β-catenin and NF-κB Signaling Pathways

    Lucidone Promotes the Cutaneous Wound Healing Process via Activation of the PI3 K/AKT, Wnt/β-catenin and NF-κB Signaling Pathways Hsin-Ling Yang, Yu-Cheng Tsai, Mallikarjuna Korivi, Chia-Ting Chang, You-Cheng Hseu PII: DOI: Reference:

S0167-4889(16)30284-1 doi:10.1016/j.bbamcr.2016.10.021 BBAMCR 17969

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BBA - Molecular Cell Research

Please cite this article as: Hsin-Ling Yang, Yu-Cheng Tsai, Mallikarjuna Korivi, ChiaTing Chang, You-Cheng Hseu, Lucidone Promotes the Cutaneous Wound Healing Process via Activation of the PI3 K/AKT, Wnt/β-catenin and NF-κB Signaling Pathways, BBA - Molecular Cell Research (2016), doi:10.1016/j.bbamcr.2016.10.021

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Research article Revised Manuscript - Unmarked (R2)

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Lucidone Promotes the Cutaneous Wound Healing Process via Activation of the PI3K/AKT,

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Wnt/β-catenin and NF-κB Signaling Pathways

a

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Hseub,c*

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Hsin-Ling Yanga, Yu-Cheng Tsaib, Mallikarjuna Korivia, Chia-Ting Changa, You-Cheng

Institute of Nutrition, College of Biopharmaceutical and Food Sciences, China Medical

b

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University, Taichung 40402, Taiwan Department of Cosmeceutics, College of Biopharmaceutical and Food Sciences, China Medical University, Taichung 40402, Taiwan Department of Health and Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan

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*Corresponding author: Dr. You-Cheng Hseu

Professor, Department of Cosmeceutics College of Biopharmaceutical and Food Sciences, China Medical University 91 Hsueh Shih Road, Taichung 40402, Taiwan. Tel.: 886-4-2205-3366 ext 7503; Fax: 886-4-22062891. Email: [email protected]

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Abbreviations CDK, cyclin dependent kinase; COX, cyclooxygenase; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; GSK3β, glycogen synthase kinase 3 beta; ICAM, intercellular

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adhesion molecule; iNOS, inducible nitric oxide synthase; LY294002, (4-morpholinyl)-8phenyl-4H-l-benzopyran-4-one; MMPs, matrix metalloproteinases; NF-κB, nuclear factor κB;

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PCNA, proliferating cell nuclear antigen; PI3K, phosphatidylinositol 3-kinase; Tcf/Lcf, T-cell

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factor/lymphoid enhancer factor; TGFβ, transforming growth factor beta; TIMPs, tissue inhibitors of metalloproteinases; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end

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labeling; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator

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receptor, VEGF; vascular endothelial growth factor.

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ABSTRACT Lucidone, which comprises a naturally occurring cyclopentenedione, has been investigated for its in vitro and in vivo wound healing properties, and the underlying molecular signaling

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cascades in the wound healing mechanism have been elucidated. We demonstrated the cell-/dosespecific responses of lucidone (0.5-8 µM) on proliferation and migration/invasion of

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keratinocyte HaCaT and fibroblast Hs68 cells. In keratinocytes, lucidone-induced nuclear translocation of β-catenin was accompanied by increased transcriptional target genes, including

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c-Myc and cyclin-D1, through GSK3β-dependent pathway. Correspondingly, lucidone promoted the cell-cycle by increasing PCNA/CDK4 and decreasing p21/p27 expressions. Lucidone

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induced EMT through the downregulation of epithelial (E-cadherin/occludin) and upregulation of mesenchymal (vimentin/Twist/Snail) marker proteins. Activated MMP-9/-2 and uPA/uPAR as

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well as suppressed TIMP-1/-2 and PAI-1 expressions by lucidone may promote the migration/invasion of keratinocytes. Notably, lucidone activated NF-κB signaling via IKKmediated-IκB degradation, and its inhibition abolished MMP-9 activation and keratinocyte Inhibition

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PI3K/AKT

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migration.

signaling

impaired

the

lucidone-induced

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proliferation/migration with corresponding suppression of β-catenin/c-Myc/cyclin-D1 and NFκB/MMP-9 expressions. Results indicate that lucidone-induced PI3K/AKT signaling anchored diminished

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the β-catenin/NF-κB-mediated healing mechanism. β-catenin knockdown substantially lucidone-induced

keratinocyte

migration.

Furthermore,

lucidone

increased

endothelial cell proliferation/migration and triggered angiogenesis (MMP-9/uPA/ICAM-1). In macrophages, lucidone-activated NF-B-mediated inflammation (COX-2/iNOS/NO) and VEGF, which may contribute to the growth of keratinocytes/fibroblasts and endothelial cells. Punched wounds on mice were rapidly healed with the topical application of lucidone (5 mM) compared with control ointment-treated mice. Taken together, lucidone accelerates wound healing through the cooperation of keratinocyte/fibroblast/endothelial cell growth and migration and macrophage inflammation via PI3K/AKT, Wnt/-catenin and NF-κB signaling cascade activation. Keywords: Wound healing, lucidone, Wnt/β-catenin, NF-κB, E-cadherin, MMP

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1. Introduction Skin is the most frequently injured tissue compared with other tissues, and wound healing is a dynamic and sophisticated multistep process to repair and restructure the injured tissue. The

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wound healing process is divided into sequential, yet overlapping phases of hemostasis,

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inflammation, proliferation and remodeling [1, 2]. Tremendous progress has been made in recent years to identify the key cellular and molecular events responsible for wound healing. Cells,

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including keratinocytes, fibroblasts, macrophages and endothelial cells, are involved in the healing process, particularly during the inflammatory and proliferation stages of wound healing.

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Molecules such as MMPs, cytokines and enzymes play a critical role in the completion of the healing process. Both cells and molecules functionally coordinate in wound contraction, re-

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epithelialization and maturation processes [3-6].

Activation of the Wnt/ß-catenin signaling pathway plays a prominent role in the proliferative phase of wound healing. At the molecular level, ß-catenin is a subunit of the cadherin protein

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complex, which has been implicated as an integral component of the canonical Wnt signaling

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pathway. Wnt plays a specific role in the regulation of ß-catenin function [7, 8]. Wnt signaling is mediated by a multi-protein complex, including GSK3β, which targets β-catenin degradation by

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ubiquitination and subsequently prevents its nuclear translocation. However, the inhibition of ubiquitin-mediated β-catenin degradation in the presence of Wnt stimuli (inactivated GSK3β) results in cytoplasmic accumulation and subsequent nuclear translocation of β-catenin. Nuclearly localized β-catenin then binds to the Tcf/Lcf family to activate several target genes, including cMyc and cyclin D1, which are involved in cell proliferation [8-10]. E-cadherin has been demonstrated to prevent nuclear localization of β-catenin by binding to it on the cell surface [11]. E-cadherin is an epithelial marker protein that mediates cell-cell adhesion and differentiation. The loss of E-cadherin is a hallmark of the EMT, which is characterized by increased cell migration, particularly during re-epithelialization. The occurrence of the EMT is integral in development and wound healing, which is finely regulated by the activation and suppression of several molecular proteins, such as MMPs, Twist, Snail, E-cadherin and occludin [12, 13]. PI3K/AKT signaling is required for the EMT and cell migration, and dysfunction of PI3K/AKT signaling has been reported to prevent the EMT [14] and proliferation and impair the wound healing process [15]. In addition, AKT stimulates MMPs via the activation of NF-κB, and this activation induces Snail expression to promote the EMT [16]. NF-κB plays a key role in the

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inflammatory phase of wound healing by producing inflammatory mediators and VEGF, an angiogenic cytokine [17, 18]. Wnt or PI3K/AKT signaling may promote the EMT via the inhibition of GSK3β to stabilize ß-catenin, which subsequently translocates to the nucleus to

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engage transcriptional factors [13].

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Lucidone (Fig. 1A) is a naturally occurring cyclopentenedione that is isolated from the dried fruits of Lindera erythrocarpa Makino (Lauraceae); it widely grows in Taiwan, Japan, Korea and

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China. The dried fruits of L. erythrocarpa have been used in traditional Chinese medicine as a digestive, analgesic, diuretic, antidote and antibacterial substance [19]. Among several

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phytochemicals isolated from L. erythrocarpa¸ lucidone has been popular because of its potent pharmacological values, including antioxidant, anti-inflammatory, neuroprotective and anti-vital

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efficacies [19-21]. Lucidone has been reported to inhibit free radical-induced oxidative stress and inflammation in human skin HaCaT cells [22]. Recently, we have demonstrated that lucidone protects human skin keratinocytes against UVA-induced DNA damage and mitochondrial

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dysfunction [23]. These dermato-protective properties of lucidone appear to be associated with

not been investigated.

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increased antioxidant genes. However, the role of lucidone on the wound healing mechanism has

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Accumulating evidence has confirmed that the activations of the PI3K/AKT, Wnt/β-catenin, EMT/E-cadherin and NF-κB/MMP signaling pathways are critically involved in cell migration/proliferation and wound healing [1, 13]. The stringent control of these molecular expressions and topographic activities is critical to the effective healing of wounds, and dysregulation leads to impaired keratinocyte proliferation/migration, which eventually results in persistent chronic wounds and severe pathology [4]. Therefore, targeting key elements in this cascading network may lead to novel therapeutic approaches for healing chronic wounds. As a result of its potent dermato-protective properties, we assume that lucidone may be a valuable candidate to investigate the wound healing properties. Thus, the key events involved in the wound healing process, including proliferation, migration/invasion and inflammation, and the molecular signaling cascades, such as PI3K/AKT-GSK3β, Wnt/β-catenin, EMT/E-cadherin and NF-κB/MMPs, which sequentially operate these events, were investigated after lucidone treatment. Along with these in vitro experiments on keratinocytes, fibroblasts, macrophages and endothelial cells, suitable in vivo studies on mice were also performed to confirm the potent wound healing properties of lucidone.

