Ursonic acid exerts inhibitory effects on matrix metalloproteinases via ERK signaling pathway

Journal Pre-proof Ursonic acid exerts inhibitory effects on matrix metalloproteinases via ERK signaling pathway Juhyeon Son, Sang Yeol Lee PII:

S0009-2797(19)31773-9

DOI:

https://doi.org/10.1016/j.cbi.2019.108910

Reference:

CBI 108910

To appear in:

Chemico-Biological Interactions

Received Date: 23 October 2019 Revised Date:

23 November 2019

Accepted Date: 27 November 2019

Please cite this article as: J. Son, S.Y. Lee, Ursonic acid exerts inhibitory effects on matrix metalloproteinases via ERK signaling pathway, Chemico-Biological Interactions (2019), doi: https:// doi.org/10.1016/j.cbi.2019.108910. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.

ERK X

CREB

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c-Fos

X

X

MMP-2/MMP- 9

MMP-1

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X

Invasion

Skin Aging

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Ursonic acid exerts inhibitory effects on matrix metalloproteinases via

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ERK signaling pathway

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Juhyeon Son and Sang Yeol Lee*

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Department of Life Sciences, College of BioNano Technology, Gachon University,

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Seongnam, Gyeonggi 13120, Korea

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* To whom correspondence should be addressed:

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Sang Yeol Lee, Department of Life Science, Gachon University, San 65, Bokjeong-Dong,

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Sujeong-Gu, Seongnam-Si, Gyeonggi-Do, 461-701, Korea, Tel: (8231) 750-8732; E-mail:

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[email protected]

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Abstract

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Ursonic acid is a pentacyclic triterpenoid compound that can be extracted from

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Ziziphus jujuba Mill., a traditional medicine. Matrix metalloproteinases (MMPs) are involved

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in cancer metastasis and skin aging. Regulation of various MMPs is closely associated with

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mitogen-activated protein kinases (MAPKs), including ERK, p38, and JNK MAPKs. In this

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study, we investigated the possibility of ursonic acid as an anti-cancer/anti-skin aging agent

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targeting MMPs. Cytotoxic effects of ursonic acid were analyzed by cell counting kit-8

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(CCK-8) assay. Invasive abilities of ursonic acid-treated A549 and H1299 non-small cell

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lung cancer (NSCLC) cells were tested with Boyden chamber assay. Effects of ursonic acid

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on MMPs were analyzed by zymography assays and quantitative real time polymerase chain

34

reaction (qRT-PCR). We also conducted flow cytometry and western blot analysis to

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elucidate the mechanisms of MMP regulation by ursonic acid. Our results revealed that

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ursonic acid inhibited transcriptional expression of gelatinases (MMP-2 and MMP-9) via

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inhibition of ERK and CREB signaling pathways in NSCLC cells. Moreover, ursonic acid

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reduced mRNA levels of collagenase (MMP-1) via suppression of ERK and c-Fos signaling

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pathways in HaCaT keratinocytes. These results suggest that ursonic acid could be a potential

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candidate for development of an effective novel anti-cancer and anti-wrinkle agent.

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Keywords: Ursonic acid; MAPK; ERK; Matrix metalloproteinase; Non-small cell lung

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cancer; Keratinocyte

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Abbreviations: NSCLC (Non-small-cell lung cancer); MMP (Matrix metalloproteinase);

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MAPK (Mitogen-activated protein kinase)

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1. Introduction

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Ziziphus jujuba Mill., commonly called as jujube or Da Zao, is a plant cultivated mainly

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in China for thousands of years and its fruits are used as herbal medicine to treat diseases [1,

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2]. Ziziphus jujuba Mill. is a main ingredient of a traditional chinese botanical formulation

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PHY906 (Huang Qin Tang), which is used in clinical studies as adjuvant theraphy for cancer

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patients [3, 4]. Other publications have demonstrated that Ziziphus jujuba Mill. alone also has

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an anti-cancer potential [5-7]. Ursonic acid is a pentacyclic triterpenoid which is a major

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constituent of Ziziphus jujuba Mill. (Fig. 1a) [8]. Several studies have reported that ursonic

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acid shows cytotoxicity against human cancer cells and induces apoptosis in human gastric

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and prostatic cancer cells [9, 10].

