ARE signaling pathway

Accepted Manuscript Antioxidant properties of 5-hydroxy-4-phenyl-butenolide via activation of Nrf2/ARE signaling pathway Yosuke Tabei, Kazutoshi Murotomi, Aya Umeno, Masanori Horie, Yoshio Tsujino, Bumbu Masutani, Yasukazu Yoshida, Yoshihiro Nakajima PII:

S0278-6915(17)30356-3

DOI:

10.1016/j.fct.2017.06.039

Reference:

FCT 9146

To appear in:

Food and Chemical Toxicology

Received Date: 1 February 2017 Revised Date:

21 June 2017

Accepted Date: 22 June 2017

Please cite this article as: Tabei, Y., Murotomi, K., Umeno, A., Horie, M., Tsujino, Y., Masutani, B., Yoshida, Y., Nakajima, Y., Antioxidant properties of 5-hydroxy-4-phenyl-butenolide via activation of Nrf2/ ARE signaling pathway, Food and Chemical Toxicology (2017), doi: 10.1016/j.fct.2017.06.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Antioxidant properties of 5-hydroxy-4-phenyl-butenolide via activation of Nrf2/ARE signaling pathway

Bumbu Masutanic, Yasukazu Yoshidaa, Yoshihiro Nakajimaa,*

Health Research Institute, National Institute of Advanced Industrial Science and

SC

a

RI PT

Yosuke Tabeia, Kazutoshi Murotomia, Aya Umenoa, Masanori Horiea, Yoshio Tsujinob,

Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1

M AN U

b

Asahidai, Nomi, Ishikawa 923-1292, Japan c

Kojun Japan Co., Ltd., 1-5-20 Tenma, Kita-ku, Osaka 530-0043, Japan

*

TE D

Corresponding author (Y. Nakajima)

Address: Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan

EP

E-mail: [email protected]. Tel: +81-87-869-3525

AC C

Fax: +81-87-869-4178

1

M AN U

5H4PB

Nrf2 ARE

AC C

Nucleus

H2O2

ROS

Keap1

EP

Keap1

TE D

Cytoplasm

Nrf2

SC

RI PT

ACCEPTED MANUSCRIPT

Oxidative stress

Ho-1 Cat Sod-1 Antioxidant enzymes

Cell death

ACCEPTED MANUSCRIPT

Keywords: 5-hydroxy-4-phenyl-butenolide; Nrf2/ARE signaling pathway; oxidative stress; reactive oxygen species; cytoprotective effect

RI PT

Abbreviations:

AAPH, 2,2ʹ-azobis(2-amidinopropane) dihydrochloride; ARE, antioxidant response element;

CAT,

catalase;

DABCO,

1,4-diazabicyclo[2,2,2]octane;

DAPI,

SC

4,6-diamidino-2-phenylindole; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GPx,

M AN U

glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; HO-1, heme oxygenase-1; H2O2, hydrogen peroxide; 5H4PB, 5-hydroxy-4-phenyl-butenolide; Keap1, Kelch-like ECH-associated protein 1; MI-MAC, multi-integrase mouse artificial chromosome; Nrf2, nuclear factor erythroid 2-related factor 2; PPARγ, peroxisome

TE D

proliferator-activated receptor-gamma; ROS, reactive oxygen species; SLG, Stable Luciferase Green; SLR3, Stable Luciferase Red3; SOD, superoxide dismutase; TK,

AC C

EP

thymidine kinase; Trolox, 2,5,7,8-tetramethyl-6-chromanol

4

ACCEPTED MANUSCRIPT

1. Introduction Oxidative stress, arising from an imbalance between the antioxidant system and the reactive oxygen species (ROS) generation, is usually thought to be the cause of

RI PT

disorders and diseases. Although ROS at low levels act as signaling molecules that promote cell proliferation and survival, a marked increase in ROS levels is associated with damage to a wide range of molecules including lipids, protein, and nucleic acids

SC

(McCord, 2000). In addition, oxidative stress plays a major role in the pathogenesis of a variety of disorders and diseases (Giasson et al., 2002; Roberts and Sindhu, 2009). It is

M AN U

therefore widely believed that attenuation of oxidative stress by antioxidants can help prevent various diseases or that antioxidants exert protective effects that lower the risk of various cancers (Uttara et al., 2009).

Antioxidants can be classified into direct and indirect antioxidants on the basis

TE D

of their mechanisms of action. Direct antioxidants are redox active and directly scavenge ROS (Dinkova-Kostova and Talalay, 2008). On the other hand, indirect antioxidants exert their effects by inducing antioxidant and cytoprotective enzymes,

EP

including heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) (Holtzclaw et al., 2004). The expression of antioxidant

AC C

enzymes is mainly controlled by the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway. Under normal conditions, Nrf2 is largely bound to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm (Ito et al., 1999). During periods of oxidative stress, Nrf2 is released from Keap1, is translocated into the nucleus, and subsequently binds to the AREs in the regulatory regions of target genes and activates the transcription of antioxidant enzymes. The Nrf2/ARE pathway therefore plays a key role in a cellular defense system against

5

ACCEPTED MANUSCRIPT

oxidative stress, and has been considered as a potential therapeutic target for the prevention of oxidative stress-evoked disorders and diseases (Jeong et al., 2006; Scapagnini et al., 2011). Based on these well-established concepts, many novel inducers

RI PT

of the Nrf2/ARE pathway, which include natural and synthetic products, have been uncovered (Forman et al., 2014).

Butenolides are a class of lactones with a four-carbon heterocyclic ring

SC

structure, and their derivatives are widely present in many natural products (Mukku et al., 2000; Beck et al., 2001). Butenolide-ring-containing compounds have attracted

M AN U

much attention owing to their structural diversity and biological activities including antimicrobial (Husain et al., 2010), anti-inflammatory (Ali et al., 2015), and anticancer activities (Wang et al., 2011). 5-Hydroxy-4-phenyl-butenolide (5H4PB) (Fig. 1) was isolated from Epichloe typhina on Phleum pratense for the first time as an antifungal

TE D

compound (Koshino et al., 1992). It seems likely that 5H4PB is produced via the interaction between E. typhina and P. pretense, and the 5H4PB plays an important role in the induction of resistance to fungal pathogens (Dickschat, 2017). More recently, it

EP

has also been isolated from fragrant vinegars as a peroxisome proliferator-activated receptor-gamma (PPARγ) activator (Tsujino, 2017). In addition, an in vivo study

AC C

suggested that 5H4PB has an anti-obesity effect in mice (Masutani et al., 2016). Thus, although this compound may have beneficial potentials for prevention of disorders or diseases, the underlying mechanism by which 5H4PB modulates intracellular signaling pathways, including the oxidative stress pathway, is unknown. Here, we elucidated 5H4PB as a new Nrf2/ARE pathway activator that upregulates the expression of antioxidant genes using a cell-based assay system as well as subsequent biochemical studies. Moreover, we also demonstrated that 5H4PB plays a

6

ACCEPTED MANUSCRIPT

key role in the cytoprotection against cell damage caused by oxidative stress in both cultured mouse fibroblasts and mouse primary hepatocytes. To the best of our

AC C

EP

TE D

M AN U

SC

effect of this butenolide-ring-containing compound.

RI PT

knowledge, this is the first report of Nrf2/ARE pathway activation and the antioxidant

7

ACCEPTED MANUSCRIPT

2. Materials and methods 2.1. 5-Hydroxy-4-phenyl-butenolide (5H4PB) 5H4PB was chemically synthesized and provided by Takasago International

RI PT

Co., Ltd. (Tokyo, Japan). 5H4PB was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) at 200 mM as a stock solution and stored at -80 °C.