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2. Materials and methods 2.1. Reagents and Chemicals Fetal bovine serum (FBS), Dulbecco’s modified Eagle medium (DMEM), glutamine and

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penicillin/streptomycin were purchased from Invitrogen/GIBCO BRL (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from

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Sigma-Aldrich (St. Louis, MO, USA). MMP-2, c-Myc, p21, p27, β-catenin, COX-2, iNOS, I-

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κBα, PCNA, IKKα/β, VEGF, TIMP-1, TIMP-2, uPA, uPAR, PAI-1, vimentin, Occludin, Twist and β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg,

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Germany). p65, MMP-9, cyclin D1, CDK4, GSK3β, p-GSK3β, p-β-catenin, p-IKKα/β, Ecadherin, p-PI3K, PI3K, p-AKT, AKT and histone antibodies were obtained from Cell Signaling

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Technology, Inc. (Danvers, MA, USA). Snail antibody was obtained from Gene Tex, Inc. (Irvine, CA, USA). 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Calbiochem (La Jolla, CA, USA). All other chemicals were of the highest grade commercially

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available and were supplied by Merck (Darmstadt, Germany) or Sigma Chemical Co (St. Louis,

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MO, USA).

2.2. Isolation and characterization of lucidone by HPLC

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Lucidone was isolated from the fruits of Lindera erythrocarpa as previously described [19, 23]. Briefly, 2.0 kg of dried fruits were extracted with 2 L of ethanol to produce an ethanolic extract. The total crude L. erythrocarpa extract was concentrated under a vacuum to yield a residue (124.3 g). Then, 100 g of the crude residue were suspended in 100 mL distilled water and partitioned with n-hexane (n-hex) and ethyl acetate (EA), which yielded an n-hex-soluble fraction, EA-soluble fraction and EA-insoluble fraction, with total yields of 16.0%, 45.6% and 34.3%, respectively. The biologically active EA-soluble fraction was further chromatographed using a silica gel (300 g) column, eluted with a gradient of n-hex/EA (95/5 to 100/0) to obtain 12 subfractions (EA1-EA12). The active fraction (EA5) was further separated via semi-preparative high-performance liquid chromatography (HPLC) using a Cosmogel column (Comosil Co., 250 mm × 10 mm) eluted with an n-hex/dichloromethane/EA solvent system to obtain the following four major compounds: lucidone (1), cis/trans-methyllucidone (2), methyl linderone (3), and linderone (4). The amount of active lucidone in the ethanol extract was further analyzed via HPLC, and a purity of lucidone of >99% was verified via HPLC and 1H-NMR. For the aqueous solution preparation, the powder samples were solubilized with 10 mM sodium phosphate buffer

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(pH 7.4) that contained 0.15 M sodium chloride (PBS) at 25 C. The stock solution (10 mg/mL) was stored at -20 C prior to the analysis of its wound healing properties. The chemical structure

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of lucidone is presented in Fig. 1A. 2.3. Cell culture

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Four types of cell lines, including human keratinocyte (HaCaT), human fibroblast (Hs68), murine macrophage (RAW 264.7) and human vascular endothelial (EA.hy926) cells, were

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obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). HaCaT cells were grown in DMEM medium supplemented with 10% heat-inactivated FBS, 2 mM

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glutamine, and 1% penicillin/streptomycin. Hs68 cells were maintained in DMEM medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, and 1% penicillin/streptomycin.

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RAW 264.7 cells were cultured in DMEM that contained 4 mM glutamine and 10% heatinactivated FBS. EA.hy926 cells were grown in DMEM supplemented with 15% FBS, HAT (100 mM sodium hypoxanthine, 0.4 mM aminopterin, and 16 mM thymidine), 1% glutamine, and 1%

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penicillin-streptomycin-neomycin. The cells were maintained in a humidified atmosphere with

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(Marienfeld, Germany).

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5% CO2. Cultures were harvested, and the cell number was determined using a hemocytometer

2.4. Cell proliferation assay

Cell proliferation was assayed via the MTT colorimetric method. The cells (1 × 105 cells/well, in a 12-well cell culture plate) were treated with the indicated concentrations of lucidone for 24 or 48 h. Following lucidone treatment, the cells were incubated with 400 μL of 0.5 mg/mL MTT in PBS for 2 h. The culture supernatant was removed and re-suspended with 400 μL of isopropanol to dissolve the MTT formazan, and the absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek Instruments, Winooski, VT, USA). Cell proliferation (%) was calculated as follows: (A570 of treated cells/A570 of untreated cells) × 100. The assay was performed in triplicate at each concentration. 2.5. Preparation of total, cytosolic and nuclear fractions Cells (1 × 106 cells/dish) in a 10-cm dish were grown in DMEM that contained 10% FBS to a nearly confluent monolayer. The cells were washed with cold PBS and re-suspended in lysis buffer that contained 10 mM HEPES (pH 8.0), 0.1 mM EDTA, 10 mM KCl, 100 µM EGTA, 1 mM DTT, 500 µM PMSF, 2.0 µg/mL leupeptin, 2.0 µg/mL aprotinin and 500 µg/mL

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benzamidine. The cells were allowed to swell on ice for 15 min. NP-40 [10% (v/v), 15 µL] was subsequently added to the cell suspension, and the samples were vortexed every 5 min for 20 min. The homogenates were centrifuged for 20 min at 12,000 × g, and the supernatant was used

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as the cytosolic extract. The nuclear pellet was re-suspended in cold extraction buffer that contained 20 mM HEPES (pH 8.0), 1 mM EDTA, 400 mM NaCl, 1 mM EGTA, 1 mM DTT, 1

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mM PMSF, 2.0 µg/mL leupeptin, 20 µg/mL aprotinin and 500 µg/mL benzamidine. The samples

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were centrifuged at 15,000 × g for 30 min, and the obtained supernatant was used as the nuclear extract. The protein content in the cytoplasmic and nuclear fractions was determined by a Bio-

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Rad protein assay reagent using BSA as a standard (Bio-Rad, Hercules, CA, USA). All protein fractions were stored at -80 °C until use.

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2.6. Western blot analysis

For Western blot, equal amounts of protein fractions were reconstituted in sample buffer (62 mM Tris–HCl, 2% SDS, 10% glycerol and 5% β-mercaptoethanol), and the mixture was boiled

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at 97 °C for 6 min. Equal amounts (60 μg) of the denatured protein samples were loaded into

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each lane, separated by SDS-PAGE on an 8–15% polyacrylamide gradient gel and subsequently transferred overnight onto polyvinylidene difluoride (PVDF) membranes. The membranes were

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blocked with 5% non-fat dried milk in PBS that contained 1% Tween-20 for 1 h at room temperature, followed by incubation with the primary antibodies for 2 h, and horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies overnight. The blots were detected using an ImageQuant™ LAS 4000 mini (Fujifilm) with the SuperSignal West Pico chemiluminescence substrate (Thermo Scientific Inc., Rockford, IL, USA). 2.7. Luciferase reporter assay

To examine the promoter activity of β-catenin or NF-κB, we used a dual-luciferase reporter assay system (Promega, Madison, WI, USA). Following lucidone treatment, cells cultured in 24well plates that had reached 70-80% confluence were incubated for 6 h with serum-free DMEM that did not contain antibiotics. The cells were subsequently transfected with a pcDNA vector or a β-catenin/NF-κB plasmid with β-galactosidase using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Following incubation with the vector or plasmid, the cells were lysed, and their luciferase activity was measured using a luminometer (Bio-Tek Instruments Inc., Winooski, VA, USA). The luciferase activity was normalized to the β-galactosidase activity in the cell lysates, and the data were expressed as the average of three independent experiments.

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2.8. Immunofluorescence staining HaCaT cells/RAW264.7 macrophages (2/1×104 cells/well) were cultured in DMEM that contained 10% FBS on glass 8-well Tek chambers (Nalge Nunc Intl., Naperville, IL, USA). The

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cells were subsequently fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, washed and blocked with 10% FBS in PBS, and then incubated for 24

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h with anti-β-catenin, anti-p65, anti-E-cadherin or anti-vimentin primary antibodies in 1.5% FBS.

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The cells were subsequently incubated with FITC (488 nm) secondary antibodies for an additional hour in 6% BSA. After incubation with the secondary antibodies, the cells were

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stained with 1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. The stained cells were washed with PBS and visualized using a confocal microscope (Leica TCS SP2, Heidelberg,

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Germany) at 630× magnification.

2.9. In vitro wound healing repair assay (Cell migration) HaCaT, Hs68 cells (2 × 104 cells/well) or EA.hy926cells (1 × 104 cells/well) were cultured on

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a 1% gelatin-coated 12-well plate and incubated with the indicated concentrations of lucidone in

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1% FBS-medium. At confluence, the monolayers were wounded using a 200 L micropipette tip, washed twice with PBS and incubated for 12, 24 or 48 h. The cells were subsequently washed

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twice with PBS, fixed with 100% methanol, and stained with Giemsa Stain solution (Merck, Darmstadt, Germany). The cultures were photographed using a phase-contrast microscope (100× magnification) to monitor the migration of cells into the wounded area, and the closure of the wounded area was calculated using Image-Pro® Plus software (Media Cybernetics, Inc., Bethesda, MD, USA).