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Matrix metalloproteinases (MMPs) are enzymes that require calcium and zinc to

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breakdown the extracellular matrix (ECM) [11]. Several MMPs have been characterized, and

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each MMP is capable of degrading different substrates, such as gelatin, collagen, and elastin,

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which constitute the ECM. MMPs play a central role in embryonic development and tissue

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remodeling. Overproduction of MMPs can result in substantial loss of ECM and lead to

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inflammation, arthritis, angiogenesis, cancer metastasis, and skin aging [12-15]. MMP-2 and

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MMP-9 are the major gelatinases that destroy gelatins. Gelatinases are often upregulated in

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lung cancer cells and can contribute to cancer cell metastasis [16]. MMP-1, a major type of

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collagenase, can cleave native collagens and several ECM components. It has been

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previously reported that extracellular stimuli such as ultraviolet (UV) radiation, epidermal

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growth factor (EGF), and tobacco smoking can induce MMP-1 expression and accelerate skin

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aging [15, 17, 18].

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Mitogen-activated protein kinases (MAPKs) are enzymes that phosphorylate serine or

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threonine residues of specific proteins [19]. Extracellular signal–regulated kinases1/2

3

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(ERK1/2), p38, and c-Jun N-terminal kinases (JNK) are the three major classes of MAPKs.

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Ras, Raf, MEK, and ERK form a signaling cascade that efficiently transmits and regulates

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signals from diverse stimuli, such as growth factors, hormones, and cytokines. This cascade

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reaction phosphorylates several transcription factors, including c-Jun, cAMP response

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element binding (CREB), and ETS1 [20, 21]. ERK pathway regulates cell proliferation, cell

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migration, cell differentiation, and apoptosis. MMP expression highly correlates with ERK

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pathway. A previous study reported that inhibition of ERK/CREB pathway downregulates

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the mRNA levels of gelatinases in SKOV-3 ovarian cancer cells [22]. Moreover, several

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reports have demonstrated that, under strong stimuli such as UV radiation, reactive oxygen

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species (ROS) can initiate a series of ERK cascade reactions that lead to elevated MMP-1

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secretion [23, 24].

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It is still unclear whether ursonic acid regulates ERK pathway and MMP expression in

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human lung cancers and keratinocytes. In the present study, we evaluated the effects of

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ursonic acid on ERK signaling and MMP regulation in A549 and H1299 human non-small

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cell lung cancer (NSCLC) cell lines and HaCaT keratinocytes.

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2. Materials and Methods

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2.1. Cell culture

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A549 human NSCLC cells and HaCaT human keratinocytes were grown in Dulbecco's

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modified Eagles' medium (DMEM, HyClone, Logan, UT, USA) supplemented with

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10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 1%

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streptomycin/penicillin (HyClone). H1299 human NSCLC cells were grown in Roswell Park

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Memorial Institute (RPMI) - 1640 medium (HyClone) containing 10% FBS and 1%

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streptomycin/penicillin. Cells were grown in a CO2 incubator at 37 °C and under 5% CO2.

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Dimethyl sulfoxide (DMSO) was added to dilute ursonic acid (Chemfaces, Wuhan, Hubei,

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China), PD98059 (Selleckchem, Houston, TX, USA), and AZD6244 (Selleckchem) to

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prepare stock solutions of 10 mM.

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2.2. Cell viability assay

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A549, H1299, and HaCaT cells were seeded into 96-well dishes (1 × 104 cells per well)

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and maintained overnight. Next day, the cells were treated with ursonic acid at various

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concentrations for 24 h. Media were exchanged with fresh media containing 10% cell

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counting kit-8 (CCK-8) (Dojindo, Rockville, MD, USA) solutions and incubated for 1 h at 37

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°C. Relative viability of cells was determined using EZ Read 400 (Biochrom Ltd.,

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Cambridge, UK) by measuring absorbance at 450 nm.

