European Journal of Pharmacology 591 (2008) 114–123

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European Journal of Pharmacology

j ourna l homepage: www.e lsev ie r.com/ locate /e jphar

Eckol protects V79-4 lung fibroblast cells against γ-ray radiation-induced apoptosisvia the scavenging of reactive oxygen species and inhibiting of thec-Jun NH2-terminal kinase pathway

Rui Zhang a, Kyoung Ah Kang a, Mei Jing Piao a, Dong Ok Ko a, Zhi Hong Wang a, In Kyung Lee b,Bum Joon Kim b, Il Yun Jeong c, Taekyun Shin d, Jae Woo Park e, Nam Ho Lee f, Jin Won Hyun a,⁎a Department of Biochemistry, College of Medicine and Applied Radiological Science Research Institute, Cheju National University, Jeju-si 690-756, Republic of Koreab Department of Microbiology and Caner Research Institute, Seoul National University College of Medicine, Seoul 110-799, Republic of Koreac Radiation Research Center for Innovative Technology, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185, Republic of Koread Department of Veterinary Medicine, Cheju National University, Jeju-si 690-756, Republic of Koreae Department of Nuclear and Energy Engineering, Cheju National University, Jeju-si 690-756, Republic of Koreaf Department of Chemistry, College of Natural Sciences, Cheju National University, Jeju-si 690-756, Republic of Korea

⁎ Corresponding author. Tel.: +82 64 754 3838; fax: +E-mail address: jinwonh@cheju.ac.kr (J.W. Hyun).

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.06.086

A B S T R A C T

A R T I C L E I N F O

Article history:

The radioprotective effect of Received 26 January 2008Received in revised form 12 May 2008Accepted 22 June 2008Available online 28 June 2008

Keywords:EckolRadiationOxidative stressReactive oxygen speciesApoptosisJNK pathway

eckol against γ-ray radiation-induced oxidative stress and its possible protectivemechanisms were investigated. Eckol was found to reduce the intracellular reactive oxygen speciesgenerated by γ-ray radiation. Moreover, eckol also protected against radiation-induced cellular DNA damageand membrane lipid peroxidation, which are the main targets of radiation-induced damage. In addition,eckol recovered the cell viability damaged by radiation via the inhibition of apoptosis. Irradiated cells witheckol treatment reduced the expression of bax, the activation of caspase 9 and caspase 3, which were inducedby radiation. However, irradiated cells with eckol recovered the expression of bcl-2 and mitochondrialcytochrome c which were decreased by radiation. The anti-apoptotic effect of eckol exerted via the inhibitionof mitogen-activated protein kinase kinase-4 (MKK4/SEK1)-c-Jun NH2-terminal kinase (JNK)-activatorprotein 1 (AP-1) cascades induced by radiation. In summary, the results suggest that eckol protects cellsagainst the oxidative stress induced by radiation via the reduction of reactive oxygen species and theattenuation of activation in SEK1-JNK-AP-1 pathway.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Ionizing radiation is known to generate reactive oxygen species inirradiated tissue and cells. Because cells contain 80% water, theprimary damage caused by radiation is due to aqueous free radicals,which are generated by the action of radiation on water. These freeradicals react with cellular macromolecules such as DNA, protein, lipidmembrane, and cause cell dysfunction and mortality (Tominaga et al.,2004; Mansour, 2006). These reactions occur both in tumor andnormal cells when exposed to radiation (Nair et al., 2001). The lung isreported to be especially sensitive to oxidative stress (Murray et al.,2004). And radiation therapy is regarded as an important treatmentfor multiple thoracic malignancies. Incidental irradiation of the lungs,which are particularly susceptible to injury, is unavoidable and oftendose-limiting. The reactive oxygen species generated by radiationtherapy are directly toxic to lung cells and initiate a cascade of

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molecular events which alter the cytokine milieu of the microenvir-onment, leading to inflammation, fibrosis, and chronic oxidative stress(Ghafoori et al., 2008). Although biological modifiers, which targetoxidative stress damage for radioprotection, have been studied fordecades, there is a need for better and more potent compounds;especially from natural products to boost antioxidant defense.