2.10. Matrigel invasion assay

An HaCaT/Hs68 cell invasion assay was performed using BD MatrigelTM invasion chambers (Bedford, MA, USA). For this assay, 10 µL Matrigel (25 mg/50 mL) were applied to polycarbonate membrane filters (8 µm), and the bottom chamber of the apparatus contained a standard medium. Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor that is rich in extracellular matrix (ECM) proteins. Briefly, the top chambers were seeded with cells (1 × 105 cells/well) in serum-free medium (500 µL), and the cells were incubated with lucidone (0.5-4 µM). The cells were placed in the bottom chambers (750 µL), which were filled with a serum-free medium, and allowed to

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migrate for 24 h at 37 °C. Following the incubation period, the non-migrated cells on the membrane surface were removed with a cotton swab. The migrated cells on the bottom side of the membrane were fixed in cold 100% methanol for 8 min and washed twice with PBS. The

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cells were stained with Giemsa stain solution and subsequently de-stained with PBS. Images were obtained using an optical microscope (200 × magnification); invading cells were quantified

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by manual counting. The invading cells were quantified and expressed in untreated cells (control)

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thereby representing 1-fold. 2.11. Gelatin zymography assay

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The activities of MMP-9 in the medium released from the cells were measured via a gelatin zymography protease assay. HaCaT cells (1 × 106 cells/well) were seeded into 6-well culture

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dishes and grown in medium with 10% FBS to a nearly confluent monolayer. The cells were resuspended in 1% FBS medium and subsequently incubated with various concentrations of lucidone (0.5-2 μM) for 24 h. After treatment, collected media with an appropriate volume

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(adjusted by the vital cell number) were prepared using SDS sample buffer, without boiling or

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reduction and were subjected to 1 mg/mL gelatin-8% SDS-PAGE electrophoresis. Following electrophoresis, the gels were washed with 2.5% Triton X-100 and subsequently incubated in the

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developing buffer (50 mM Tris-base, 200 mM NaCl, 5 mM CaCl2 and 0.02% Brij 35) at 37 °C for 24 h. The gels were stained with Coomassie brilliant blue R-250. The relative changes in the MMP-9 activities were quantified using Matrix Inspector 2.1 software (AlphaEase, Genetic Technology, Inc., Miami, FL, USA). 2.12. Immunoprecipitation

Cells were treated with 2 µM of lucidone for 24 h and subjected to an immunoprecipitation assay. One mg of the protein samples was precleared with protein A-sepharose beads for 1 h and subsequently incubated with 2 mg of anti-E-cadherin or anti-GSK3β antibodies overnight. The immunoprecipitated complex was washed 5 times with RIPA buffer and then denatured with SDS sample buffer. The immunoprecipitated product or the total cell lysate (50 mg) were separated via SDS-PAGE and electrophoretically transferred to a PVDF membrane. After blotting with 5% skim milk for 30 min, the membrane was incubated with specific primary antibodies overnight and further incubated with HRP-conjugated secondary antibodies for 1 h. The plots were visualized using ECL reagents (Millipore).

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2.13. siRNA transfection The human β-catenin small interfering RNA (siRNA) was transfected with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For

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the transfection, HaCaT cells were grown in DMEM medium that contained 10% FBS and plated in 6-well plates to yield a 40−60% confluence at the time of transfection. On the next day, the

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culture medium was replaced with 500 μL of Opti-MEM (Invitrogen), and the cells were

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transfected using RNAiMAX transfection reagent (Invitrogen). For each transfection, 5 μL RNAiMAX were mixed with 250 μL of Opti-MEM and incubated for 5 min at room temperature.

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In a separate tube, siRNA (100 pM for a final concentration of 100 nM in 1 mL of Opti-MEM) was added to 250 μL of Opti-MEM, and the siRNA solution was added to the diluted RNAiMAX

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reagent. The resulting siRNA/RNAiMAX mixture (500 μL) was incubated for an additional 25 min at room temperature to enable complex formation. The solution was subsequently added to the cells in the 6-well plates, which resulted in a final transfection volume of 1 mL. After

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incubation for 6 h, the transfection medium was replaced with 2 mL of standard growth medium,

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and the cells were cultured at 37 °C. The cells were replaced with growth medium after transfection for 24 h. After lucidone treatment (2 μM) for 30 min or 24 h, the cells were

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subjected to Western blot, migration and immunofluorescence assays. 2.14. Determination of NO levels in culture media RAW264.7 cells were pretreated with lucidone (1-4 μM) for 24 h. The concentration of NO in the culture media was determined based on the accumulation of nitrite, a major stable product of NO, using Griess reagents (Sigma-Aldrich, St. Louis, MO, USA). Culture supernatants (100 µL) were mixed with an equivalent volume of Griess reagents, and the absorbance of the mixture was measured at 540 nm using an ELISA micro-plate reader. A standard curve was constructed using known concentrations of sodium nitrate. 2.15. Animal care and sample treatment BALB/c mice aged 6-8 weeks were purchased from The National Laboratory Animal Center (NLAC, Taipei, Taiwan). The mice were maintained in the Animal Center of China Medical University in a pathogen-free isolation facility with a 12 h light/dark cycle. The mice had ad libitum access to water and rodent chow (Oriental Yeast Co Ltd., Tokyo, Japan). All animal experiments strictly followed “The Guidebook for the Care and Use of Laboratory Animals” published by the Chinese Society of Animal Science, Taiwan. The animal protocols were

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approved by the Institutional Animal Care and Use Committee of China Medical University. 2.16. In vivo wound healing studies 2.16.1. Wound biopsy and measurement of wound closure area

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Mice (BALB/c) were anesthetized with 2% Rompun solution (0.1 mL/20 g body weight;

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Bayer, Leverkusen, Germany). The back of the mouse was gently shaved and then sterilized using an alcohol swab. A sterile biopsy punch (5 mm diameter, Miltex Inc., PA, USA) was used

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to punch through the full thickness of the back skin below the shoulder blades. A wound placed in this area cannot be reached by the mouse, which therefore prevents self-licking. Special care

2.16.2. Lucidone treatment on wound healing

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was used in animal handling and wound creation for the mice.

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Control ointment (100 mg pure white petrolatum jelly [Vaseline], n = 6) alone or ointment that contained lucidone (5 mM, n = 6) was applied to the wound every day. The healing of the

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wounds following ointment application was carefully observed and recorded. Wounds from individual mice were digitally photographed on day 0, day 3 and day 5. For all measurements,

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the wound area was quantified using Scion software (Scion Corporation).

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2.16.3. Histopathological examination

During the process of wound closure, skin samples (approximately 1 x 1 cm2) that contained the wound areas were collected on day 5 after post-wounding and fixed in 4% formaldehyde for histological study. The samples were embedded in paraffin and cut into 7 mm-thick sections from the middle part of the wounds. The sections were subsequently stained with hematoxylin and eosin (H & E) for 5 and 1 min, respectively. The H & E stained slides were visualized using a bright-field optical microscope. Rapid healing of a wound or an unhealed wound was defined by the distance between two opposite edges of the wound from the images. 2.17. Statistical analyses Data are presented as the mean±SD of at least three independent experiments (n＝3). Oneway analysis of variance (ANOVA) followed by Dunnett’s test were performed to determine the significant differences between groups. SPSS version 12.0 (Chicago, IL, USA) was used for the statistical analyses, and significance was defined as p<0.05 for all tests.

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3. Results 3.1. Lucidone promotes proliferation, migration and invasion of keratinocyte HaCaT and fibroblast Hs68 cells as a wound healing mechanism

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Keratinocytes and fibroblasts, which comprise the first cells to respond to injury,

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migrate/invade towards wound margins to initiate re-epithelialization, and increased cell proliferation promotes migration/invasion into wound margins [6, 24]. We initially investigated

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the effect of lucidone on the proliferation of keratinocyte HaCaT and fibroblast Hs68 cells via treatment with different concentrations of lucidone (0.5-4 and 1-8 μM) for 24 and 48 h. A low

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concentration of lucidone significantly enhanced the HaCaT (0.5-2 μM) and Hs68 (1-4 μM) cell proliferation in a time- and dose-specific fashion (Fig. 1B). The effect of lucidone (0.5-2 and 1-4

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μM) on the migration of HaCaT and Hs68 cells followed by a scratch was subsequently examined using an in vitro wound closure assay. The results demonstrated that the opened wound area was progressively closed by lucidone in a time- and dose-dependent manner. Strikingly, the

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cells with 48 h lucidone incubation migrated inwardly and covered a larger area of the wound,

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whereas the control cells without lucidone remained with a larger wound area (Fig. 1C & D). The effect of lucidone on the invasion ability was subsequently determined via a BD Matrigel

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chamber. Lucidone (0.5-2 and 1-4 μM, 24 h) significantly increased the invasion of both HaCaT and Hs68 cells in a dose-dependent fashion (Fig. 1E & F). These findings indicate that lucidone promotes an in vitro wound healing mechanism through increased cell migration, proliferation and invasion.

3.2. Lucidone activates Wnt/β-catenin signaling in HaCaT cells and triggers cell-cycle regulatory genes to promote proliferation Wnt/β-catenin signaling is well-known to regulate the dermal cell proliferation rate, motility, invasiveness and wound size [25]. HaCaT cells exposed to lucidone (0.5-2 µM) exhibited a substantial increase in the β-catenin promoter activity (dose-dependently), as assayed by a luciferase (TOP) reporter assay (Fig. 2A). Immunofluorescence data further demonstrated that lucidone treatment (2 µM) for 24 h increased the nuclear translocation of β-catenin (Fig. 2B), which correlated with increased promoter activity. The functional role of lucidone on β-catenin activation was subsequently determined by monitoring the β-catenin levels in whole cells (total/ phosphorylated) and cytosolic and nuclear fractions of HaCaT cells. The Western blot results indicated that lucidone (0.5-2 µM, 24 h) dose-dependently and evenly upregulated both the total-

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β-catenin and nuclear β-catenin levels, whereas the cytosolic β-catenin and phosphorylated βcatenin levels were downregulated (Fig. 2C). The phosphorylated β-catenin targets for ubiquitination were subsequently degraded in the cytosol.