106 107

2.3. Boyden chamber assay

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Polycarbonate membrane with 8 µm pores (Neuro Probe, USA) was covered with gelatin

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using a solution consisting of 0.1% gelatin and 0.1 g/L acetic acid. A549 and H1299 cells

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were harvested using trypsin and resuspended in 0.1% FBS media. Medium containing 3%

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FBS and ursonic acid (0, 2.5, or 5 µM) was used as chemoattractant (30 µL) in the lower

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chamber of a 48-well Micro Chemotaxis Chamber (Neuro Probe) and medium containing

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0.1% FBS was used as the negative control. Cells were seeded in the upper chamber (5 ×

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104 cells per well in 50 µL medium) and incubated for 24 h. The membrane was fixed with

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4% formaldehyde and stained with a solution consisting of 1% crystal violet and 20%

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methanol. Non-invading cells were removed and numbers of cells that had invaded the

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membrane were calculated by a Leica DM IL LED (Leica Microsystems).

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2.4. Gelatin and collagen zymography

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Cells were treated with media containing 0.1% FBS, ursonic acid, PD98059, and

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AZD6244 for 48 h. Conditioned media were harvested and secretory proteins were separated

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by SDS-PAGE. We used 8% polyacrylamide gels containing gelatin for A549 and H1299

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cells and collagen for HaCaT cells. Gels were rinsed with 2.5% Triton-X 100 for 30 min at

124

room temperature (RT) to remove SDS and renature proteins. Triton-X 100 solution was

125

decanted and gels were incubated with developing buffer containing 50 mM Tris-HCl (pH

126

7.6) and 5 mM CaCl2 for 24 h at 37 °C. Gels were stained with Coomassie Blue R250 for 30

127

min. Next, the gels were washed with destaining solution containing 20% methanol and 10%

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glacial acetic acid, until white bands of the degraded areas were visible.

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2.5. qRT-PCR analysis 6

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Cells were treated with media containing 0.1% FBS, ursonic acid for 48 h. Total RNA

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from A549, H1299, and HaCaT cells was isolated using HiGene Total RNA Prep kit

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(Biofact, Korea), according to the manufacturer's protocol. cDNA was synthesized using

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HiSenScript RH(-) RT PreMix Kit (Intronbio, Seongnam, Korea). qRT-PCR was performed

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with QuantiSpeed SYBR Kit (Philekorea, Daejeon, Korea) to obtain cycle threshold (Ct)

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values. Relative mRNA level of each group was calculated using 2-∆∆Ct method.

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Table 1. List of primer sequences used for qRT-PCR Gene

Sequence Forward primer: 5′- GAGATCATCGGGACAACTCTCCTT-3′,

MMP-1

Reverse primer: 5′- GTTGGTCCACCTTTCATCTTCATCA-3′ Forward primer: 5′- TTGACGGTAAGGACGGACTC -3′,

MMP-2 Reverse primer: 5′- ACTTGCAGTACTCCCCATCG -3′ Forward primer: 5′- GAGACCGGTGAGCTGGAT -3′, MMP-9 Reverse primer: 5′- TACACGCGAGTGAAGGTGAG-3′ Forward primer: 5′- TGCACCACCAACTGCTTAGC -3′, GAPDH Reverse primer: 5′- GGCATGGACTGTGGTCATGAG -3′ Forward primer: 5′- CCTGCATTGCTCGCTGTGTG -3′, RECK Reverse primer: 5′- CTCGTGGTTTGGGTATGCACCTT -3′ 138 139 140

2.6. Western blotting

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Cells were treated with ursonic acid, PD98059, and AZD6244 for 24 h. Harvested cells

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were lysed by using RIPA buffer mixed with protease and phosphatase inhibitor cocktail

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(Gendepot, USA). The concentrations of each protein sample were measured by 7

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bicinchroninic acid (BCA) protein assay kit (Thermo Fisher, USA). Proteins were separated

145

using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) (Millipore, Billerica,

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MA) membranes. The PVDF membranes were incubated with a blocking buffer tris-buffered

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saline (TBS)-T mixed with 5% skimmed milk for 1 h at RT. The PVDF membranes were

148

incubated with primary antibodies (1:1000) at 4 °C overnight. The PVDF membranes were

149

washed and incubated with secondary antibodies, conjugated with horseradish peroxidase

150

(1:10000), for 2 h at RT. Bands of proteins were detected using enhanced chemiluminescence

151

(ECL) method. We purchased antibodies against phospho-ERK (sc-7383), ERK (sc-94), JNK

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(sc-571), phospho-JNK (sc-6254), phospho-p38 (sc-17852-R), p38 (sc-7972), phospho-

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CREB (sc-7978-R), actin (sc-1615), β-catenin (sc-7199), NF-κB (sc-372), phospho-c-Fos

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(sc-81458), c-Fos (sc-166940), and ETS1 (sc-350) from Santa Cruz Biotechnology (Santa

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Cruz, CA, USA). We obtained antibody against CREB (9104) from Cell Signaling

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Technology (Beverly, MA, USA), and antibody against MMP-1 (MAB901) from R&D

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Systems (Minneapolis, MN, USA).