Eckol is a trimeric phloroglucinol with a dibenzeno-1, 4-dioxinskeleton, and is one of the major phlorotannins isolated from Eckloniacava. E. cava is a brown alga (Laminariaceae) that is abundant in thesubtidal regions of Jeju island, Korea. It has been reported that theEcklonia species exhibits radical scavenging activity (Kang et al.,2003, 2004a,b), cytoprotective properties against oxidative stress(Kang et al., 2005a,b, 2006a,b; Moon et al., 2007), and hemeoxygenase-1 inducing activity (Kang et al., 2007). Reactive oxygenspecies, including the superoxide anion, hydroxyl radical, singleoxygen, and hydrogen peroxide, are oxygen containing moleculeswith unpaired electrons or abstract electrons from other molecules.These reactive oxygen species can lead to functional damage in lipid,proteins and DNA, which can eventually result in cell death (Halliwelland Gutteridge, 1990). Gamma-ray radiation is known to induce

Fig. 1. Chemical structure of eckol.

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oxidative stress via the generation of reactive oxygen species in cells(Ewing and Jones, 1987; Mikkelsen andWardman, 2003), and in manycases, radiation-induced cell death has been identified as apoptosis(Chen et al., 1996a,b; Lee et al., 2007).

Recently, we reported that eckol, which was isolated from E. cava,attenuated H2O2-induced cell damage through the activation of anantioxidant system (Kang et al., 2005b). Since the cells irradiated by γ-ray generate reactive oxygen species, we speculated that eckol, withits reactive oxygen species scavenging effect, may provide cytopro-tective properties against γ-ray-induced V79-4 lung fibroblast celldamage. Consequently, we investigated the protective effects of eckolon lung cell damage induced by γ-ray radiation and the possiblemechanisms providing cytoprotection.

2. Materials and methods

2.1. Materials and reagents

Eckol (Fig. 1) was obtained from Professor Nam Ho Lee of ChejuNational University, Korea. The purity of eckolwas assessed byHPLC andwas N 90%. Eckol was freshly dissolved in dimethyl sulfoxide (DMSO),yielding a final concentration, which did not exceed 0.1%. 2′, 7′-dichlorodihydrofluorescein diacetate (DCF-DA), propidium iodide, andHoechst 33342 were purchased from the Sigma Chemical Company(St. Louis, MO, USA). 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) was purchased from theInvitrogen Corporation (Carlsbad, CA, USA), and thiobarbituric acid fromBDH Laboratories (Poole, Dorset, UK). The primary anti-Bcl-2, Bax,cytochrome c, caspase 9, caspase 3, PARP, JNK, phospho JNK, SEK1, andphospho SEK1 antibodies were purchased from Cell Signaling Technol-ogy (Beverly, MA, USA), and the primary anti-phospho histone H2A.Xantibody from Upstate Biotechnology (Lake Placid, NY, USA). Theplasmid containing the AP-1 binding site-luciferase construct wasa generous gift from Professor Young Joon Surh of Seoul NationalUniversity, Korea.

2.2. Cell culture and irradiation

Chinese hamster lung fibroblasts (V79-4) cells from the AmericanTypeCulture Collection (Rockville,MD,USA)weremaintained at 37 °C inan incubator with a humidified atmosphere of 5% CO2 and cultured inDulbecco's modified Eagle's medium, containing 10% heat-inactivatedfetal calf serum, streptomycin (100 μg/ml) and penicillin (100 units/ml).The cells were exposed to γ-ray irradation at 1.5 Gy/min from a 60Co γ-ray source (MDS Nordion C-188 standard source, Cheju NationalUniversity, Jeju, Korea).

2.3. Intracellular reactive oxygen species measurement

The V79-4 cells were treated with eckol at 10 μg/ml and wereexposed to γ-ray radiation an hour later. The cells were incubated foran additional 24 h at 37 °C. After adding 25 μMof DCF-DA solution, the

fluorescence of 2′, 7′-dichlorofluorescein was detected using a PerkinElmer LS-5B spectrofluorometer and a flow cytometer (BectonDickinson, Mountain View, CA, USA), respectively (Rosenkranz et al.,1992). The image analysis for the generation of intracellular reactiveoxygen specieswas achieved by seeding the cells on a cover-slip loadedsix well plate at 2 × 105 cells/well. Sixteen hours after plating, the cellswere treated with eckol at 10 μg/ml. An hour following eckoltreatment, the plate was irradiated with 10 Gy of γ-ray. Twenty fourhours later, 100 μM of DCF-DA was added to each well and wasincubated for an additional 30min at 37 °C. Afterwashingwith PBS, thestained cells were mounted onto a microscope slide in mountingmedium (DAKO, Carpinteria, CA, USA). The microscopic images werecollected using the Laser Scanning Microscope 5 PASCAL program(Carl Zeiss, Jena, Germany) on a confocal microscope.