SC

2.2. Cell culture

Mouse fibroblast A9 cells harboring the multi-integrase mouse artificial

M AN U

chromosome (MI-MAC) vector (Takiguchi et al., 2014) (a gift from Dr. M. Oshimura of Tottori University) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS, HyClone, Thermo Scientific, Waltham, MA) at 37 °C in an atmosphere of

TE D

5% CO2.

2.3. Plasmid construction

EP

The following beetle luciferases were used: green-emitting Stable Luciferase Green (SLG; TOYOBO, Osaka, Japan; Nakajima et al., 2005) from Rhagophthalmus

AC C

ohbai (Ohmiya et al., 2000) and red-emitting Stable Luciferase Red3, (SLR3) from Phrixotrix hirtus (Viviani et al., 1999; Nakajima et al., 2005). To generate a reporter plasmid carrying ARE, a synthesized oligonucleotide containing five tandem repeats of ARE

from

the

NAD(P)H

quinone

oxidoreductase

gene

(5’-TCACAGTGACTCAGCAAAATT-3’) (Dhakshinamoorthy and Jaiswal, 2000) and the herpes simplex thymidine kinase (TK) promoter was ligated into the XhoI/NcoI site of pNF-κB-TK-SLR3 (unpublished construction), from which the NF-κB-TK cassette

8

ACCEPTED MANUSCRIPT

was removed, resulting in pARE-TK-SLR3. An expression cassette containing ARE, the TK promoter, SLR3, and a poly A signal (pA) was excised with XhoI and NcoI from pARE-TK-SLR3, and ligated into the XhoI/NcoI site of pNFκB-TK-SLR3-pENTR

RI PT

(unpublished construction) from which an expression cassette was removed, resulting in pARE-TK-SLR3-pENTR. An expression cassette containing the TK promoter, SLG, and pA was PCR-amplified with pTK-SLG (TOYOBO) as a template using

SC

5’-CACCATCAATGTATCTTATCATGTCTGCTCGAG-3’ as the forward primer and 5’-GCGGATACATATTTGAGGATCC-3’ as the reverse primer. The amplified fragment

M AN U

was cloned into pENTR-D-TOPO (Invitrogen, Carlsbad, CA), resulting in pARE-TK-SLG-pENTR. The expression cassettes containing ARE-TK-SLR3-pA and TK-SLG-pA were recombined into pNeo-ϕC31 attB (Yamaguchi et al., 2011) and pBsd-R4 attB (Yamaguchi et al., 2011) by the LR reaction using LR Clonase II Plus Mix

(Invitrogen),

resulting

in

pARE-TK-SLR3-ϕC31-Neo

and

TE D

Enzyme

pTK-SLG-R4-Bsd, respectively. These constructions were inserted by homologous recombination into multi-integration sites of the MI-MAC vector (Takiguchi et al.,

EP

2014) as described below.

AC C

2.4. Generation of stable cell line The A9 cells harboring the MI-MAC vector were seeded in six-well plates at 6

× 105 cells per well one day before transfection. Three micrograms of pTK-SLG-R4-Bsd was cotransfected with 1 µg of the R4 recombinase expression plasmid pCMV-R4 (Yamaguchi et al., 2011) (a gift from Dr. T. Ohbayashi) using Lipofectamine PLUS (Invitrogen) according to the manufacturer’s instruction. The transfected cells were seeded onto 10 cm dishes three days after transfection, and

9

ACCEPTED MANUSCRIPT

subcultured for selection with 6 µg/ml blasticidin S (Wako) over 2-3 weeks. After that, pARE-TK-SLR3-ϕC31-Neo was cotransfected into the generated A9 cells with 1 µg of the ϕC31 recombinase expression plasmid pCMV-ϕC31 (Yamaguchi et al., 2011) (a gift

RI PT

from Dr. T. Ohbayashi) as described above, and subcultured for selection with 800 µg/ml G418 (Nacalai Tesque, Kyoto, Japan). Integration of the transgene into the

SC

corresponding site on the MAC vector was confirmed by genomic PCR.

2.5. Real-time bioluminescence measurement of 5H4PB-induced ARE activation

M AN U

The generated A9 stable cell lines were seeded on a 24-well plate at 1.5 × 105 cells/well. After one-day incubation, the medium was replaced with DMEM without phenol red (Gibco-BRL, Grand Island, NY) containing 10% FBS, 4 mM L-glutamine, 25 mM HEPES (pH 7.0), 400 µM

D-luciferin

potassium salt (RESEM BV, Linden,

TE D

Netherland), and 5H4PB at various concentrations. Bioluminescence was recorded in real time for 10 s at 13 min intervals in the absence or presence of the R62 long-pass filter (HOYA, Tokyo, Japan) at 37 °C in 5% CO2 atmosphere under saturated humidity

EP

using a microplate-type luminometer (WSL-1560, ATTO, Tokyo, Japan). SLG and SLR3 luminescence intensities were calculated as described previously (Noguchi et al.,

AC C

2010) using the following equation:

 F ( − )   1 . 0 1 .0   SLG    =     ,  F ( + )   κ SLG κ SLR 3   SLR 3 

(1)

where SLG and SLR3 are the SLG and SLR3 luminescence intensities, F(-) is the total luminescence count measured in the absence of the R62 long-pass filter, F(+) is the count of luminescence that passed through the filter, and κSLG and κSLR3 are the filter transmission coefficients for the two luciferases.

10

ACCEPTED MANUSCRIPT

2.6. Cytotoxicity assay Cell viability was determined using a Premix WST-1 Cell Proliferation Assay

RI PT

System (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. Cell membrane damage was determined with a Cytotoxicity Detection KitPLUS (LDH)

SC

(Roche Diagnostics GmbH, Mannheim, Germany).

2.7. Immunocytochemistry

M AN U

To detect Nrf2 localization in cultured cells after the treatment with 5H4PB, luciferase-expressing A9 cells were seeded onto Lab-Tek II chamber slides (Thermo Fischer Scientific). After an overnight incubation, the cells were treated with 50 µM 5H4PB for 4 h. Then, the cells were fixed with 4% paraformaldehyde for 15 min and

TE D

incubated with 0.25% Triton X-100 in PBS (PBST) for 30 min. The cells were blocked with Blocking One Histo (Nacalai Tesque) at room temperature for 10 min, washed with PBST for 5 min, and incubated overnight with rabbit monoclonal anti-Nrf2 antibody (1:

EP

400; Cell Signaling Technologies, MA, USA) at 4 °C. After washing three times with PBST, the cells were incubated with goat anti-mouse Alexa Fluor 546 (1: 500; Thermo Scientific)

for

AC C

Fischer

1

1,4-diazabicyclo[2,2,2]octane

h,

and

this

(DABCO,

was

followed

by

Sigma-Aldrich)

mounting

with

containing

4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). Fluorescence was detected by using a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan).

2.8. Isolation of total RNA and quantitative real-time PCR analysis Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA,

11

ACCEPTED MANUSCRIPT

USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 500 ng of total RNA using a ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO) according to the manufacturer’s instructions. PCR was performed

RI PT

with KOD SYBR qPCR Mix (TOYOBO) using a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). The following primers were used: Ho-1, 5’-GCTCGAATGAACACTCTGG-3’ and 5’-GTTCCTCTGTCAGCATCAC-3’; Cat,

5’-GAGACCTGGGCAATGTGACT-3’

5’-GTTTACTGCGCAATCCCAAT-3’;

5’-ACTACACCGAGATGAACGA-3’

M AN U

Gpx-1,

and

SC

5’-AGATGGAGAGGCAGTCTATT-3’ and 5’-AGATCTCGGAGGCCATAAT-3’; Sod-1,

5’-GACGTACTTGAGGGAATTCAG-3’;

and

and β-actin,

5’-TTCTTTGCAGCTCCTTCGTT-3’ and 5’-GACCAGCGCAGCGATATC-3’. Cycling conditions were as follows: initial denaturation at 98 °C for 2 min, followed by 40

TE D

cycles of 98 °C for 10 s, 60 °C for 10 s, and 68 °C for 30 s. After amplification, one cycle of a linear temperature gradient from 65 to 95 °C was performed to assess the specificity of the PCR product. Relative expression levels of transcripts were

EP

determined after normalization to the corresponding sample expression level of β-actin.