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2.7. Flow cytometry assay

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A549, H1299, and HaCaT cells were plated onto 6-well dishes (1 × 105 cells/well). Cells

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were treated with ursonic acid for 24 h and harvested by trypsinization. Cells were washed

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using PBS and stained with 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (10 µM

163

in HBSS) for 15 min at 37 °C. Stained cells were centrifuged and washed with PBS.

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Fluorescence of cells was detected using flow cytometry.

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2.8. Statistical analysis 8

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All quantitative data provided in the graphs are marked as the mean ± SD of three

168

independent experiments (n = 3). Statistical results were analyzed by ANOVA and Tukey’s

169

test (*p < 0.05, **p < 0.01).

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3. Results

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3.1. Ursonic acid inhibits the invasive abilities of A549 and H1299 NSCLC cell lines

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Cell viability was determined to investigate cytotoxicity of ursonic acid in A549 and

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H1299 NSCLC cell lines. The survival rate of A549 cells decreased to ~ 50% after treatment

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with 40 µM ursonic acid (Fig. 1b). IC50 value of ursonic acid for A549 cells was calculated to

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be 45.2 µM. In addition, H1299 cell viability reduced to ~80% at 100 µM ursonic acid

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treatment (Fig. 1c). IC50 value of ursonic acid for H1299 cells was calculated to be >200 µM.

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Figure 1. Cytotoxicity of ursonic acid to A549 and H1299 NSCLC cells. (a) Chemical

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structure of ursonic acid. (b & c) A549 and H1299 NSCLC cells were seeded onto 96-well

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plate and treated with ursonic acid (0 – 200 µM) for 24 h. Cell viability was determined using

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CCK-8 assay.

182 183

Furthermore, we estimated the effect of ursonic acid on invasive abilities of A549

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and H1299 cells. In A549 cells, the number of invaded cells, which reached lower chamber,

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decreased after treatment with ursonic acid in a dose-dependent manner (Fig. 2a). Invasion of

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H1299 cells was also suppressed by ursonic acid (Fig. 2b). The results showed that ursonic

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acid treatment effectively blocked invasion of NSCLC cells.

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Figure 2. Ursonic acid inhibits cell invasion of A549 and H1299 NSCLC cells. Invasion of

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A549 (a) and H1299 (b) NSCLC cells was analyzed using Boyden chamber. 3% FBS

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containing ursonic acid (0, 2.5, 5 µM) was used as a chemoattractant and 0.1% FBS was used

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for negative control (-). Invaded cells from the lower part of gelatin-coated membrane were

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counted.

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3.2. Ursonic acid inhibits proteolytic activities of gelatinases in A549 and H1299 NSCLC

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cells

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We hypothesized that the repressed invasive abilities of A549 and H1299 NSCLC

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cells might be because of inhibition of MMP-2 and MMP-9 genes. We detected the changes

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in gelatinolytic activities of the two proteinases in A549 and H1299 NSCLC cell lines by

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ursonic acid. In A549 cells, degradation of gelatin by MMP-2 significantly decreased,

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whereas degradation by MMP-9 was not affected (Fig. 3a). The proteolytic activities of both

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MMP-2 and MMP-9 were markedly attenuated in H1299 cells (Fig. 3b). This suggested that

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ursonic acid can hinder NSCLC cell invasion through MMP inhibition.