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The effect of lucidone (0.5-2 µM, 24 h) on cell-cycle regulatory components was subsequently investigated via the assessment of changes in growth promoting (c-Myc, cyclin D1,

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PCNA and CDK4) and growth restraining (p21 and p27) signals. c-Myc, an inducer of cyclin D1

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and a negative regulator of p27, has been reported to be activated by Wnt/β-catenin signaling [26, 27]. In our study, lucidone-induced upregulated c-Myc in HaCaT cells appears to be transcribed

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by β-catenin activation (Fig. 2D). Similarly, PCNA, a protein involved in cell proliferation, was dose-dependently upregulated after lucidone treatment (Fig. 2D). It has been demonstrated that

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cyclin D isoforms interact with CDK4/6, and cyclin-CDK complex activation regulates the orderly progression through the G1 phase of the cell-cycle. Inhibitors of the CDK family, including p21 and p27, contain characteristic motifs at their N-terminal moieties that enable them

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to bind both CDK and cyclins [27]. Here, we demonstrated that lucidone tremendously increased

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both cyclin D1 and CDK4 expressions in a dose-dependent manner, which indicates the capability of lucidone to drive the progression of cells through the G1 phase (Fig. 2D). However,

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the p21 and p27 expressions were dose-dependently decreased by lucidone (Fig. 2D), which may explain the favorable context for cell proliferation. These findings indicate that lucidone increased nuclear localization of β-catenin may play a sensible role to drive cell proliferation via alterations in key cell-cycle regulatory proteins. 3.3. Lucidone-induced GSK3β phosphorylation and loss of E-cadherin (EMT) promotes nuclear translocation of β-catenin Without Wnt stimulation, β-catenin is constantly degraded by proteasome, and this degradation strictly depends on β-catenin phosphorylation, which is performed by GSK3β in complex with other proteins [10]. We demonstrated that lucidone treatment (0.5-2 µM) dosedependently increased p-GSK3β with a concomitant degradation of total GSK3β protein in keratinocytes (Fig. 3A). Immunoprecipitation data for GSK3β activity further confirms that lucidone (2 µM) inhibited GSK3β in the sample (Fig. 3B). The decreased GSK3β in combination with the decreased cytosolic β-catenin may explain the increased nuclear accumulation of βcatenin, which subsequently transcribes genes that favor cell proliferation. These findings imply that GSK3β signaling is required for lucidone-induced β-catenin activation in HaCaT cells.

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E-cadherin is an epithelial marker protein that mediates cell-cell adhesion, differentiation and migration. The loss of E-cadherin is a hallmark of the EMT characterized by an increased migration of cells during wound re-epithelialization [12]. Lucidone treatment (0.5-2 µM) of

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HaCaT cells led to a dose-dependent loss of E-cadherin, which indicates the loss of cell-cell adhesion (Fig. 3C). We subsequently performed dual immunofluorescence staining to identify

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the subcellular localization of E-cadherin. Corresponding to the decreased protein levels, the

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cells after lucidone treatment (2 µM, 24 h) were visualized with the loss of E-cadherin appearance. The control cells without lucidone exhibited immunoreactivity of E-cadherin,

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subcellular localization and cell-cell adhesions (Fig. 3D). The immunoprecipitation results indicated that the cells with lucidone were represented by significantly decreased E-cadherin and

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β-catenin expressions compared with the control cells (Fig. 3E). Occludin, a structural component of tight junctions [28], was dose-dependently inhibited by lucidone along with the loss of E-cadherin (Fig. 3C). Furthermore, decreased E-cadherin was accompanied by a dose-

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dependent increase in vimentin, a mesenchymal marker protein (Fig. 3C). Images from a

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confocal microscope indicated the increased subcellular accumulation of vimentin followed by lucidone incubation (2 µM, 24 h) (Fig. 3F). These findings suggest that lucidone induces the

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EMT via the suppression of E-cadherin and/or activation of vimentin in keratinocytes. Twist and Snail family transcription factors are reported to play a major role in the downregulation of E-cadherin/EMT [29, 30]. Therefore, to strengthen our findings that lucidone potently degraded E-cadherin, we determined the Twist and Snail expressions in keratinocytes. We determined that lucidone substantially increased both Twist and Snail proteins in a dosedependent manner (0.5-2 µM), in which E-cadherin was decreased with the same dose of lucidone (Fig. 3C). This inverse correlation between Twist/Snail and E-cadherin further emphasizes the capability of lucidone to induce the EMT and promote keratinocyte migration/invasion, which are required in the wound healing process. 3.4. Lucidone activates NF-κB signaling via IKK-mediated-IκBα degradation in HaCaT cells NF-κB signaling is essential during angiogenesis, cell migration and inflammation to promote wound healing [17, 31]. Lucidone treatment (0.5-2 µM) to HaCaT cells remarkably increased the luciferase reporter activity of NF-κB in a dose-dependent manner (Fig. 4A). Increased luciferase activity was associated with an increased nuclear translocation of p65, a major subunit of NF-κB (Fig. 4B-D). Immunofluorescence images further confirmed the increased nuclear localization of

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p65 following lucidone incubation (2 µM). NF-κB in the control cells was tethered in the cytoplasm, which implies sequestered NF-κB activity in the absence of lucidone (Fig. 4B). The time-dependent studies (15-240 min) indicated that lucidone (2 µM) increased the p65

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expression in nuclear fractions (p65 localization into nucleus), which peaked at 30 min following treatment (Fig. 4C). The activation of NF-κB is regulated by the degradation of its inhibitory

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protein IκB, which leads to dissociate free NF-κB subunits from the IκB/NF-κB complex. The

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estimated cytosolic IκBα protein in HaCaT cells was degraded by lucidone (0.5-2 µM), which was accompanied by a substantial increase in the p-IκBα and p-IKK levels. The dose-dependent

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increase in IKK/IκBα phosphorylations and the subsequent degradation of IκBα with lucidone explain the increased nuclear localization of NF-κB in HaCaT cells (Fig. 4D).

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3.5. Lucidone induces MMP activation through NF-κB signaling in HaCaT cells MMPs comprise a family of proteolytic enzymes cleaved into active forms that promote ECM degradation and play a central role in the inflammation/re-epithelialization phases of

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wound healing. TIMPs restrain the activity of specific MMPs and suppress excessive proteolytic

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ECM degradation [32]. We demonstrated that lucidone treatment (0.5-2 µM, 24 h) profoundly upregulated MMP-9/MMP-2, whereas it downregulated TIMP-1/TIMP-2 expressions in a dose-

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dependent fashion (Fig. 5A). The MMP-9 activity was subsequently estimated using a gelatin zymography assay, which indicated that lucidone (0.5-2 µM) substantially increased the MMP-9 activity and was approximately 5-fold with 2 µM lucidone (Fig. 5B). uPA comprises a glycoprotein initially secreted as an inactive pro-enzyme; it binds to uPAR and is activated by proteolytic cleavage, which consequently activates MMPs [33]. Interesting results demonstrated that lucidone (0.5-2 µM) dose-dependently increased both the uPA and uPAR expressions, whereas it suppressed PAI-1, an antagonist or negative regulator of uPA cleavage (Fig. 5A). Our findings suggest that lucidone-induced MMP activation and proteolytic activation of uPA may accelerate keratinocyte migration/proliferation. It has been suggested that NF-κB signaling may trigger MMP expressions following stimulation [34]. To address this phenomenon, cells were pretreated with an NF-κB inhibitor (Celastrol, 0.3 M) for 2 h, followed by lucidone (2 M), and changes in the MMP-9 protein and activity were determined. We identified fascinating results in which the lucidone-induced substantial increases in the MMP-9 protein and activity levels (Fig. 5D & E) were tremendously suppressed by the inhibition of NF-κB signaling in keratinocytes. Successful blocking of NF-κB

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signaling was confirmed by reporting the diminished luciferase activity and protein expressions of NF-κB/p65 with Celastrol treatment, even in the presence of lucidone (Fig. 5C & D). These findings elucidate that lucidone-activated NF-κB signaling regulates MMP activation in HaCaT

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cells.

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3.6. PI3K/AKT signaling involved in lucidone-induced migration/proliferation of HaCaT cells PI3K/AKT signaling is involved in keratinocyte migration and epithelialization during the

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wound healing process, and its inhibition abolished cell migration and impaired the healing mechanism [15]. To demonstrate the involvement of PI3K/AKT signaling that underlies the

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lucidone-induced migration, dose- and time-dependent changes in p-PI3K/PI3K and p-AKT/AKT proteins were initially assessed. Following lucidone incubation, the p-PI3K levels were

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prominently increased in a dose- (0.5-2 µM) and time-dependent (15-90 min) fashion (Fig. 6A & B). Interestingly, p-AKT was substantially increased with lucidone in the dose-dependent studies (0.5-2 µM) (Fig. 6A); however, the time-dependent elevation was confined to 60 min (2 µM)

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followed by lucidone treatment (Fig. 6B). We subsequently examined the influence of PI3K/AKT

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signaling on the increased proliferation and migration of keratinocytes via treatment with the specific PI3K inhibitor LY294002 (30 µM). The blockade of PI3K was confirmed by Western

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blot, in which the lucidone-induced p-AKT elevation was substantially inhibited (Fig. 6C). We subsequently demonstrated that the lucidone-induced proliferation (Fig. 6D) and migration (Fig. 6E) of keratinocytes were predominantly (p<0.001) abolished in the cells pretreated with LY294002. These findings clearly demonstrated that lucidone induced keratinocyte migration/proliferation through the activation of the PI3K/AKT signaling pathway. 3.7. Lucidone-induced β-catenin and NF-κB activation anchored by PI3K/AKT signaling in HaCaT cells Our other key findings indicated that lucidone accelerated the wound healing mechanism through the activation of β-catenin, NF-κB and PI3K/AKT signaling, which are crucially involved in cell proliferation and migration/invasion. To elucidate this phenomenon, HaCaT cells were incubated with a specific inhibitor of PI3K/AKT (LY294002), and the changes in βcatenin, GSK3β, cyclin D1, c-Myc and NF-κB levels were measured with or without lucidone treatment (2 µM). We demonstrated that the pretreatment of cells with LY294002 (30 µM) resulted in a striking loss of β-catenin levels (Fig. 7A) and blunted the β-catenin promoter activity (p<0.001, Fig. 7B) against the lucidone-induced elevation. Moreover, the loss of GSK3β

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by lucidone was further exacerbated in the presence of LY294002 (Fig. 7C). Importantly, cyclin D1 and c-Myc, key regulators of the cell-cycle, were substantially downregulated by the blockade of PI3K/AKT signaling (Fig. 7C). The inhibition of PI3K/AKT signaling further

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diminished lucidone-induced NF-κB/p65 translocation (Fig. 7D) and luciferase activity (Fig. 7E). This occurrence with LY294002 was led by a vanished MMP-9 expression against lucidone (Fig.