AC C

2.9. Measurement of CAT and SOD activities CAT activity was measured with a Catalase Assay Kit (Cayman Chemical Co.,

Ann Arbor, MI, USA) according to the manufacturer’s instructions. SOD activity was measured with an SOD Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Protein concentration of was determined with a Bio-Rad Protein Assay Kit (Hercules, CA, USA), where BSA was used as the standard.

12

ACCEPTED MANUSCRIPT

2.10. Measurement of intracellular ROS level The

level

of

intracellular

ROS

was

detected

using

2’,

RI PT

7’-dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich). DCFH-DA was dissolved in DMSO at 5 mM as a stock solution and stored at -20 °C. After incubation of cells with 5H4PB or H2O2, the medium was changed to serum-free DMEM

SC

containing 10 µM DCFH-DA, and the cells were further incubated for 30 min at 37 °C. Then, the cells were collected, washed, and resuspended in PBS. Cell samples were

M AN U

excited with a 488 nm argon laser in a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ). The emission of DCF was recorded at 525 nm. Data were collected from at least 5000 gated events. For intracellular ROS imaging, the cells were subjected to fluorescence measurement using a BZ-X710 all-in-one fluorescence

TE D

microscope (Keyence).

2.11. Measurement of radical-scavenging activity of 5H4PB

EP

We obtained 2,5,7,8-tetramethyl-6-chromanol (Trolox) from Cayman Chemical. Hydrophilic 2,2ʹ-azobis(2-amidinopropane) dihydrochloride (AAPH) was obtained from

AC C

Wako; AAPH was used to generate free radicals at a constant and controlled rate. We purchased fluorescein from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The experiments were carried out using Trolox as a reference material for comparison. Trolox is a vitamin E mimic and water-soluble antioxidant and its kinetic information is available. Solutions of 5H4PB and Trolox were prepared at the concentration of approximately 1 mM in methanol and DMSO (9:1, vol/vol). AAPH (0.5 M) and fluorescein (1 mM) stock solutions were prepared in PBS. Quartz cells (4 ml) were used

13

ACCEPTED MANUSCRIPT

as reactors. Portions of stock solutions and fluorescein were first added to PBS in the quartz cells. We used a UV-vis absorption spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) equipped with a thermostatted cell maintained at 37 °C. The reaction was

RI PT

started by adding AAPH, and the rate of reaction of fluorescein with free radicals in PBS was monitored by measuring the decay of absorption at 494 nm. The lag phase was obtained graphically by extrapolating the slope of maximal probe decay to the

SC

intersection with the slope of minimal probe decay at the initial stage of the reaction (Umeno et al., 2015). The lag phase is expressed as follows (Takashima et al., 2012): (2)

M AN U

Lag phase = n[IH]/Ri ,

where n and Ri are the stoichiometric number and the rate of radical flux, respectively. The reported stoichiometric number of Trolox is two (Wayner et al., 1985).

TE D

2.12. Determination of reduced and oxidized glutathione levels

Reduced and oxidized glutathione levels were measured with a GSSG/GSH Quantification Kit (Dojindo Laboratories) according to the manufacturer’s instructions.

EP

GSH level was calculated using following formula: (3)

AC C

GSH (µmol/L) = Total glutathione (µmol/L) – 2 × GSSG (µmol/L)

2.13. Alkaline comet assay Alkaline comet assay was performed according to the manufacturer’s

instructions (Comet Assay, Trevigen, Gaithersburg, MD, USA). In brief, cells pretreated with 5H4PB were exposed to H2O2 for 6 h, and then washed and resuspended in PBS. The cell suspension was mixed with LMagarose and immediately transferred onto CometSlides. After cell lysis at 4 °C, the CometSlides were treated with an alkaline

14

ACCEPTED MANUSCRIPT

unwinding solution (0.2 M NaOH and 1 mM EDTA, pH > 13) for 60 min. Electrophoresis was performed at 20 V for 30 min at 4 °C in the dark, and then the separated products were stained with a silver-staining kit (Trevigen). The comet tail

RI PT

length was defined as the distance between the leading edge of the nucleus and the end of the tail. Data were collected from 150 cells per experiment in triplicate (50

SC

cells/slide/culture).

2.14. Isolation of primary hepatocytes

M AN U

To isolate primary hepatocytes, we used female ICR mice aged from 10 to 13 weeks. Primary hepatocytes were isolated by a two-step collagenase perfusion method according to the method of Guenthner et al. (2014) with some modifications. After an ICR mouse was anesthetized with 3% isoflurane gas using a small animal anesthetizer

TE D

(MK-AT200, Muromachi Kikai, Tokyo, Japan), the liver was perfused with 64 ml of EGTA buffer (in g/l: 0.1902 EGTA, 8.0 NaCl, 0.4 KCl, 6.0 HEPES, 0.06 KH2PO4, 0.35 NaHCO3, and 1.0 D-glucose; pH 7.4) at a rate of 8 ml/min via the hepatic portal vein

EP

and simultaneously the abdominal inferior vena cava was cut to bleed. After the liver was freed of blood, EGTA buffer was replaced with 100 ml of collagenase solution,

AC C

which contained low-glucose DMEM (Gibco-BRL) supplemented with 6 mM HEPES (pH, 7.2-7.5), antibiotics (penicillin and streptomycin), and 100 U/ml collagenase in equal units of Type III collagenase and Type IV collagenase (Worthington Biochemical Corporation, NJ). Collagenase solution was heated at 37 °C for 90 min before use. The temperature of perfusate buffer was maintained at approximately 40 °C during the entire procedure. After perfusion, the liver was rapidly transferred to a sterile petri dish and shredded in a cold hepatocyte wash medium (Gibco-BRL). After washing by low-speed

15

ACCEPTED MANUSCRIPT

centrifugation at 50 g for 2 min at 4 °C, the viability of hepatocytes was assessed by trypan blue exclusion, and suspensions with viability of over 70% were used. The animal experimental protocols were approved by the Institutional Animal Care and Use

RI PT

Committee of the National Institute of Advanced Industrial Science and Technology. All animal experiments were carried out in accordance with the approved protocols.

For the following experiments, the hepatocytes were suspended in a hepatocyte

SC

maintenance medium (PromoCell GmbH, Heidelberg, Germany) and seeded at 3 × 104 cells/well in a 96-well plate coated with Type I-A collagen (Nitta Gelatin, Osaka, Japan),

M AN U

which was air dried for 2-3 h before seeding. After incubation for 6 h in a humidified atmosphere containing 5% CO2 at 37 °C, half volume of the cultured medium was replaced with the hepatocyte maintenance medium containing a twofold higher

TE D

concentration of 5H4PB or H2O2, and cell viability was measured as described above.

2.15. Statistical analysis

All assays were conducted in at least triplicate. Data are expressed as means

EP

with standard deviation (SD). The results were subjected to analysis of variance and the post-hoc Tukey test to analyze the significance of difference. P values < 0.05 were

AC C

considered statistically significant.