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Figure 3. Ursonic acid inhibits gelatinase activities of MMP-2 and MMP-9 in A549 and

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H1299 NSCLC cells. (a & b) A549 and H1299 NSCLC cells were treated with media

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consisting of 0.1% FBS and ursonic acid (0, 2.5, 5 µM) for 48 h. Conditioned media were

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harvested and used for gelatin zymography to observe gelatinase activities of MMP-2 and

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MMP-9. (c & d) qRT-PCR was performed to analyze mRNA amounts of MMP-2 and MMP-

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9 in A549 (c) and H1299 (d) cells after treatment of ursonic acid (0, 2.5, 5 µM) for 48 h. (e &

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f) qRT-PCR was performed to analyze mRNA amounts of RECK in A549 (e) and H1299 (f)

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cells after treatment of ursonic acid (0, 2.5, 5 µM) for 48 h. 13

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To investigate whether this inhibitory effect was transcriptionally regulated, we

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assessed the relative mRNA levels of the two gelatinases in ursonic acid-treated A549 and

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H1299 NSCLC cells. In A549 cells, the transcriptional level of MMP-2 was downregulated

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(Fig. 3c), and the mRNA levels of both MMP-2 and MMP-9 were reduced in H1299 cells

218

(Fig. 3d). Taken together, the results showed that ursonic acid can transcriptionally inhibit

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MMP expression in NSCLC cells. It has been reported that the membrane-anchored protein

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RECK not only suppresses the transcriptional expression of MMP-9 but also attenuates the

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proteolytic activity and secretion of MMP-9 [25, 26]. Therefore, we also investigated the

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effect of ursonic acid on the transcriptional expression of RECK in A549 and H1299 cells.

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As seen in Fig. 3 e & f, RECK mRNA level was downregulated in A549 cells, whereas it was

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upregulated in H1299 cells. This indicated why the gelatinase activity and transcriptional

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expression of MMP-9 were decreased only in H1299 cells.

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3.3. Inhibitory effects of ursonic acid on MMP-2 and MMP-9 via ERK and CREB

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signaling pathways in A549 and H1299 NSCLC cell lines

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To determine the molecular mechanisms underlying the effects of ursonic acid,

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MAPK signaling was considered a major target for MMP regulation. MAPKs are widely

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reported to be involved in the transcriptional expression of MMPs [24, 27]. We analyzed the

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phosphorylation levels of ERK, p38, and JNK, which represented the active forms of each

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MAPK, after ursonic acid treatment. In both A549 and H1299 NSCLC cells, ursonic acid

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significantly downregulated p-ERK (an active form of ERK) (Fig. 4a & b), whereas JNK and

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p38 expression levels remained unchanged (Fig. S1a & b).

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Figure 4. Ursonic acid inhibits expression of MMP-2 and MMP-9 through suppressing ERK

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and CREB in A549 and H1299 NSCLC cells. A549 (a) and H1299 (b) NSCLC cells were

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treated with ursonic acid (0, 2.5, 5 µM) for 24 h. Extracted proteins were used for western

240

blotting and phosphorylation levels of ERK and CREB were analyzed. Relative levels are p-

241

ERK/total-ERK and p-CREB/total-CREB ratios.

242 243

Transcription of MMPs is regulated by several transcription factors such as CREB,

244

β-catenin, and nuclear factor-κB (NF-κB) [28, 29]. CREB is one of the major proteins that is

245

activated by ERK and induces MMP-2 and MMP-9 expressions [22]. We used western blot

246

analysis to investigate whether ursonic acid inhibited CREB phosphorylation. CREB 15

247

activation was reduced in both A549 and H1299 cells by ursonic acid (Fig. 4a & b). ERK

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signaling and MMP induction are associated with transcription factors other than CREB [30];

249

therefore, we determined whether the protein levels of β-catenin and NF-κB could be

250

downregulated by ursonic acid. However, ursonic acid did not affect the expression levels of

251

β-catenin and NF-κB (Fig.S1a & b). To confirm that the suppressive effects of ursonic acid

252

on MMPs are exerted via ERK inhibition, we selected ERK inhibitor, PD98059, and MEK

253

inhibitor, AZD6244. Then, we performed key experiments using A549 and H1299 cells. In

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A549 cells, inhibition of ERK decreased activity of MMP-2 (Fig. 5a & c) and

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phosphorylation of CREB (Fig. 5e & g). Also, ERK and MEK inhibitors downregulated

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MMP-2, MMP-9 (Fig. 5b & d) and CREB in H1299 cells (Fig. 5f & h). The results indicated

257

that MMP-2 and MMP-9 inhibition is associated with ERK inhibition and subsequent CREB

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inhibition in A549 and H1299 cells.