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catenin/NF-κB-mediated cell proliferation or migration.

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7D). These findings confirmed that lucidone stimulated PI3K/AKT signaling is essential for β-

3.8. Knockdown of β-catenin (siRNA) suppresses lucidone-induced migration of HaCaT cells

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To further confirm that lucidone-induced Wnt/β-catenin plays a central role in wound healing, β-catenin was knocked-down by the transfection of siRNA against β-catenin, and the expressions

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of β-catenin and migration of HaCaT cells were subsequently assayed. Lucidone increased the nuclear localization of β-catenin in the control cells, in contrast to the β-catenin knockdown cells, as evidenced by the Western blot data (Fig. 8A). The immunofluorescence results indicated that

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the cells transfected with siRNA against β-catenin were represented by sheared β-catenin

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localization. Nevertheless, the control siRNA transfected cells exhibited increased nuclear localization of β-catenin after lucidone treatment (Fig. 8B). Consistent with the β-catenin

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obliteration, the lack of β-catenin resulted in the restraining of lucidone-induced HaCaT cell migration. This finding was evidenced by significantly decreased migration ability in the si-βcatenin transfected cells (Fig. 8C). This additional evidence affirmed that the activation of Wnt/β-catenin signaling by lucidone promotes the wound healing mechanism, and silencing of the β-catenin gene diminished the lucidone-induced migration of HaCaT cells. 3.9. Lucidone triggers angiogenesis and migration of human endothelial (EA.hy926) cells Successful cutaneous wound healing requires the coordination of various cell types, including endothelial cells. Therefore, we extended our studies to delineate the wound healing promotional effects of lucidone on human endothelial EA.hy926 cells. Of various concentrations (5-20 µM, 24 h), only 10 µM lucidone promoted the proliferation of EA.hy926 cells (Fig. 9A). The Western blot results indicated that lucidone treatment (10 µM) considerably increased the MMP-9, uPA and ICAM expressions, which indicates the activation of angiogenesis (Fig. 9B). The effect of lucidone (10 µM) on migration was subsequently evaluated following 12 and 24 h of treatment. The results demonstrated that increased endothelial cell migration towards the scratch area was prominent at 24 h compared with 12 h after treatment (Fig. 9C). Taken together, these findings

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support that lucidone promotes angiogenesis and migration, which are essential to providing sufficient nutrients to newly proliferated cells and completing the re-epithelialization process. 3.10. Lucidone induces inflammatory responses via NF-κB-mediated COX-2/iNOS induction

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in murine RAW264.7 macrophages

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As a result of its anchoring role in the activation of the NF-κB-mediated wound healing mechanism in HaCaT cells, we investigated whether lucidone triggers the NF-κB signaling and

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subsequent inflammatory response in murine macrophages (RAW264.7 cells). Prior to elucidating this phenomenon, we assayed the macrophage proliferation with different

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concentrations of lucidone (1-8 µM) and demonstrated that 4 µM lucidone increased the proliferation following 24 and 48 h treatment (Fig. 10A). We subsequently demonstrated that

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lucidone remarkably increased the NF-κB/p65 expression with a subsequent degradation of its inhibitory protein, IκBα (cytosolic), in a dose-dependent manner (Fig. 10C). Lucidone-induced increased p65 subsequently translocates into the nucleus as evidenced by immunofluorescence

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images (Fig. 10B). Degradation of IκBα is reasoned by the increased p-IKK and p-IκBα (Fig.

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10C), which facilitates the cleavage of the IκBα/NF-κB complex to release the free NF-κB subunits. Increased nuclear translocation of p65 is associated with a dose-dependent increase in

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COX-2 and iNOS expressions in lucidone treated (1-4 µM) macrophages (Fig. 10D). Moreover, increased NF-κB-mediated inflammatory genes further resulted in a substantial increase in NO production (Fig. 10E). Consistent with NF-κB activation, lucidone treatment dose-dependently augmented the VEGF expression in macrophages, which implies activated angiogenesis (Fig. 10D). These findings suggest that lucidone is critically involved in the inflammatory and proliferative phases of wound healing via NF-κB signaling activation. 3.11. Lucidone effectively increases in vivo wound healing process in mice. In addition to its promising in vitro wound healing abilities, the in vivo wound closure property of lucidone was also examined using an excisional wound healing mouse model. Lucidone containing ointment or control ointment was topically applied to the wounds, and the wound closure area was digitally photographed and quantified. Digital photographs were obtained on day-0 through day-5 of post-wounding; the data indicated that lucidone treatment (5 mM) accelerated the wound closure, and the wound gradually disappeared by a time-course that was faster than the control ointment (Fig. 11A). The original wound sizes in the lucidone-treated mice on days 0, 1, 2, 3, 4 and 5 were 25.0±1.7, 14.4±3.6, 6.9±3.7, 5.4±3.1, 3.3±1.7 and 3.0±1.8

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mm2, respectively. The wound sizes in the control ointment-treated mice were 25.4±0.2, 17.5±0.8, 15.9±2.3, 12.9±3.3, 9.5±2.2 and 8.0±1.5 mm2 on the respective days of the experimental group (Fig. 11B). The significant decrease in the wound size after lucidone

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application (within 5 days of post-wounding) amplified the potent cutaneous wound healing capability of lucidone. Images from histopathological studies further indicated that lucidone

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application for 5 days accelerated re-epithelialization and closed the wound margins, whereas the

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wound margins remained opened with the control ointment (Fig. 11C). 4. Discussion

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To the best of our knowledge, this investigation represents the first study to demonstrate that lucidone, a naturally occurring cyclopentenedione, effectively promotes wound healing

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mechanism through the PI3K/AKT/Wnt/β-catenin/NF-κB-mediated signaling cascades. The cutaneous wounds made on the mice were rapidly healed with topical application of the lucidone ointment. In vitro lucidone treatment to keratinocyte HaCaT and fibroblast Hs68 cells increased

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the proliferation and migration/invasion. Lucidone induced transcriptional activation of β-catenin

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through a GSK3β-dependent pathway, and activated c-Myc and cyclin D1 expressions in

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keratinocytes may have enhanced the proliferation. These results were accompanied by the upregulation of cell-cycle promoting genes (CDK4/PCNA) and the downregulation of cell-cycle restraining genes (p21/p27). Lucidone-induced EMT through the loss of epithelial marker proteins

(E-cadherin/occludin)

and

increased

mesenchymal

marker

proteins

(vimentin/Twist/Snail), which may promoted keratinocyte migration/invasion. Increased migration was further supported by upregulated MMP-9/-2, uPA and uPAR and downregulated TIMP-1/-2 and PAI-1 expressions. In addition, lucidone appears to influence the inflammatory phase of wound healing through NF-κB activation via IKK-mediated-IκB degradation. The blockade of NF-κB signaling diminished the lucidone-induced MMP-9 activation, which indicates NF-κB activation is essential for keratinocyte migration/proliferation. Next, we demonstrated that the inhibition of lucidone-activated PI3K/AKT signaling substantially diminished the proliferation/migration and β-catenin nuclear accumulation followed by decreased cyclin D1 and c-Myc genes in keratinocytes. The PI3K/AKT pathway was demonstrated to be a key signaling regulator in the induction of NF-κB activation and NF-κBmediated MMP-9 activation following lucidone treatment. Furthermore, the knockdown of βcatenin abolished the lucidone-induced wound healing mechanism, as evidenced by sheared β-

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catenin and impaired keratinocyte migration. Moreover, lucidone triggered angiogenesis (MMP9, uPA and ICAM) in endothelial cells and increased the NF-κB-mediated inflammatory response (COX-2, iNOS and NO) and VEGF in macrophages, which may facilitate the growth

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and survival of newly proliferated cells.

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Keratinocyte proliferation, differentiation and migration are the inevitable steps following skin injuries. During the early stages of healing, a greater number of keratinocytes migrate to

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wound margins, integrate into the neoepidermis and initiate the re-epithelialization [24]. Wounding of the skin promotes keratinocyte activation that triggers migration and proliferation

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with corresponding changes in keratinocyte adhesion and cytoskeletal content [1, 6]. In contrast, impaired keratinocyte migration results in a poor wound healing process and persistence of

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chronic wounds [4, 15]. In our study, we demonstrated that lucidone treatment accelerated keratinocyte proliferation and increased migration/invasion as assessed by an in vitro wound healing assay. Increased keratinocyte migration/invasion with lucidone resulted in the closing of

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the wounding area after a scratch, which confirms the capability of lucidone to accelerate the

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wound healing process. We have recently demonstrated that lucidone treatment increased the viability of HaCaT cells and protected the cells from UVA-induced damage [23]. It has been

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claimed that keratinocyte proliferation and migration/invasion are the inevitable events in wound healing, and these events are regulated by the activation of the PI3K/AKT, Wnt/β-catenin/EMT and NF-κB/MMP signaling cascades [13, 15]. Therefore, we elucidated the functional role of lucidone on the modulation of these molecular signaling cascades. Wnt/β-catenin signaling pathways are essential in wound healing processes, which interact with a wide array of molecules that are essential for cell maintenance, motility, proliferation and re-epithelialization [1, 25]. The activation of Wnt/β-catenin signaling is reported to be achieved by the inhibition of GSK3β levels [10]. Active GSK3β associates with β-catenin in the cytoplasm and promotes β-catenin degradation by ubiquitination, which thereby prevents its nuclear translocation. However, in the presence of Wnt stimulation, GSK3β is inactivated, which subsequently facilitates the nuclear translocation of β-catenin [9]. In our study, the inactivated or phosphorylated GSK3β by lucidone led to an increased nuclear translocation of β-catenin in HaCaT cells. The increased nuclear localization of β-catenin following stimulation has been reported to associate with Tcf/Lef-1 family of transcription factors to activate various target genes that are involved in cell proliferation (c-Myc/cyclin D1) and determine the fate of cells [8,

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10]. Nuclearly translocated β-catenin by lucidone in our study may serve as a transcriptional activator to trigger downstream genes, such as c-Myc and cyclin D1, which thereby increased keratinocyte proliferation. c-Myc is essential for the transition from the G1 to S phases of the

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cell-cycle and promotes the proliferation of transit amplifying cells; the deregulation of c-Myc has been demonstrated to deplete epidermal stem cells, which disable tissue to react to the injury

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[35]. Thus, the activation of c-Myc/cyclin D1 by lucidone may influence the epidermal biology

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to promote the wound healing process.