16

ACCEPTED MANUSCRIPT

3. Results 3.1. 5H4PB activates ARE-dependent transcription To determine whether 5H4PB modulates intracellular signaling pathways,

RI PT

mouse fibroblast A9 cells, in which activation of signaling pathways including inflammatory, endoplasmic reticulum (ER) stress and heat shock pathways can be monitored using luciferase, were treated with 5H4PB, but no significant activation was

SC

observed (data not shown). Interestingly, however, the light output from A9 cells, in which activation of the oxidative stress pathway can be monitored by ARE-dependent

M AN U

transactivation of the luciferase gene, was increased by treatment with 5H4PB in a dose-dependent manner (Figs. 2A and S1). This finding strongly indicates that 5H4PB activates the Nrf2/ARE signaling pathway. Note that red-emitting luciferase (SLR3) was used for monitoring ARE-dependent transcription and green-emitting luciferase

TE D

(SLG) was used as an internal control to normalize SLR3 luminescence (Fig. 2, upper panel). As shown in Fig. S1, whereas SLR3 luminescence intensity increased following treatment with 5H4PB, SLG luminescence intensity (internal control reporter) did not

EP

significantly change, indicating that 5H4PB does not nonspecifically upregulate overall transcription but specifically activates ARE-dependent transcription, as monitored using

AC C

SLR3 in real time.

Next, we examined the cytotoxicity of 5H4PB in the luciferase-expressing A9

cells because the Nrf2/ARE pathway is also activated by cell damage induced by oxidative stress. Cytotoxicity was assessed in terms of cell viability and cell membrane damage by WST-1 assay and LDH release assay, respectively (Fig. 2B). When the A9 cells were treated with 5H4PB at various concentrations for 24 h, no cytotoxicity analyzed by WST-1 assay and LDH release assay was observed up to 100 µM and 60

17

ACCEPTED MANUSCRIPT

µM, respectively. The 5H4PB concentration used in the monitoring of activation of the Nrf2/ARE signaling pathway shown in Fig. 2A was in the range in which no

signaling pathway without any apparent cytotoxic effect.

RI PT

cytotoxicity occurred, indicating that 5H4PB specifically activates the Nrf2/ARE

To determine whether the nuclear translocation of Nrf2 is induced by the treatment with 5H4PB, intracellular localization in control and 5H4PB-treated cells was

SC

examined by immunocytochemistry (Fig. 2C). Nrf2 was predominantly localized in the cytoplasm of control cells (Fig. 2C, upper panels). Upon treatment with 5H4PB, the

M AN U

immunostaining of anti-Nrf2 (red) and DAPI (blue) almost overlapped (Fig. 2C, bottom panels), demonstrating that Nrf2 was translocated into the nucleus by the treatment with 5H4PB. These results indicate that the induction of ARE-dependent transcription in luciferase-expressing cells treated with 5H4PB is activated by the Nrf2/ARE signaling

TE D

pathway.

3.2. 5H4PB increases expression levels of endogenous ARE-driven genes

EP

To verify whether the activation of luciferase activity by 5H4PB reflects the expression of endogenous ARE-driven genes, we performed quantitative real-time PCR

AC C

analysis of Ho-1, Cat, Sod-1, and Gpx-1, which are under the control of the ARE enhancer (Nguyen et al., 2003). The luciferase-expressing A9 cells were treated with 50 µM 5H4PB for 4, 12, and 24 h, and the mRNA expression levels of these genes were determined. As shown in Fig. 3, the mRNA expression level of Ho-1 markedly increased after 4 h treatment with 5H4PB, and gradually decreased after prolonged incubation. Cat and Sod-1 mRNA expression levels also increased after treatment with 5H4PB, consistent with the increase of enzymatic activities (Fig. S2). On the other hand,

18

ACCEPTED MANUSCRIPT

no change in the Gpx-1 mRNA expression level was observed, indicating that 5H4PB selectively activated the ARE of Ho-1, Cat, and Sod-1. These findings suggest that

the Nrf2/ARE signaling pathway.

3.3. 5H4PB reduces intracellular ROS levels induced by H2O2

RI PT

5H4PB increases the mRNA expression levels of antioxidant-related genes by triggering

SC

To investigate whether 5H4PB reduces oxidative stress-induced ROS production, the luciferase-expressing A9 cells were pretreated with 5H4PB at various

M AN U

concentrations for 24 h and then exposed to H2O2 for an additional 2 h. Intracellular ROS level was measured using a fluorescent dye, DCFH-DA. As shown in Fig. 4A, whereas a bright signal of DCF, which shows enhanced fluorescence during ROS generation, was observed in the H2O2-exposed cells (right upper panel), the signal was

TE D

effectively diminished by the pretreatment with 20 µM 5H4PB (right bottom panel). We noted that the 24-h treatment with 20 µM 5H4PB did not cause any appreciable variation in the basal levels of ROS (left bottom panel). To quantify the effect of 5H4PB

EP

on H2O2-induced increase in intracellular ROS levels, ROS levels were measured as an increase in DCF fluorescence intensity by flow cytometry. The incubation of

AC C

non-pretreated cells with 4 mM H2O2 for 2 h significantly increased intracellular ROS production dose-dependently up to approximately fivefold (Fig. 4B). On the other hand, the H2O2-induced ROS production was dose-dependently attenuated by the pretreatment with 5H4PB, suggesting that the cellular antioxidant capacity was enhanced by the pretreatment with 5H4PB, which may be due to the increase in the antioxidant gene expression levels, as shown in Fig. 3. To verify that the reduction in H2O2-induced ROS production is not due to radical

19

ACCEPTED MANUSCRIPT

scavenging by 5H4PB, 5H4PB antioxidant capacity was assessed by reducing the consumption of fluorescein, which reacts with peroxyl radicals in the presence of AAPH. As shown in Fig. S3A, 5H4PB did not significantly inhibit fluorescein decay even at

RI PT

high concentrations, whereas Trolox, which has a markedly high radical-scavenging capacity as a water-soluble vitamin E mimic compound, produced a clear lag phase (Fig.

that 5H4PB has no radical-scavenging activity.

M AN U

3.4. 5H4PB reduces oxidized glutathione levels

SC

S3B) in a concentration-dependent manner (Fig. S3C). These findings clearly indicate

Next, we determined whether 5H4PB increases antioxidant capacity by determining the level of cellular reduced glutathione (GSH). GSH is involved in the protection against oxidative stress, and the ratio of reduced to oxidized glutathione

TE D

levels (GSH/GSSG) represents antioxidant capacity (Espinosa-Diez et al., 2015). The luciferase-expressing A9 cells were pretreated with 5H4PB at various concentrations for 24 h and then exposed to 4 mM H2O2 for an additional 4 h. As shown in Fig. S4,

EP

although exposure to H2O2 decreased the GSH/GSSG ratio, the decrease was attenuated dose-dependently by the pretreatment with 5H4PB, consistent with the finding that the

AC C

GSH/GSSG ratio increased when 5H4PB alone was applied. This observation was due to mainly the inhibition of the decrease in GSH values rather than the increase in GSSG values (data not shown). This finding suggests that 5H4PB improves the intracellular antioxidant capacity against H2O2-induced oxidative stress. Thus, it may be reasonable to assume that the reduction in H2O2-induced ROS production by 5H4PB (Fig. 4) is caused by the synergy between the improvement of intracellular antioxidant capacity and the increase in the expression levels of antioxidant-related genes.