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Figure 5. Expression of MMP-2 and MMP-9 is regulated through ERK and CREB signaling

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in NSCLC cells. (a & c) A549 NSCLC cells were treated with media consisting of 0.1% FBS, 16

262

PD98059 (a), and AZD6244 (c) for 48 h. Collected media were used for gelatin zymography

263

to observe gelatinase activities of MMP-2. (b & d) H1299 NSCLC cells were treated with

264

media consisting of 0.1% FBS, PD98059 (b) and AZD6244 (d). Collected media were used

265

for gelatin zymography to observe gelatinase activities of MMP-2 and MMP-9. (e & g) A549

266

cells were treated with PD98059 (e) and AZD6244 (g) for 24 h. Harvested proteins were used

267

for western blotting to analyze phosphorylation levels of ERK and CREB. (f & h) H1299

268

cells were treated with PD98059 (f) and AZD6244 (h) for 24 h. Harvested proteins were used

269

for western blotting to analyze phosphorylation levels of ERK and CREB.

270 271

3.4. Ursonic acid inhibits MMP-1 expression in HaCaT keratinocytes

272

We hypothesized that ursonic acid might also inhibit MMP expression in non-cancer

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cells. MMPs promote skin aging as well, and therefore, we investigated the effect of ursonic

274

acid in HaCaT keratinocytes. Our results revealed that ursonic acid had a cytotoxic effect in

275

HaCaT cells (Fig. 6a). IC50 value of ursonic acid for HaCaT cells was calculated to be 38.3

276

µM. Since MMP-1 is a major collagenase expressed in keratinocytes, we observed the

277

changes in collagen degradation upon ursonic acid treatment [15]. Our results revealed that

278

ursonic acid inhibited the collagenase activity of MMP-1 in HaCaT cells (Fig. 6b). Similarly,

279

the MMP-1 mRNA level was reduced upon ursonic acid treatment (Fig. 6c). Analysis of the

280

culture media reported a notable decrease in MMP-1 secretion (Fig. 6d). These data together

281

implicated that the inhibitory effect of ursonic acid on MMP-1 is exerted via the suppression

282

of transcriptional expression of MMP-1.

17

283 284

Figure 6. Ursonic acid inhibits MMP-1 in HaCaT keratinocytes (a) HaCaT keratinocytes

285

cells were seeded onto 96-well plate and were treated with ursonic acid (0 - 40µM) for 24 h

286

and cell viability was measured using CCK-8 assay. (b) HaCaT cells were treated with media

287

consisting of 0.1% FBS and ursonic acid (0, 5, 10 µM) for 48 h. Conditioned media were

288

harvested and used for collagen zymography to detect collagenase activity of MMP-1. (c)

289

HaCaT cells were treated with ursonic acid (0, 5, 10 µM) for 48 h and mRNA levels of

290

MMP-1 was measured using qRT-PCR. (d) Conditioned media were used for western

291

blotting to detect amounts of secreted MMP-1. Actin from cell lysates was used as a loading

292

control.

293 294

3.5. Inhibitory effect of ursonic acid on transcriptional expression of MMP-1 is exerted

295

via ROS regulation in HaCaT keratinocytes

18

296

Since reactive oxygen species (ROS) are reported to upregulate MMP-1 expression, we

297

detected the ROS level changes after ursonic acid treatment in HaCaT keratinocytes. Ursonic

298

acid significantly decreased the ROS level(s) in HaCaT cells (Fig. 7a), whereas ROS levels

299

remained unchanged in A549 and H1299 cell lines (Fig. S2), which indicated that the

300

antioxidant effect of ursonic acid is cell line-dependent. To confirm the relatedness of

301

inhibition of MMP-1 production and ROS regulation, ursonic acid-treated HaCaT cells were

302

treated with hydrogen peroxide (H2O2) and qRT-PCR was performed. As shown in Figure 7b,

303

ursonic acid-induced suppression of MMP-1 transcription was ameliorated by H2O2 treatment

304

in HaCaT cells. The results suggested that ursonic acid suppresses transcriptional expression

305

of MMP-1 via ROS regulation.