Another important finding of this study is that activated β-catenin signaling by lucidone may

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be involved in the acceleration of cell-cycle at the G0 phase via the upregulation of cyclin D1 and its related protein kinase (CDK4) and/or the downregulation of its inhibitory proteins

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(p21/p27). The complex cyclin D/CDK4 has been reported to phosphorylate pRb, which subsequently releases E2F to transcribe the genes essential for cell-cycle progression [27]. Lucidone-induced cyclin D1 in keratinocytes may interact with CDK4 to drive the progression of

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cells through the G1 phase. It has been demonstrated that increased PCNA, a protein synthesized

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in the early G1 and S phases by β-lapachone, was associated with increased proliferation of keratinocytes [31]. Our findings indicated a substantial increase of PCNA and a corresponding

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decrease of p21/p27 genes in lucidone treated keratinocytes. The anti-mitotic signals p21 and p27 not only bind to and inhibit CDKs, which are required for the initiation of the S phase but also bind to PCNA and inhibit PCNA-dependent DNA replication [36]. Nevertheless, the upregulation of p21/27 genes results in the inhibition of cell-cycle progression from the G1 to S phases of the cell-cycle [37]. Therefore, the inhibition of both p21 and p27 genes by lucidone has no/less ability to bind to and inhibit cyclin D1/CDK4-mediated signaling cascades, which subsequently contribute to endorse keratinocyte proliferation. The EMT may promote migration/invasion and is essential in the wound healing process; thus, we expected that lucidone may trigger the EMT by altering the key molecular proteins concerned in the EMT. The loss of E-cadherin is a hallmark of EMT, which facilitates the migration of cells during wound healing and promotes re-epithelialization [12, 13]. E-cadherin and occludin are the epithelial marker proteins that are involved in cell-cell adhesion, migration/invasion and maintenance of tissue integrity [12, 28]. In our study, the loss of Ecadherin/occludin with lucidone corroborates the increased keratinocyte migration and invasion. It has been indicated that the blockade of E-cadherin function with an antibody caused uneven

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wound margins and disruption of actin cytoskeleton reorganization in mouse epidermis [38]. In mouse epidermis, E-cadherin expression was decreased 3 days after wounding (full-thickness incisional or excisional wound models), which may depend on the activity of migration and

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mitosis [39]. The cleavage of E-cadherin has been demonstrated to promote disassembly of the E-cadherin/β-catenin adhesion complex from the cytosol, which thus increases the soluble β-

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catenin levels [40]. Free cytosolic β-catenin is a potent regulator of Wnt signaling, which

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subsequently translocates to the nucleus, where it complexes with Lef-1/Tcf transcription factors to activate the expression of downstream target genes, including c-Myc and cyclin D1 [8, 41].

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Taken together, our findings demonstrate that the lucidone-induced loss of E-cadherin and increase of β-catenin regulates the motility and proliferation of keratinocytes via the Wnt/β-

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catenin signaling pathways.

Next, to provide sufficient evidence regarding the lucidone-induced EMT or E-cadherin degradation, we investigated other molecular proteins that are responsible for the EMT and E-

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cadherin stabilization. Evidence accumulated that Twist and Snail family transcription factors

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(mesenchymal markers) play key roles in E-cadherin suppression and thereby promote invasiveness [29, 30]. Twist overexpression causes transcriptional downregulation of E-cadherin,

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binds to the E-cadherin promoter and downregulates promoter activity [42]. The Snail family transcription factors have been reported to be involved in the remodeling of chromatin structure and silencing of E-cadherin expression [43]. Therefore, it is possible that lucidone-induced increased Twist and Snail genes may contribute to E-cadherin degradation, which subsequently functions in a sequential manner to initiate EMT. Furthermore, vimentin is an intermediate-sized filament highly expressed in mesenchymal cells that actively participates in the EMT, and increased vimentin expression is positively correlated with the increased invasiveness [44]. Another line of evidence has demonstrated that increased subcellular localization and protein expression of vimentin after lucidone treatment may imply the activation of the EMT and invasion. During wound healing, macrophages represent a significant bridge between the inflammatory phase and proliferative phase by producing various growth factors and cytokines, which, in turn, orchestrate the successful completion of wound healing [3, 18]. The inflammatory phase is mediated by multiple inflammatory mediators, mainly NF-κB and MMPs [17, 34]. NF-κB activation is reported to be sufficient to induce the EMT and Snail expression through AKT

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signaling [16]. Lucidone-induced NF-κB activation in keratinocytes/macrophages appears to be regulated via IKK-mediated IκB degradation, which cleaves the IκB/NF-κB complex, and the resulting free NF-κB subunits translocate to the nucleus. Increased NF-κB signaling may activate

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various genes, including iNOS and COX-2, which are responsible for the secretion of proinflammatory cytokines [45]. It has been claimed that one of the many ways in which the

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inflammatory process is linked to the proliferative phase of wound healing is through cytokine

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activation [3]. Increased iNOS/COX-2 and NO levels by lucidone may participate in the promotion of cell proliferation and migration. As an inflammatory cytokine, NO has been

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demonstrated to induce VEGF production in macrophages, and this induction is dependent on the cell density [46]. It is also indicated that the cells involved in the healing process may release

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cytokines and growth factors that act as paracrine factors for VEGF induction [47]. Increased VEGF with lucidone indicates the triggering of angiogenesis to re-establish the capillary network and provides nutrients to newly proliferative cells. NF-κB and PI3K signaling is involved in the

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induction of VEGF [18]; thus, lucidone-induced activation of NF-κB and PI3K signaling may

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further support our findings.

Keratinocytes express several proteins, including uPA, MMPs and TIMPs, which facilitate

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ECM degradation and thereby clear the path for cell migration and remodeling [3, 33]. uPA comprises an important protein in this process, which cleaves plasminogen to active protease plasmin. The uPA receptor (uPAR) regulates the plasminogen activation system by binding the uPA (active), and its zymogen (pro-uPA) forms [33]. Activated plasmin cleaves a range of ECM components essential for degradation, stimulates growth factors and activates MMPs essential for cell motility [48]. Accelerated cell-associated plasminogen activation by uPA–uPAR may facilitate cell migration [3, 33]. We demonstrated that lucidone regulates this entire signaling pathway to promote keratinocyte migration. In agreement with this finding, we identified increased expressions of uPA/uPAR analogous to MMP-2/MMP-9 activation with lucidone. Notably, PAI-1/SERPINE1, which antagonizes the proteolytic activity of uPA and the plasminogen activation system [49], was manipulated after lucidone treatment. The activation of MMPs is essential during the inflammatory and re-epithelialization phases of wound healing, whereas TIMPs restrain the activity of specific MMPs to inhibit excessive ECM degradation [32]. The increased MMP-2 and MMP-9 with lucidone corroborates the decreased TIMP-1 and TIMP-2 expressions in keratinocytes. It has been demonstrated that both

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MMP-2 and MMP-9 together may influence the formation of tubules via the migration of endothelial cells [3]. However, the blockade of NF-κB signaling suppressed the lucidone-induced MMP-9 activation, which indicates that NF-κB-mediated inflammation is essential in the

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activation of MMP-9 and angiogenesis. We subsequently identified a substantial increase in the MMP-9, uPA and ICAM expressions on endothelial cells with lucidone, which indicates the

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activation of angiogenesis. Increased angiogenesis following inflammation is correlated with

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increased MMP-9, ICAM and NF-κB expressions in endothelial cells [50]. In our study, lucidone-induced keratinocyte/endothelial cell migration and NF-κB/MMP-9 activation may

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have contributed to the formation of new capillaries, which thereby provide nutrients to newly proliferated cells. Furthermore, occludin, a substrate for gelatinases, including MMP-2 and

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MMP-9 [28], has been demonstrated to be degraded by lucidone. Presumably, lucidone-induced overexpression of MMP-2/MMP-9 may make more use of occludin, in which cleavage subsequently facilitates the loosening of tight junctions. These findings indicate that lucidone

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operates most signaling cascades involved in cell migration and the wound healing process.