20

ACCEPTED MANUSCRIPT

3.5. 5H4PB reduces DNA damage induced by H2O2 Generation of intracellular ROS and the consequent oxidative stress could

RI PT

cause much DNA damage including single- and double-strand breaks, and base modifications (Finkel, 2003). On the basis of the above findings, we hypothesized that pretreatment with 5H4PB may reduce H2O2-induced DNA damage via increased

SC

antioxidant capacity. The luciferase-expressing A9 cells were pretreated with 5H4PB for 24 h and then exposed to 4 mM H2O2 for an additional 6 h. DNA single-strand breaks

M AN U

were examined by alkaline comet assay. As shown in Fig. S4, although exposure to H2O2 significantly increased the number of DNA strand breaks by approximately 2.6-fold that of untreated control cells (upper middle panel), the H2O2-induced DNA damage was dose-dependently attenuated by the pretreatment with 5H4PB (Fig. S5A).

TE D

On the other hand, treatment with 5H4PB after H2O2 exposure did not reduce the H2O2-induced DNA damage (Fig. S5B), indicating that 5H4PB confers the genoprotective effect against H2O2-induced DNA damage, but does not activate the

EP

DNA repair capacity.

AC C

3.6. 5H4PB protects against H2O2-induced cell death in fibroblasts The results obtained above led us to speculate that 5H4PB could confer

tolerance to oxidative stress. We determined whether 5H4PB exerts cytoprotective effect against H2O2-induced cell death. The luciferase-expressing A9 cells were pretreated with 5H4PB for 24 h and then exposed to H2O2 for an additional 24 h. As shown in Fig. 5, when the A9 cells alone were exposed to H2O2, their viability significantly decreased in a dose-dependent manner as compared with that of the

21

ACCEPTED MANUSCRIPT

untreated control cells. On the other hand, the pretreatment of the cells with 5H4PB provided significant cytoprotection against cell death induced by subsequent treatment with H2O2. The cytoprotective effect of 5H4PB was not observed in the simultaneous

RI PT

treatment with 5H4PB and H2O2 (data not shown), suggesting that the intracellular accumulation of antioxidant enzymes by pre-treatment with 5H4PB is essential for cytoprotection against H2O2. These results clearly suggest that pretreatment with

SC

5H4PB confers a significant antioxidant effect against H2O2-induced cytotoxicity.

M AN U

3.7. 5H4PB protects mouse primary hepatocytes against H2O2-induced cell death Finally, we determined whether the cytoprotective effect of 5H4PB shown in fibroblast cells also occurs in other physiological cell types. The liver is a major organ that performs a wide range of functions such as carbohydrate metabolism and

TE D

detoxification, and oxidative stress contributes to initiation and progression of liver injury. We therefore investigated the cytoprotective effect of 5H4PB against H2O2-induced cell death using mouse primary hepatocytes. Primary hepatocytes were

EP

isolated by the two-step collagenase perfusion method as described in Materials and methods. Hepatocytes were pretreated with 5H4PB for 24 h and then exposed to H2O2

AC C

for an additional 24 h. As shown in Fig. 6, when primary hepatocytes were exposed to H2O2 alone, the viability of these hepatocytes decreased as compared with that of the untreated control cells. On the other hand, the pretreatment of hepatocytes with 5H4PB for 24 h provided a significant protective effect against H2O2-induced cell death (Fig. 6). In addition, it was confirmed that the mRNA expression level of Ho-1 markedly increased after 4 h treatment with 5H4PB (Fig. S6), but no significant differences in Cat, Sod-1, and Gpx-1 mRNA expression levels were observed (data not shown). These

22

ACCEPTED MANUSCRIPT

findings strongly suggest that 5H4PB has a hepatoprotective effect against oxidative stress induced by H2O2 and the capability to induce Ho-1 via the activation of the

AC C

EP

TE D

M AN U

SC

RI PT

Nrf2/ARE signaling pathway.

23

ACCEPTED MANUSCRIPT

4. Discussion In this study, we found that 5H4PB activates the Nrf2/ARE signaling pathway, followed by the significant upregulation of cytoprotective genes. Furthermore, we

RI PT

showed that although H2O2 exposure decreased cell viability and induced DNA damage through ROS generation, the pretreatment with 5H4PB attenuated the H2O2-induced DNA damage and cell death by the reduction in intracellular ROS generation. compounds

are

broadly

found

SC

Butenolide-ring-containing

in

natural

compounds, and they show a wide variety of biological activities (Mukku et al., 2000;

M AN U

Beck et al., 2001). However, there are no reports regarding the direct activation of the Nrf2/ARE signaling pathway by these compounds, although some of these compounds were reported to increase contrarily intracellular ROS levels (Wang et al., 2006; Yang et al., 2010). The Nrf2/ARE signaling pathway can be activated by not only cellular stress,

TE D

such as oxidative stress and ER stress, but also chemical inducers that form both endogenous and exogenous sources that trigger the activation of the Nrf2/ARE signaling pathway by binding to the active cysteine residues of Keap1 (Kensler et al.,

EP

2007). As shown in Fig. 1A, real-time luciferase monitoring revealed that 5H4PB induces ARE-dependent transactivation in a dose-dependent manner in the

AC C

concentration range in which no cytotoxicity is induced, as measured by WST-1 and LDH release assays, indicating that the 5H4PB-induced activation of the Nrf2/ARE signaling pathway is not triggered indirectly by cellular stress. Indeed, we observed no significant fluorescence signals of DCF (Fig. 4) or ER stress in the 5H4PB-treated fibroblasts monitored by luciferase assay (data not shown). In addition, although 5H4PB acts as an agonist for PPARγ, it has never been reported that PPARγ agonist activates the Nrf2/ARE signaling pathway. Thus, it may be reasonable to assume that 5H4PB

24

ACCEPTED MANUSCRIPT

directly activates the Nrf2/ARE signaling pathway by binding to Keap1, although further studies are needed to clarify the mechanism in detail. As shown in Figs. 3 and S6, the mRNA expression level of Ho-1 significantly

RI PT

increased during the pretreatment with 5H4PB in both cultured fibroblasts and primary hepatocytes. HO-1, a ubiquitous stress-responsive protein, catalyzes the degradation of pro-oxidative heme to biliverdin, ferrous iron, and carbon monoxide. Biliverdin is

SC

further converted to bilirubin, which plays key roles in the scavenging of free radicals and protection against oxidative stress (Llesuy and Tomaro, 1994). Therefore, HO-1 has

M AN U

attracted significant attention for its therapeutic effects on cardiovascular and hepatic diseases (Barbagallo et al., 2013). In addition, the statistical significance of the induction of Cat and Sod-1 mRNAs was observed in mouse cultured fibroblasts by the treatment with 5H4PB (Fig. 3). CAT, contains four porphyrin heme iron groups,

TE D

accelerates the decomposition of H2O2 to molecular oxygen (O2) and water, and plays important roles in cellular protection against oxidative stress-induced cell damage (Kang et al., 2006). SOD, which catalyzes the dismutation of superoxide anion into O2

EP

and H2O2, is also one of the most important antioxidant proteins. Therefore, antioxidant enzymes regulated via the Nrf2/ARE signaling pathway may comprehensively

AC C

participate in the expression of the cytoprotective effect of 5H4PB. Here, we also used primary hepatocytes to investigate the cytoprotective effect

of 5H4PB against H2O2-induced cell death. The liver contains a family of detoxifying enzymes, including aldehyde dehydrogenase and the cytochrome P450 enzyme system, and metabolizes various compounds that produce large amounts of ROS. Oxidative stress plays a key role in the pathogenesis of both xenobiotic and drug-induced hepatotoxicity (Jaeschke et al., 2012). Our results clearly revealed that the pretreatment

25

ACCEPTED MANUSCRIPT

with 5H4PB induced the expression of Ho-1 (Fig. S6) and conferred tolerance to H2O2-induced hepatotoxicity (Fig. 6). We also demonstrated that the pretreatment with 5H4PB attenuated

RI PT

H2O2-induced DNA damage (Fig. S5). Oxidative damage induced by accumulation of intracellular ROS results in DNA damage including single- and double-strand breaks, and base modifications, many of which can trigger mutagenesis and carcinogenesis

SC

(Finkel, 2003). From our results, we concluded that the pretreatment with 5H4PB has a genoprotective effect against H2O2-induced DNA damage (Fig. S5). In the present study,

M AN U

however, when cells were treated with the 5H4PB after H2O2 exposure, no reduction of DNA damage was observed, indicating that DNA repair was not activated by 5H4PB. Although the free radical scavenging activity of butenolide-ring-containing compounds has been reported (Zhang et al., 2015), no free radical scavenging activity was detected

TE D

in 5H4PB (Fig. S3). Therefore, these findings suggest that the intracellular accumulation of antioxidant enzymes induced by 5H4PB prior to the generation of ROS plays an important role in maintaining DNA integrity.