19

306 307

Figure 7. Inhibited transcription of MMP-1 is mediated by downregulation of ROS in HaCaT

308

keratinocytes. (a) HaCaT keratinocytes were treated with ursonic acid (0, 5, 10 µM) for 24 h

309

and stained with DCFH-DA. ROS levels were measured using flow cytometry. (b) HaCaT

310

cells were treated with ursonic acid (10 µM) in the presence or absence of hydrogen peroxide

311

(100 µM) for 48 h. mRNA levels of MMP-1 was analyzed using qRT-PCR.

312 313

3.6. Ursonic acid inhibits MMP-1 via ERK and c-Fos signaling pathways in HaCaT

314

keratinocytes 20

315

Similar to other MMPs, MMP-1 expression was regulated by MAPK signaling. In

316

HaCaT keratinocytes, band intensities of p-ERK were decreased by ursonic acid in a dose-

317

dependent manner, whereas the phosphorylated forms of p38 and JNK remained unchanged

318

(Fig. 8). Contrary to A549 and H1299 NSCLC cell lines, ursonic acid did not downregulate

319

CREB activation in HaCaT cells (Fig. S1c). Since ursonic acid lowered the ROS level(s) in

320

HaCaT cells, we investigated other transcription factors that were related to the redox

321

regulation and ERK signaling. c-Fos, a component of AP-1 has been reported to interact with

322

c-Jun, and ROS has been observed to induce c-Fos expression [31]. In addition, c-Fos can be

323

activated by ERK and can stimulate MMP-1 expression [32]. Hence, it was hypothesized that

324

ursonic acid inhibits c-Fos via downregulation of ERK. Thus, c-Fos phosphorylation was

325

suppressed by ursonic acid treatment (Fig. 8).

326 327

Figure 8. Ursonic acid inhibits ERK and c-Fos in HaCaT keratinocytes. HaCaT cells were

328

treated with ursonic acid (0, 5, 10 µM) for 24 h. Isolated proteins were used for western

329

blotting to analyze phosphorylation levels of ERK (a) and c-Fos (b). Relative levels are p-

330

ERK/total-ERK and p-c-Fos/total-c-Fos ratios.

331

21

332

Taken together, ursonic acid attenuates MMP-1 transcription via downregulation of

333

ERK and c-Fos signaling pathways in HaCaT keratinocytes. We also performed selected

334

assays using PD98059 (ERK inhibitor) and AZD6244 (MEK inhibitor) (Fig. 9). The results

335

indicated that MMP-1 suppression is associated with ERK inhibition and subsequent c-Fos

336

inhibition in HaCaT cells.

337 338

Figure 9. Expression of MMP-1 is regulated through ERK and c-Fos signalings in HaCaT

339

keratinocytes. (a & b) HaCaT keratinocytes were treated with media consisting of 0.1% FBS,

340

PD98059 (a) and AZD6244 (b) for 48 h. Collected media were used for collagen zymography

341

to observe collagenase activities of MMP-1. (c & d) HaCaT cells were treated with PD98059

342

(c) and AZD6244 (d) for 24 h. Harvested proteins were used for western blotting to analyze

343

phosphorylation levels of ERK and c-Fos.

344 345

22

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4. Discussion

347 348

MMPs are multidomain proteins that are specialized in degradation of ECM. MMPs

349

constitute several classes depending on their structure and substrate specificity [11]. For

350

instance, collagenases such as MMP-1 and MMP-8 mainly digest collagens, whereas

351

gelatinase A and B (MMP-2 and MMP-9, respectively) largely cleave gelatins. Since each

352

MMP degrades a different protein, they are involved in various cellular processes. Controlled

353

activity of MMPs is necessary as ECM degradation is important for tissue remodeling,

354

wound healing, and cell development [11]. However, aberrant expression of MMPs can cause

355

several diseases. ECM can act as a barrier to block cell invasion, and therefore, MMPs play a

356

major role in cancer metastasis. Overexpression of gelatinases by Skp2 has been reported to

357

result in enhanced invasive phenotypes of the lung cancer cells [33]. Moreover, high

358

collagenase levels reportedly mediate UV-induced photo-aging or arthritis [12, 23].