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Another key finding of our study is that lucidone activates PI3K/AKT signaling, which is implicated in keratinocyte migration, proliferation and epithelialization during the wound healing

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process through the activation of Wnt/β-catenin and inhibition of GSK3β [15, 51]. To address whether PI3K/AKT signaling is functionally required for keratinocyte proliferation/migration, we blocked PI3K/AKT signaling with a pharmacological inhibitor, LY294002. We demonstrated that substantial inhibition of the p-AKT levels by LY294002 significantly diminished the lucidoneinduced proliferation and migration. Previous studies have reported that the inhibition of PI3K/AKT signaling resulted in decreased keratinocyte proliferation/migration and impaired the wound healing mechanism [15]. We further demonstrated that the inhibition of PI3K/AKT signaling abolished the lucidone-induced β-catenin activation and subsequent cell-cycle regulatory proteins (c-Myc/cyclin D1), which may be responsible for the impaired migration/proliferation of keratinocytes. In the inflammatory phase, we demonstrated the inhibition of PI3K/AKT signaling was led by suppressed NF-κB activation and NF-κB-mediated MMP-9 activation. Our findings are in agreement with previous studies that demonstrated PI3K/AKT signaling regulated Wnt/β-catenin/NF-κB activation in wound healing [51, 52]. Valproic acid is an antiepileptic drug reported to promote the in vivo wound healing process through the enhancement of the HaCaT keratinocyte motility via Wnt/β-catenin and PI3K/AKT

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signaling pathway activation [15]. To further confirm the importance of Wnt/β-catenin signaling in wound healing, the β-catenin expression was silenced by siRNA transfection, and the lucidone-induced keratinocyte migration and β-catenin levels were determined. The functional

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role of Wnt/β-catenin signaling in wound healing was repeatedly confirmed by the diminution of lucidone-induced migration of cells towards the wound margins. These findings provide

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additional evidence that β-catenin not only regulates the proliferative stage but also modulates

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the inflammatory stage of the healing process.

In accordance with the in vitro findings, evidence from an in vivo study on BALB/c mice

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confirmed the potent wound healing properties of lucidone. The topical application of lucidone on cutaneous wounds may enhance keratinocyte migration and proliferation, which subsequently

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commence re-epithelialization and thereby decrease the wound size. The re-epithelialization process is initiated with a wedge-shaped mass of keratinocytes that move across the granulation tissue and continues until keratinocytes from opposing sides of the wound reestablish contact [3].

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Mice treated with lithium, a known stimulator of β-catenin-mediated transcription, have been

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demonstrated to heal larger sized wounds through increased transcriptional activation and protein levels of β-catenin. It is also indicated that wound size in mice is correlated with the protein

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levels of β-catenin [25]. During wound healing, the antioxidant capacity of the skin is crucial to address excessive ROS, which specifically act as secondary messengers to trigger signals that promote cell proliferation and differentiation [53]. The topical application of virgin coconut oil on dermal wounds has been demonstrated to rapidly heal and complete the epithelialization process potentially via the cumulative effects of intra/extracellular matrix components and the antioxidant profile in rats [54]. Similarly, lucidone has been reported to protect skin cells against UVA-induced ROS production, mitochondrial dysfunction and cell death through increased antioxidant genes [23]. Taken together, the well-established antioxidant properties of lucidone may contribute to accelerate the wound healing process. 5. Conclusions To the best of our knowledge, this investigation represents the first study to demonstrate that lucidone promoted the cutaneous wound healing process in vivo and in vitro by the activation of PI3K/AKT/Wnt/β-catenin/NF-κB

signaling-mediated

keratinocyte

proliferation

and

migration/invasion. Cutaneous wounds in mice were effectively healed by lucidone as visualized

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by a decreased wound area size and histopathological images. At the molecular level, increased Wnt/β-catenin signaling appears to be initiated by a GSK3β-dependent pathway, which subsequently contributed to the activation of c-Myc and cyclin D1 genes. Lucidone increased

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cell-cycle regulatory proteins to accelerate keratinocyte proliferation and triggered EMT/MMP activation to facilitate the loosening of tight junctions and promote migration/invasion. Lucidone

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was further involved in the inflammatory phase of wound healing through NF-κB-mediated

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iNOS/COX-2/NO production and VEGF expression in macrophages. PI3K/AKT signaling anchored the activation of β-catenin/NF-κB cascades following lucidone treatment. The

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inhibition of PI3K/AKT or the knockdown of β-catenin signaling diminished the β-catenin/NFκB activation and impaired the lucidone-induced migration/proliferation. Furthermore, lucidone

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promotes cell growth via the activation of angiogenesis and inflammation in endothelial cells and macrophages, respectively. Taken together, lucidone is functionally involved in the regulation of sequential events in the wound healing process through the proper coordination of events among

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keratinocytes, fibroblast, endothelial cells and macrophages. Our findings suggest that the

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inclusion of lucidone in the preparation of wound healing ointments may comprise a suitable

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strategy for the effective treatment of chronic wounds. Acknowledgements

This work was supported by grants MOST-104-2320-B-039-040-MY3, MOST-103-2320-B039-038-MY3,

NSC-103-2622-B-039-001-CC2,

CMU102-ASIA-17

and CMU102-ASIA-

22 from the Ministry of Science and Technology (MOST), Asia University, and China Medical University (CMU), Taiwan. Conflict of interest All authors declare there are no conflict of interests.

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Figure Legends Fig. 1. Lucidone (low concentration) promotes proliferation, migration and invasion of human keratinocyte HaCaT and fibroblast Hs68 cell lines.

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(A) Chemical structure of lucidone. (B) HaCaT and Hs68 cells were treated with lucidone (0.5-8

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μM) for 24 or 48 h, and proliferation was determined by MTT assay. Cell proliferation (%) was calculated as follows: (A570 of treated cells/A570 of untreated cells) × 100. (C-D) Cells were

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treated with lucidone (0.5-4 M). Scratched migration was observed using a phase-contrast microscope (100 × magnification) at 0, 24 and 48 h (C), and the closure of the area was

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calculated using commercially available software (D). (E-F) Cells were treated with lucidone (0.5-4 M) for 24 h, and invading cells under the membrane were photographed (200 ×

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magnification). Invasion ability was monitored via a BD Matrigel chamber assay (E). Cell invasion was determined by counting cells in three microscopic fields per sample. (F) Quantified migrating/invading cells were expressed on the basis that untreated cells (control) represented 1-

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fold. The results represent the mean ± SD of three assays. The results are significant at *p < 0.05;

p < 0.001 compared with 48 h control cells.

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###

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**p < 0.01; ***p < 0.001 compared with 24 h control cells; significant at #p < 0.05; ##p < 0.01;

Fig. 2. Lucidone enhances nuclear translocation and transcriptional activation of β-catenin in HaCaT cells to promote cell-cycle regulatory proteins. (A) Luciferase reporter assay of β-catenin. HaCaT cells were co-transfected with luciferase reporter or β-catenin (TOP/FOP) and subsequently treated with lucidone (0.5-2 µM) for 6 h. Luciferase activity was determined, normalized by β-gal activity and presented as the relative luciferase activity. (B) Immunofluorescence assay for β-catenin nuclear localization. After lucidone treatment (2 M, 24 h), cells were incubated with an anti-β-catenin antibody, followed by a FITC-conjugated secondary antibody (green), and visualized under a confocal microscope (630  magnification). (C) Nuclear translocation of β-catenin. After lucidone treatment (0.5-2 M, 24 h), changes in the β-catenin levels in the whole cell (total/phosphorylated), cytosolic and nuclear fractions of HaCaT cells were determined by Western blot. (D) Lucidone upregulates cell-cycle regulatory genes, including c-Myc, cyclin D1, PCNA and CDK4, and downregulates p21/p27 expressions in HaCaT cells, determined by Western blot following lucidone treatment

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(0.5-2 M) for 24 h. The results represent the mean ± SD of three assays and were significant at *p < 0.05; **p < 0.01; ***p < 0.001 compared with control cells.

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Fig. 3. Lucidone activates Wnt/β-catenin via downregulation of E-cadherin/GSK3β signaling and upregulation of EMT-related proteins in HaCaT cells.

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(A-F) Cells were treated with lucidone (0.5-2 µM) for 24 h. (A) Lucidone decreases the total GSK3β and increases the p-GSK3β levels. Whole cells were subjected to Western blot, and the

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total GSK3β/p-GSK3β levels were determined. (B) Determination of GSK3β protein by immunoprecipitation. Equivalent amounts of proteins were immunoprecipitated with an anti-

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GSK3β/anti-β-catenin antibodies and subjected to Western blot with GSK3β/β-catenin antibodies. Relative changes in proteins were measured by densitometric analysis. (C) Lucidone induces the

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EMT in keratinocytes. Following lucidone treatment, cells were subjected to Western blot, and changes in E-cadherin, occludin, vimentin, Twist and Snail expressions were estimated. (D) Immunofluorescence staining indicates subcellular localization of E-cadherin after lucidone

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treatment. Cells were incubated with an anti-E-cadherin antibody, followed by a FITC-labeled

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secondary antibody, and changes in E-cadherin appearance were visualized using confocal

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microscopy (630 × magnification). (E) Immunoprecipitation determines the changes in Ecadherin protein. Equivalent amounts of proteins were immuno-precipitated with an anti-Ecadherin antibody and subjected to Western blot with E-cadherin/β-catenin antibodies. (F) Immunofluorescence staining indicates subcellular localization of vimentin after lucidone treatment. Cells were incubated with an anti-vimentin antibody, followed by a FITC-labeled secondary antibody, and the vimentin appearance was visualized using confocal microscopy (630 × magnification). The results are significant at *p < 0.05; **p < 0.01; ***p < 0.001 compared with control cells. Fig. 4. Lucidone activates NF-κB signaling through IKK-mediated-IκBα degradation in HaCaT cells. (A) Luciferase assay of NF-κB promoters. Cells were co-transfected with luciferase reporters of NF-κB and subsequently treated with lucidone (2 μM) for 15 min. Luciferase promoter activity was determined, normalized by β-gal activity, and presented as the relative luciferase activity. (B) Immunofluorescence staining indicates the changes in NF-κB (p65) in lucidone (2 μM) treated cells for 30 min. Cells were incubated with an anti-p65 antibody, followed by a FITC-labeled

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secondary antibody. The subcellular localization of p65 was visualized using confocal microscopy (630 ×magnification). (C-D) Lucidone promotes nuclear translocation of NF-κB. (C) Cells were treated with lucidone (2 μM) for 15-240 min, and time-dependent changes in p65

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expressions in nuclear fractions were determined by Western blot. (D) Cells were treated with lucidone (0.5-2 μM) for 30 min, and protein changes in nuclear or cytoplasmic fractions were

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determined using anti-p65, anti-IκB, anti-p-IκB, anti-p-IKKα and anti-IKKα antibodies,

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respectively. The results are significant at *p < 0.05; **p < 0.01; ***p < 0.001 compared with control cells.