EP

In conclusion, we have demonstrated that the pretreatment with 5H4PB confers cytoprotection against H2O2-induced cell injury. The 5H4PB-induced cytoprotective

AC C

effect depends on the ARE-driven induction of antioxidant genes. Recently, there has been increased interest in the therapeutic use of antioxidants in the treatment of diseases and disorders associated with oxidative stress. 5H4PB may have the potential to be developed as a therapeutic agent, such as vitamin E and edaravone, for reducing or preventing oxidative-stress-induced disorders and diseases.

26

ACCEPTED MANUSCRIPT

Acknowledgements We are grateful to Dr. M. Oshimura and Dr. T. Ohbayashi of Tottori University for the generous gift of A9 cells harboring the MI-MAC vector and

AC C

EP

TE D

M AN U

SC

Fujita, and A. Tada of AIST for excellent technical assistance.

RI PT

recombinase expression plasmids. We also thank T. Iwaki, N. Ohnishi, S. Sugino, Y.

27

ACCEPTED MANUSCRIPT

References Ali, Y., Alam, M.S., Hamid, H., Husain, A., Shafi, S., Dhulap, A., Hussain, F., Bano, S., Kharbanda, C., Nazreen, S., Haider, S., 2015. Design and synthesis of

RI PT

butenolide-based novel benzyl pyrrolones: Their TNF-ɑ based molecular docking with in vivo and in vitro anti-inflammatory activity. Chem. Biol. Drug Des. 86, 619-625.

SC

Barbagallo, I., Galvano, F., Frigiola, A., Cappello, F., Riccioni, G., Murabito, P., D’Orazio, N., Torella, M., Gazzolo, D., Li Volti, G., 2013. Potential therapeutic

Redox Signal. 18, 507-521.

M AN U

effects of natural heme oxygenase-1 inducers in cardiovascular diseases. Antioxid.

Beck, B., Magnin-Lachaux, M., Herdtweck, E., Domling, A., 2001. A novel three component butenolide synthesis. Org. Lett. 3, 2875-2878.

TE D

Chen, C., Kong, A.N., 2004. Dietary chemopreventive compounds and ARE/EpRE signaling. Free Radic. Biol. Med. 36, 1505-1516. Dhakshinamoorthy, S., Jaiswal, A.K., 2000. Small maf (MafG and MafK) proteins

EP

negatively regulate antioxidant response element-mediated expression and antioxidant induction of the NAD(P)H:Quinone oxidereductase1 gene. J. Biol.

AC C

Chem. 22, 40134-40141.

Dickschat, J.S., 2017. Fungal volatiles – a survey from edible mushrooms to moulds. Nat. Prod. Rep. 34, 310-328.

Dinkova-Kostova, A.T., Talalay, P., 2008. Direct and indirect antioxidant properties of inducers of cytoprotective proteins. Mol. Nutr. Food Res. 52, S128-S138. Espinosa-Diez, C., Miguel, V., Mennerich, D., Kietzmann, T., Sánchez-Pérez, P., Cadenas, S., Lamas, S., 2015. Antioxidant responses and cellular adjustments to

28

ACCEPTED MANUSCRIPT

oxidative stress. Redox Biol. 6, 183-197. Finkel, T., 2003. Oxidant signals and oxidative stress. Curr. Opin. Cell Biol. 15, 247-254.

RI PT

Forman, H.J., Davies, K.J., Ursini, F., 2014. How do nutritional antioxidants really work: nucleophilic tone and para-hormesis versus free radical scavenging in vivo. Free Radic. Biol. Med. 66, 24-35.

SC

Giasson, B.I., Ischiropoulos, H., Lee, V.M., Trojanowski, J.Q., 2002. The relationship between oxidative/nitrative stress and pathological inclusions in Alzheimer’s and

M AN U

Parkinson’s diseases. Free Radic. Biol. Med. 32, 1264-1275.

Guenthner, C.J., Luitje, M.E., Pyle, L.A., Molyneux, P.C., Yu, J.K., Li, A.S., Leise, T.L., Harrington, M.E., 2014. Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures. PLoS One 9, e87573.

TE D

Holtzclaw, W.D., Dinkova-Kostova, A.T., Talalay, P., 2004. Protection against electrophile and oxidative stress by induction of phase 2 genes: the quest for the elusive sensor that responds to inducers. Adv. Enzyme Regul. 44, 335-367.

EP

Husain, S., Alam, M.M., Shaharyar, M., Sukhbir, L., 2010. Antimicrobial activities of some synthetic butenolide and their pyrrolone derivatives. J. Enzyme Inhib. Med.

AC C

Chem. 25, 54-61.

Ito, K., Wakabayashi, N., Katoh, Y., Ishii, T., Igarashi, K., Engel, J.D., Yamamoto, M., 1999. Keap1 represses nuclear activation of antioxidant response elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 13, 76-86. Jaeschke, H., McGill, M.R., Ramachandran, A., 2012. Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity. Drug Metab. Rev. 44, 88-106.

29

ACCEPTED MANUSCRIPT

Jeong, W.S., Jun, M., Kong, A.N., 2006. Nrf2: a potential molecular target for cancer chemoprevention by natural compounds. Antioxid. Redox Signal. 8, 99-106. Kang, K.A., Lee, K.H., Chae, S., Zhang, R., Jung, M.S., Ham, Y.M., Baik, J.S., Lee,

RI PT

N.H., Hyun, J.W., 2006. Cytoprotective effect of phloroglucinol on oxidative stresses induced cell damage via catalase activation. J. Cell. Biochem. 97, 609-620. Kensler, T.W., Wakabayashi, N., Biswal, S., 2007. Cell survival responses to

SC

environmental stress via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47, 89-116.

M AN U

Koshino, H., Yoshihara, T., Okuno, M., Sakamura, S., Tajimi, A., Shimanuki, T., 1992. Gamahonolides A, B, and Gamahorin, novel antifungal compounds from stromata of Epichloe typhina on Phleum pretense. Biosci. Biotech. Biochem. 56, 1096-1099. Llesuy, S., Tomaro, M.L., 1994. Heme oxygenase and oxidative stress. Evidence of

TE D

involvement of bilirubin as physiological protector against oxidative damage. Biochim. Biophys. Acta 1223, 9-14.

Masutani, B., Tsujino, Y., An, J., Yamatsu, A., Yamashita, Y., Harada, S., 2016. PPARγ

EP

activating agent. WO2016006548A1.

McCord, J.M., 2000. The evolution of free radicals and oxidative stress. Am. J. Med.

AC C

108, 652-659.