2.4. Electron spin resonance (ESR) measurement

Radicalswere generated by a Nordion gamma cell exactor. The doserate was 1.8 cGy/s at 21 °C, and the applied absorbed dose was 10 Gy.The ESRmeasurementswere carried out following the irradiationwiththe gamma cell exactor. The ESR spectra of control (0 Gy) and eckolwere recorded at 21 °C and at − 80 °C for 10 Gy and eckol treatment at10 Gy on a JEOL JES-FA 200 electron spin resonance (ESR) spectrometerwith the following parameters: microwave frequency of 9.19 GHz,microwave power at 1 mW, a modulation amplitude of 2 mT, and atime constant of 30 ms. After 1.5 min, the relative intensity of theradical was determined.

2.5. Single cell gel electrophoresis (Comet assay)

A comet assay was performed to determine the degree of oxidativeDNA damage (Singh, 2000; Rajagopalan et al., 2003). The cellsuspension was mixed with 75 μl of 0.5% low melting agarose (LMA)at 39 °C, and spread on a fully frostedmicroscopic slide pre-coatedwith200 μl of 1% normal melting agarose (NMA). After the solidification ofthe agarose, the slide was covered with another 75 μl of 0.5% LMA andthen immersed in a lysis solution (2.5 M NaCl, 100 mM Na-EDTA,10 mM Tris, 1% Trion X-100, and 10% DMSO, pH 10) for 1 h at 4 °C. Theslides were then placed in a gel-electrophoresis apparatus containing300 mM NaOH and 10 mM Na-EDTA (pH 13) for 40 min to allow forDNAunwinding and the expression of the alkali labile damage. Next, anelectricalfieldwas applied (300mA, 25V) for 20min at 4 °C to draw thenegatively charged DNA toward an anode. After the electrophoresis,the slides were washed three times for 5 min at 4 °C in a neutralizingbuffer (0.4M Tris, pH 7.5), followed by stainingwith 75 μl of propidiumiodide (20 μg/ml). The slides were observed with a fluorescencemicroscope and image analyzer (Kinetic Imaging, Komet 5.5, UK). Thepercentage of the total fluorescence in the tail and the tail length of the50 cells per slide were recorded.

2.6. Western blot analysis

The V79-4 cells were treated with eckol at 10 μg/ml and with γ-rayradiation at 10 Gy, an hour later. Next, the cells were incubated for 48 hat 37 °C, and harvested, followed by washing twice with PBS. Theharvested cells were then lysed on ice for 30 min in 100 μl of a lysisbuffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP 40] and centrifugedat 13,000 ×g for 15 min. The supernatants were collected from thelysates and the protein concentrations were determined. Aliquotsof the lysates (40 μg of protein) were boiled for 5 min and electro-phoresed in 10% SDS-polyacrylamide gel. The blots in the gels weretransferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA,USA), and subsequently incubated with anti-primary antibodies.The membranes were further incubated with secondary anti-immunoglobulin-G-horseradish peroxidase conjugates (Pierce, Rock-ford, IL, USA), followed by exposure to X-ray film. The protein

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bands were detected using an enhanced chemiluminescence westernblotting detection kit (Amersham, Little Chalfont, Buckinghamshire,UK).

2.7. 8-hydroxy-2′-deoxyguanosine (8-OHdG) assay

The quantity of 8-OHdG in DNAwas determined using a Bioxytech8-OHdG-ELISA kit purchased fromOXIS Health Products (Portland, OR,USA) following the manufacturer's instructions. Cellular DNA wasisolated using the DNAzol reagent (Life Technologies, Grand Island, NY,USA) and quantified using a spectrophotometer.

2.8. Lipid peroxidation assay

Lipid peroxidation was assayed by a thiobarbituric acid re-action (Ohkawa et al., 1979). The V79-4 cells were treated with eckolat 10 μg/ml and with γ-ray radiation at 10 Gy an hour later. The cellswere then incubated for 24 h at 37 °C, followed by washing with coldPBS, scraping and homogenizing in ice-cold 1.15% KCl. One hundred μlof the cell lysateswasmixedwith0.2ml of 8.1% SDS,1.5ml of 20% aceticacid (adjusted to pH 3.5) and 1.5 ml of 0.8% thiobarbituric acid (TBA).Next, distilled water was added to the mixture to reach a final volumeof 4 ml, followed by heating to 95 °C for 2 h. After cooling the mixtureto room temperature, 5 ml of an n-butanol and pyridinemixture (15:1,v/v) was added to each sample, followed by gentle shaking. Aftercentrifuging the mixture at 1000 ×g for 10 min, the supernatantfraction was isolated and the absorbance was measured spectro-photometrically at 532 nm.