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Fig. 5. Lucidone-induced NF-κB signaling regulates MMP activation in HaCaT cells. (A) Lucidone upregulates MMP-9, MMP-2, uPA and uPAR and downregulates TIMP-1, TIMP-2

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and PAI-1 expressions. Cells were treated with lucidone (0.5-2 μM, 24 h) and analyzed via Western blot. (B) Cells were treated with lucidone (0.5-2 μM, 24 h), and the conditioned media

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were subjected to gelatin zymography to analyze the MMP-9 activity. The results represent the mean ± SD of three assays. Values are significant at *p < 0.05; **p < 0.01; ***p < 0.001

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compared with control cells. (C) Luciferase reporter assay for NFκB activity. Cells were co-

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transfected with the luciferase reporter of NF-κB. Luciferase activity was determined, normalized by β-gal activity and presented as the relative luciferase activity. Cells were pretreated with Celastrol (0.3 M, NFκB inhibitor) for 2 h, followed by lucidone incubation (2 M) for 15 min. The results represent the average of three independent experiments. Significant at ***p < 0.001 compared with lucidone alone treated cells. (D) Cells were pretreated with Celastrol (0.3 M) for 2 h, followed by lucidone incubation (2 M) for 30 min (NF-κB) or 24 h (MMP-9). Changes in NF-κB (p65, nuclear) and total MMP-9 were determined by Western blot. (E) Cells were pretreated with Celastrol (0.3 M) for 2 h, followed by lucidone incubation (2 M) for 24 h. The conditioned media were subjected to gelatin zymography to analyze the MMP-9 activity. Significant at ***p < 0.001 compared with lucidone alone treated cells. Fig. 6. PI3K/AKT signaling pathways are involved in lucidone-enhanced cell proliferation and migration of HaCaT cells. (A-B) Lucidone mediates PI3K/AKT activation. (A) Cells were treated with 0.5-2 μM lucidone for 15 min or (B) 2 μM lucidone for 15-90 min. Whole cell lysates were subjected to Western

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blot with specific antibodies against p-PI3K and p-AKT. Total PI3K and AKT levels were assessed as loading controls. (C) Cells were pretreated with a PI3K/AKT inhibitor (LY294002, 30 M) for 2 h, followed by lucidone incubation (2 M) for 15 min. Western blot was performed

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to detect the p-AKT levels using anti-p-AKT antibody. (D-E) LY294002 suppresses lucidoneinduced cell proliferation and migration. HaCaT cells were pretreated with LY294002 (30 M)

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for 2 h and subsequently treated with lucidone (2 M) for 24 h or 48 h. (D) Cell proliferation

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was determined by MTT assay. (E) Cell migration was assessed using a wound healing repair assay at 0, 24 or 48 h, and the closure of the wounded area was calculated. The results represent

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the mean ± SD of three assays. Significant at ***p < 0.001 compared with lucidone alone treated cells.

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Fig. 7. Inhibition of PI3K/AKT signaling diminished lucidone-induced β-catenin activation and cyclin D1/c-Myc/MMP-9 upregulation in HaCaT cells.

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(A) Cells were pretreated with a PI3K/AKT inhibitor (LY294002, 30 M) for 2 h, followed by

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lucidone incubation (2 M) for 24 h. β-catenin levels in whole cell and nuclear fractions were determined via Western blot. (B) Luciferase reporter assay for β-catenin activity. Cells were co-

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transfected with the luciferase reporter of β-catenin. Luciferase activity was determined, normalized by β-gal activity and presented as the relative luciferase activity. Cells were pretreated with LY294002 (30 M) for 2 h, followed by lucidone incubation (2 M) for 6 h. Significant at ***p < 0.001 compared with lucidone alone treated cells. (C) Cells were pretreated with LY294002 (30 M) for 2 h, followed by lucidone incubation (2 M) for 24 h; whole cells were subjected to Western blot to determine the GSK3β, cyclin D1 and c-Myc levels. (D) Cells were pretreated with LY294002 (30 M) for 2 h, followed by lucidone incubation (2 M) for 30 min (NF-κB) or 24 h (MMP-9). Changes in NF-κB (p65, nuclear) and total MMP-9 were determined by Western blot. (E) Luciferase reporter assay for NFκB activity. Cells were cotransfected with the luciferase reporter of NF-κB. Luciferase activity was determined, normalized by β-gal activity and represented as the relative luciferase activity. Cells were pretreated with LY294002 (30 M) for 2 h, followed by lucidone incubation (2 M) for 15 min. Significant at ***p < 0.001 compared with lucidone alone treated cells.

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Fig. 8. Knockdown of β-catenin (siRNA) suppresses lucidone-induced migration of HaCaT cells. HaCaT cells were transfected with a specific siRNA against β-catenin or a non-silencing control.

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(A) Cells were incubated with lucidone (2 M) for 24 h, and β-catenin in the nuclear fraction was determined by Western blot in control siRNA-transfected and si-β-catenin transfected cells.

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(B) Immunofluorescence staining for β-catenin detection. After transfection, cells were treated

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with lucidone treatment (2 M) for 24 h, incubated with an anti-β-catenin antibody, followed by a FITC-conjugated secondary antibody (green), and visualized under confocal microscopy (630

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 magnification). siβ-catenin transfected cells exhibited scrambled β-catenin accumulation after lucidone treatment in contrast to control siRNA transfected cells. (C) Non-silencing control and

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β-catenin-siRNA cells were treated with lucidone (2 M) for 24 h. Scratched migration was observed using a phase-contrast microscope (100 × magnification) at 0 and 24 h, and the closure of the wounded area was calculated. Migrating cells were quantified and expressed on the basis

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that siRNA transfected cells (control) represented 1-fold. The results are significant at **p < 0.01

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compared with control siRNA transfected cells.

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Fig. 9. Lucidone induces angiogenesis in human endothelial (EA.hy926) cells. (A) Cells were treated with lucidone (5-20 M) for 24 h, and proliferation was determined by an MTT assay. The results are significant at *p< 0.05 compared with control cells. (B) Lucidone upregulates MMP-9, uPA and ICAM expressions. Cells were treated with lucidone (10 μM) for 24 h and subsequently subjected to Western blot using β-actin as a control. (C) Cells were treated with lucidone (10 M) for 12 or 24 h, and the scratched migration was subsequently assessed using a phase-contrast microscope (100 × magnification). The closure of the wounded area was calculated (histogram) and compared with untreated control cells, which represented 1fold. The results represent the mean ± SD of three assays. Significant at *p < 0.05; **p < 0.01; ***p < 0.001 compared with 12 h control cells and significant at # p < 0.01 compared with 12 h lucidone treated cells.

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Fig. 10. Lucidone triggers NF-κB activation and COX-2/iNOS expressions in murine RAW264.7 macrophages. (A) Cells were treated with lucidone (1-8 M) for 24 or 48 h, and macrophage proliferation was significant at #p < 0.05;

##

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determined via an MTT assay. Significant at *p < 0.05 compared with 24 h control cells and p < 0.01 compared with 48 h control cells. (B) Immunofluorescence

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staining indicates changes in p65 in lucidone (4 μM, 2 h) treated cells. Cells were incubated with

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an anti-p65 antibody, followed by a FITC-labeled secondary antibody. The subcellular localization of p65 was subsequently visualized using a confocal microscope (630 ×

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magnification). (C) Cells were treated with lucidone (4 µM) for 0.5-4 h. Changes in NF-κB (p65, nuclear) and cytosolic IκB, p-IκB, p-IKKα and IKKα were determined by Western blot. (D)

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Cells were incubated with lucidone (1-4 µM) for 24 h. Total cell lysates were subjected to Western blot to monitor iNOS, COX-2 and VEGF protein expressions. (E) Lucidone (1-4 µM, 24 h) induces NO production. The NO production was determined by measuring the formation of

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nitrite, the stable end-metabolite of NO in culture media. The results represent the mean ± SD of

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three assays. Significant at **p < 0.01; ***p < 0.001 compared with control cells.

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Fig. 11. Lucidone promotes in vivo wound healing in mice. (A-B) BALB/c mice were wounded using a sterile biopsy punch through the full thickness of the back skin below the shoulder blades and were treated with lucidone (5 mM) for 1, 3 or 5 days. (A) Control ointment (n = 6) or ointment that contained lucidone (5 mM, n = 6) was applied to a wound on the back of mice for 1, 3 or 5 days, and the healing of the wounds on individual mice was digitally photographed from wounding day 1, day 3 and day 5. (B) Relative changes in the healing of the wounded area by lucidone were quantified and compared with the control. Wound sizes were measured from day 1 to day 5 after wounding (n = 6). (C) Representative histopathological images of wounded skin treated with or without lucidone ointment on day 5 of post-wounding. The unhealed wound (control) and healed wound (lucidone) were defined by the distance between two opposite edges of the wounds.

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Lucidone promoted proliferation and migration/invasion of HaCaT and Hs68 cells. Lucidone triggerd β-catenin nuclear translocation, c-Myc/cyclin D1 and cell-cycle. Lucidone-induced PI3K/AKT signaling regulates β-catenin and proliferation/migration. β-catenin knockdown diminished lucidone-induced migration and β-catenin activation. Topical application of lucidone rapidly healed the wounds made on BALB/c mice.

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