Mukku, V.J., Speitling, M., Laatsch, H., Helmke, E., 2000. New butenolides from two marine Streptomycetes. J. Nat. Prod. 63, 1570-1572.

Nakajima, Y., Kimura, T., Sugata, K., Enomoto, T., Asakawa, A., Kubota, H., Ikeda, M., Ohmiya, Y., 2005. Multicolor luciferase assay system: one-step monitoring of multiple gene expressions with a single substrate. Biotechniques 38, 891-894. Nguyen, T., Sherratt, P.J., Pickett, C.B., 2003. Regulatory mechanisms controlling gene

30

ACCEPTED MANUSCRIPT

expression mediated by the antioxidant response element. Annu. Rev. Pharmacol. Toxicol. 43, 233-260. Noguchi, T., Michihata, T., Nakamura, W., Takumi, T., Shimizu, R., Yamamoto, M.,

RI PT

Ikeda, M., Ohmiya, Y., Nakajima, Y., 2010. Dual-color luciferase mouse directly demonstrates coupled expression of two clock genes. Biochemistry 49, 8053-8061.

SC

Ohmiya, Y., Sumiya, M., Viviani, V., Ohba, N., 2000. Comparative aspects of a luciferase molecule from the Japanese luminous beetle, Rhagophthalmus ohbai. Sci.

M AN U

Rep. Yokosuka City Mus. 47, 31-38.

Roberts, C.K., Sindhu, K.K., 2009. Oxidative stress and metabolic syndrome. Life Sci. 84, 705-712.

Scapagnini, G., Vasto, S., Abraham, N.G., Caruso, C., Zella, D., Fabio, G., 2011. of Nrf2/ARE pathway by food

polyphenols:

a nutritional

TE D

Modulation

neuroprotective strategy for cognitive and neurodegenerative disorders. Mol. Neurobiol. 44, 192-201.

EP

Takashima, M., Horie, M., Shichiri, M., Hagihara, Y., Yoshida, Y., Niki, E., 2012. Assessment of antioxidant capacity for scavenging free radicals in vitro: a rational

AC C

basis and practical application. Free Radic. Biol. Med. 52, 1242-1252.

Takiguchi, M., Kazuki, Y., Hiramatsu, K., Abe, S., Iida, Y., Takehara, S., Nishida, T., Ohbayashi, T., Wakayama, T., Oshimura, M., 2014. A novel and stable mouse artificial chromosome vector. ASC Synth. Biol. 3, 903-914. Tsujino, Y., 2017. A new agonist for peroxisome proliferation-activated receptor γ (PPARγ),

fraglide-1

from

Zhenjiang

fragrant

vinegar:

Screening

characterization based on cell culture experiments. J. Oleo Sci. 66, 615-622.

31

and

ACCEPTED MANUSCRIPT

Umeno, A., Takashima, M., Murotomi, K., Nakajima, Y., Koike, T., Matsuo, T., Yoshida, Y., 2015. Radical-scavenging activity and antioxidative effects of olive leaf components oleuropein and hydroxytyrosol in comparison with homovanillic

RI PT

alcohol. J. Oleo Sci. 64, 793-800.

Uttara, B., Singh, A.V., Zamboni, P., Mahajan, R.T., 2009. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant

SC

therapeutic options. Curr. Neuropharmacol. 7, 65-74.

Viviani, V.R., Bechara, E.J., Ohmiya, Y., 1999. Cloning, sequence analysis, and

M AN U

expression of active Phrixothrix railroad-worms luciferases: relationship between bioluminescence spectra and primary structures. Biochemistry 38, 8271-8279. Wang, Y.M., Peng, S.Q., Zhou, Q., Wang, M.W., Yan, C.H., Yang, H.Y., Wang, G.Q., 2006.

Depletion

of intracellular

glutathione

mediates

butenolide-induced

TE D

cytotoxicity in HepG2 cells. Toxicol. Lett. 164, 231-238.

Wang, X.J., Xu, H.W., Guo, L.L., Zheng, J.X., Xu, B., Guo, X., Zheng, C.X., Liu, H.M. 2011. Synthesis and in vitro antitumor activity of new butenolide-containing

EP

dithiocarbamates. Bioorg. Med. Chem. Lett. 21, 3074-3077. Wayner, D.D., Burton, G.W., Ingold, K.U., Locke, S., 1985. Quantitative measurement

AC C

of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation. The important contribution made by plasma proteins. FEBS Lett. 187, 33-37.

Yamaguchi, S., Kazuki, Y., Nakayama, Y., Nanba, E., Oshimura, M., Ohbayashi, T., 2011. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector. PLoS One 6, e17267. Yang, H.Y., Wang, Y.M., Peng, S.Q., 2010. Metallothionein-I/II null cardiomyocytes are

32

ACCEPTED MANUSCRIPT

sensitive to Fusarium mycotoxin butenolide-induced cytotoxicity and oxidative DNA damage. Toxicon 55, 1291-1296. Zhang, P., Li, X.M., Wang, J.N., Li, X., Wang, B.G., 2015. New butenolide derivatives

AC C

EP

TE D

M AN U

SC

scavenging activity. Phytochem Lett 11, 85-88.

RI PT

from the marine-derived fungus Paecilomyces variotii with DPPH radical

33

ACCEPTED MANUSCRIPT

Figure captions Fig. 1. Chemical structure of 5-hydroxyl-4-phenyl-butenolide (5H4PB).

RI PT

Fig. 2. Effects of 5H4PB on ARE-driven luciferase reporter activity and viability of luciferase-expressing A9 cells. (A) Effects of 5H4PB on ARE-dependent transcription. The cells were seeded in a 24-well plate and treated with 5H4PB at various

SC

concentrations. Bioluminescence was recorded in real time for 10 s at 13 min intervals for 24 h. The time-dependent change in ARE activation was expressed as fold change

M AN U

where the relative bioluminescence intensity (SLR3/SLG) of the 5H4PB-treated cells was normalized to that (SLR3/SLG) of the untreated control cells at each time point. Values are means ± SD (n = 4). (B) Effects of 5H4PB on cell viability and cell membrane. Cell viability and cell membrane damage were measured by WST-1 assay

TE D

and LDH release assay, respectively, and the results are expressed as percent of untreated controls. The red dotted vertical line indicates the 5H4PB concentration at 50 µM. Values are means ± SD (n = 3). * P < 0.05, ** P < 0.01, compared with untreated

EP

control. (C) Localization of Nrf2 in 5H4PB-treated cells. The cells were treated or not treated with 50 µM 5H4PB for 4 h, and then fixed, permeabilized, and immunostained

AC C

with anti-Nrf2 antibody (Alexa Fluor 546, red). Nuclei were stained with DAPI (blue). Merged images appear in the right panels. Scale bar = 50 µm. Magnified images of 5H4PB-treated cells marked by a white arrowhead are shown in the upper right corners of the panels. Schematic drawing of reporter plasmids is shown above the panels. Keys: HS4, HS4 insulator; TK, TK promoter; ARE RE, ARE response element; pA, polyA signal.

34

ACCEPTED MANUSCRIPT

Fig. 3. Effects of 5H4PB on expression of endogenous ARE-driven genes in luciferase-expressing A9 cells. The mRNA expression levels of Ho-1, Cat, Sod-1, and Gpx-1 were determined by quantitative real-time PCR analysis at the indicated time

RI PT

points after treatment of cells with 50 µM 5H4PB. Each mRNA expression level was normalized to corresponding β-actin value and presented as relative units compared with untreated control. Values are means ± SD (n = 3). * P < 0.05, ** P < 0.01,

SC

compared with untreated control.