359

ERK, JNK, and p38 are the three major types of MAPKs and are related to MMPs, as

360

phosphorylation by MAPK cascade signaling can activate downstream transcription factors

361

to express MMPs. Eukaryotic cells produce most of the ATP from oxidative phosphorylation

362

in mitochondria. In this process, mitochondrial electron transport chain is the major source of

363

cellular ROS generation. Mitochondrial ROS has been proposed to be associated with MMP

364

expression regulation. ROS can affect MMPs via transcriptional expression and proenzyme

365

modulation via growth factor regulation [34]. ROS signaling involves MAPKs and

366

transcription factors, such as AP-1 and ETS-1. Among MAPKs, ERK is a key enzyme that

367

regulates the production of several MMPs. A previous study reported that inhibition of

368

Src/FAK/ERK/ β-catenin pathway led to downregulation of MMP-2 and MMP-9 in NSCLC

23

369

cell lines [27]. Moreover, reduced phosphorylation level of ERK decreased the activity of

370

ETS-1 and MMP-1 transcription in HaCaT keratinocytes [35].

371

Although, as previously reported, ursonic acid shows cytotoxicity in several cancer cell

372

lines [9], the effects of ursonic acid on ERK pathway and MMP expression are not well

373

understood. Actually, ursonic acid is an oxidized derivative of ursolic acid, which is a well-

374

known compound for the therapeutic potential [36, 37]. While ursolic acid has an alcohol

375

group at C-3 position, ursonic acid has a ketone group instead. There are previously

376

published studies that ursolic acid has suppressed metastasis of various cancer cells via

377

downregulating MMP-2, MMP-9, and other MMP-related proteins such as NF-ĸB, CD44,

378

VEGF, and integrin αVβ5 [38-42]. On the other hand, it is also reported that ursolic acid has

379

elevated expression of MMP-1 in HSC-T6 rat liver cells, while inhibiting ERK, PI3K, Akt,

380

and p38 [43]. In this study, ursonic acid had suppressive effect(s) on various MMPs,

381

including gelatinases and collagenases. It also inhibited cell invasions via downregulation of

382

ERK/CREB signaling pathway and transcriptional expressions of gelatinases (MMP-2 and -

383

9) in A549 and H1299 NSCLC cell lines. Particularly, ursonic acid also affected transcription

384

of the MMP-9 inhibitor, RECK. Therefore, ursonic acid treatment differentially affected

385

MMP-9 in A549 and H1299 cells. The result indicated how ursonic acid remarkably

386

attenuated the invasive abilities of NSCLC cells. There is a report that 100 µg/ml of

387

Ziziphus jujuba Mill. extract has inhibited A549 NSCLC cells’ viability about 20% [7].

388

Ursonic acid, a main compound of Ziziphus jujuba Mill., has suppressed A549 cells’ survival

389

about 50% at 40 µM (= 18.188 µg/ml) in our study (Fig. 1b). This implicates that pure

390

ursonic acid may exert more effective anti-cancer activity than Ziziphus jujuba Mill. extract.

391

In addition, in HaCaT keratinocytes, it reduced the ROS levels and repressed MMP-1

392

transcription via modulation of ERK and c-Fos signaling pathways. Considering that MMP-1

24

393

is a well-known enzyme responsible for damaging ECM in human skin, ursonic acid may

394

have potential in preventing UV-induced photo-aging. Taken together, the results suggest that

395

ursonic acid could be a promising candidate as both anti-cancer and anti-wrinkle agent.

396 397

Acknowledgment

398

This research was supported by Basic Science Research Program through the

399

National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-

400

2015R1D1A1A09058494) and the Gachon University research fund of 2019 (GCU-2019-

401

0363).

402 403 404

Conflict of interest The authors declare no conflict of interest.

405 406 407

25

408

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Highlights

● Inhibitory effects of ursonic acid on various MMPs were investigated. ● Gelatinase activities of MMP-2 and MMP-9 were inhibited in A549 and H1299 human NSCLC cells. ● Collagenase activity of MMP-1 was inhibited in HaCaT human keratinocyte cells. ● Ursonic acid suppressed the transcriptional expressions of MMPs in human NSCLC and keratinocyte cells via ERK signaling axis. ● Ursonic acid may be a strong candidate as both anti-metastatic and anti-skin aging agent.

Author Contribution Statement SYL conceived and designed the project; JS performed the experiments; JS and SYL interpreted data and wrote the manuscript.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: