 HMGB1-L

14

(B) was measured by MTS assay. E-cadherin expression was detected

15

using western blot analysis (C). Polarized migration was measured with 50,

16

100, and 200 M GR for 24 h (D) and quantification (E). Shown are the

17

averages of three independent experiments. *: p < 0.05 compared with

18

control cells.

19 20

Figure 7. Proposed model for glycyrrhizin blocked the process of EMT

21

by modulating HMGB1 initiated signaling pathway. In this scenario,

22

our results demonstrated that knocking-down of gene expression,

23

pharmacological inhibition or glycyrrhizin treatment effectively suppress

24

HMGB1

25

cascade and impaired cell migration and invasion abilities elicited by EMT

26

in prostate cancer cells.

initiated

Rac1/Cdc42/GSK-3β/Snail/E-cadherin

27


signaling


(A)

(B)

(C)

(D)

(E)

(F)

(G)

(H)

(I)

Figure. 1


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(A)

(B)

(C)

Figure. 2



(A)

(B)

(C)

(D)

Figure. 3


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(A)

(B)

(C)

(D)

(E)

(F)

Figure. 4



(A)

(B)

(C)

(D)

(E)

(F)

Figure. 5


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Page 33 of 35


(A)

(C)

(B)

(D)

(E)

Figure 6.



Figure 7.


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TOC Graphic


Glycyrrhizin Attenuates the Process of Epithelial-to-Mesenchymal

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