M AN U

Fig. 4. Effects of 5H4PB on intracellular ROS production in luciferase-expressing A9 cells. The cells were pretreated with 5H4PB at various concentrations for 24 h, exposed to H2O2 for an additional 2 h and then observed under a fluorescence microscope (A). Intracellular ROS levels were measured by the DCFH method using a flow cytometer

TE D

(B). The standardized DCF fluorescence intensity of the untreated control was 1. The upper panels are fluorescence microscopy images of untreated controls and H2O2-exposed cells with or without the pretreatment with 20 µM 5H4PB. Values are

EP

means ± SD (n = 3). ** P < 0.01, compared with untreated control.

##

P < 0.01,

compared with cells exposed to H2O2 at corresponding concentrations without 5H4PB

AC C

pretreatment.

Fig. 5. Cytoprotective effect of 5H4PB against H2O2-induced cell death in luciferase-expressing A9 cells. The cells were pretreated with 5H4PB at various concentrations for 24 h and then exposed to H2O2 for an additional 24 h. Cell viability was measured by WST-1 assay, and results are expressed as percent of untreated controls. Values are means ± SD (n = 3).

#

P < 0.05,

35

##

P < 0.01, compared with cells

ACCEPTED MANUSCRIPT

exposed to H2O2 at corresponding concentrations without 5H4PB pretreatment.

Fig. 6. Cytoprotective effect of 5H4PB on mouse primary hepatocytes against

RI PT

H2O2-induced cell death. The mouse primary hepatocytes were pretreated with 10, 60, or 100 µM 5H4PB for 24 h and then exposed to H2O2 for an additional 24 h. Cell viability was measured by WST-1 assay, and results are expressed as percent of ##

P < 0.01, compared with cells

SC

untreated controls. Values are means ± SD (n = 3).

AC C

EP

TE D

M AN U

exposed to H2O2 at corresponding concentrations without 5H4PB pretreatment.

36

ACCEPTED MANUSCRIPT

Fig. S1. Real-time bioluminescence recordings of 5H4PB-treated luciferase-expressing A9 cells. Red and green circles represent ARE-TK-driven and TK-driven light outputs of SLR3 and SLG, respectively. Values are means ± SD (n = 4). Schematic drawing of

promoter; ARE RE, ARE response element; pA, polyA signal.

RI PT

the reporter plasmids is shown above the panels. Keys: HS4, HS4 insulator; TK, TK

SC

Fig. S2. Effects of 5H4PB on CAT and SOD activities in luciferase-expressing A9 cells. CAT and SOD activities were determined at the indicated time points after treatment of

compared with untreated control.

M AN U

cells with 50 µM 5H4PB. Values are means ± SD (n = 3). * P < 0.05, ** P < 0.01,

Fig. S3. Radical-scavenging activity of 5H4PB. (A and B) Radical-scavenging effects of

TE D

5H4PB and Trolox. Radicals were induced by AAPH. Fluorescein (10 µM) was used as a probe and its decay was monitored at 494 nm. (C) Plot of lag phase against Trolox

EP

concentration.

Fig. S4. Effects of 5H4PB on GSH/GSSG ratio in luciferase-expressing A9 cells. The

AC C

cells were pretreated with 5H4PB at various concentrations for 24 h and then exposed to H2O2 for an additional 4 h. GSH/GSSG ratio was determined as described in Materials and methods. Values are means ± SD (n = 3). ** P < 0.01, compared with untreated control. ## P < 0.01, compared with cells treated with H2O2 alone.

Fig. S5. Genoprotective effect of 5H4PB on DNA integrity in luciferase-expressing A9 cells. (A) The cells were pretreated with 5H4PB at various concentrations for 24 h and

37

ACCEPTED MANUSCRIPT

then exposed to H2O2 for an additional 6 h. The upper panels are comet images of untreated controls (left panel) and H2O2-exposed cells without (middle panel) or with (right panel) the pretreatment with 5H4PB. (B) The cells were treated with 5H4PB for

RI PT

24 h after the exposure to H2O2 for 6 h. The tail lengths of DNA were obtained by analyzing at least 50 random comet images for each treatment. Values are means ± SD (n = 3). ** P < 0.01, compared with untreated control. ## P < 0.01, compared with cells

SC

treated with H2O2 alone.

M AN U

Fig. S6. Effects of 5H4PB on expression of Ho-1 in mouse primary hepatocytes. mRNA expression levels were determined by quantitative real-time PCR analysis at the indicated time points after treatment of primary hepatocytes with 100 µM 5H4PB. Each mRNA expression level was normalized to corresponding β-actin value and presented

TE D

as relative unit compared with untreated control. Values are means ± SD (n = 3). ** P <

AC C

EP

0.01, compared with untreated control.

38

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 1

ACCEPTED MANUSCRIPT HS4 ARE RE TK x2

SLR3 pA HS4 x2

HS4 TK x2

SLG pA HS4 x2

(A)

50 µM 40 µM 30 µM 20 µM 10 µM

RI PT

1.5

1

0.5

(B)

WST-1

80

10 15 Time (h)

TE D

60 40

LDH release

0

AC C

1

EP

% of control

100

20

(C)

5

5H4PB (μM)

Nrf2

20

M AN U

0

SC

Fold change

2

10 5H4PB (µM)

DAPI

25

**

**

** **

* *

100

Overlay

0

50

Figure 2

ACCEPTED MANUSCRIPT

40 20 ** 12 - +h

24 - +h

4h

12 h

24 h

TE D

1

EP

*

AC C

Relative expression level

Sod-1 2

**

3 2 1

**

0 h 5H4PB 4- +

12 - +h

24 - +h

4h

12 h

24 h

M AN U

0 5H4PB 4- h+

4

RI PT

**

5

SC

**

60

Relative expression level

Cat

Relative expression level

Relative expression level

Ho-1

80

Gpx-1

2

1

0 5H4PB 4- h +

12 - +h

24 - +h

0 h 5H4PB 4- +

12 - +h

24 - +h

4h

12 h

24 h

4h

12 h

24 h

Figure 3

ACCEPTED MANUSCRIPT

(A)

H2O2 (mM) 0

RI PT

4

0

SC

5H4PB (μM)

20

M AN U

100 μm

(B)

5 μM

6 **

4 **

2 0

6

**

10 μM

6

4

## ##

TE D

DCF fluorescence intensity (rel.)

Control

6 ##

4

##

2

2

2

0

0

0

0 1 2 3 4 5

0 1 2 3 4 5

## ##

0 1 2 3 4 5

H2O2 (mM)

AC C

EP

0 1 2 3 4 5

4

20 μM

Figure 4

5 μM 100

50

100

50

0

#

0

20 μM 100

# ##

#

## ##

50

0

0 1 2 3 4 5

0 1 2 3 4 5

50

0 0 1 2 3 4 5

H2O2 (mM)

EP

TE D

0 1 2 3 4 5

M AN U

100

10 μM

AC C

Cell viability (% of control)

Control

SC

RI PT

ACCEPTED MANUSCRIPT

Figure 5

SC

RI PT

ACCEPTED MANUSCRIPT

10 μM

60 μM

M AN U

Control

100 μM ##

100

100

100

100

## ## ##

##

50

0

0

0

500 1000

## ##

50

TE D

50

0

500 1000

50

0

0 0

500 1000

0

500 1000

EP

H2O2 (µM)

AC C

Cell viability (% of control)

##

Figure 6

ACCEPTED MANUSCRIPT

Highlights 5H4PB activates the Nrf2/ARE signaling pathway. 5H4PB increases the expression levels of antioxidant genes.

RI PT

5H4PB reduces oxidative stress.

AC C

EP

TE D

M AN U

SC

5H4PB protects against hydrogen peroxide-induced cell death.

2