2.9. Cell viability

The effect of eckol on the viability of the V79-4 cells was determinedusing the [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium]bromide (MTT) assay, which is based on the reduction of a tetrazoliumsalt by mitochondrial dehydrogenase in viable cells (Carmichael et al.,1987). The V79-4 cells were treated with eckol at 10 μg/ml and with γ-ray. Fortyeight hours later, 50 μl of theMTTstock solution (2mg/ml)wasadded to each well to reach a total reaction volume of 200 μl. Afterincubating for 4 h, theplatewas centrifuged at 800 ×g for 5min followedby aspiration of the supernatants. The formazan crystals in each wellwere dissolved in 150 μl of DMSO and the A540 was read on a scanningmulti-well spectrophotometer.

2.10. Nuclear staining with Hoechst 33342

The V79-4 cells were treated with eckol at 10 μg/ml and with γ-rayradiation at 10 Gy an hour later. Next, the cells were incubated for anadditional 48 h at 37 °C. 1.5 μl of Hoechst 33342 (stock 10 mg/ml),which is a DNA-specific fluorescent dye, was added to each well andincubated for 10min at 37 °C. The stained cells were visualized under afluorescent microscope, equipped with a CoolSNAP-Pro color digitalcamera to examine the degree of nuclear condensation.

2.11. Detection of apoptotic sub-G1 hypodiploid cells

The amount of apoptotic sub-G1 hypodiploid cells was determinedusing flow cytometer (Nicoletti et al., 1991). The V79-4 cells weretreated with eckol at 10 μg/ml and with γ-ray radiation at 10 Gy, anhour later. Next, the cells were incubated for an additional 48 h at37 °C, followed by harvesting and fixing in 1 ml of 70% ethanol for30 min at 4 °C. The cells were then washed twice with PBS, andincubated for 30 min in the dark at 37 °C in 1 ml of PBS containing

Fig. 2. Effect of eckol on scavenging intracellular reactive oxygen species generated by γ-rirradiation at 10 Gy an hour later. Next, the cells were incubated for 24 h, the intracellul(B) confocal microscope, (C) flow cytometer after DCF-DA staining, and (D) ESR spectromet

100 μg of propidium iodide and 100 μg of RNase A. A flow cytometricanalysis was performed using a FACS Calibur flow cytometer. The sub-G1 hypodiploid cells were assessed based on the histograms generatedby the Cell Quest and Mod-Fit computer programs.

2.12. DNA fragmentation

Cellular DNA fragmentation was assessed by analyzing thecytoplasmic histone-associated DNA fragmentation, using a kit fromRoche Diagnostics (Portland, OR, USA) according to themanufacturer'sinstructions.

2.13. Mitochondrial membrane potential (Δψ) analysis

The cells were harvested, washed and suspended in PBS containingJC-1 (10 μg/ml). After 15 min of incubation at 37 °C, the cells werewashed, suspended in PBS and analyzed by flow cytometer (Troianoet al., 2007).

2.14. Preparation of the nuclear extract and electrophoretic mobilityshift assay

The cells were harvested, and subsequently lysed on ice with 1 mlof lysis buffer (10 mM Tris–HCl, pH 7.9, 10 mM NaCl, 3 mMMgCl2, and1% NP-40) for 4 min. After 10 min of centrifugation at 3000 ×g, thepellets were resuspended in 50 μl of extraction buffer (20 mM HEPES,pH 7.9, 20% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, and1 mM PMSF), incubated on ice for 30 min and centrifuged at 13,000 ×gfor 5min. The supernatant (nuclear protein) was stored at − 70 °C afterdetermining the protein concentration. The oligonucleotides contain-ing the transcription factor AP-1 consensus sequence (5′-CGC TTGATGACT CAG CCG GAA-3′) were annealed, labeled with [γ-32P] ATP usingT4 polynucleotide kinase and used as probes. The probes (50,000 cpm)were incubated with 6 μg of the nuclear extracts at 4 °C for 30 min, toreach a final volume of 20 μl, containing 12.5% glycerol, 12.5 mMHEPES (pH 7.9), 4 mM Tris–HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, and1 mM DTT with 1 μg of poly (dI-dC). The binding products wereresolved on 5% polyacrylamide gel, and the bands were visualized byautoradiography.

2.15. Transient transfection and AP-1 luciferase assay

The cells were transiently transfected with the plasmid harboringthe AP-1 promoter, using DOTAP as the transfection reagent, accordingto the manufacturer's instructions (Roche Diagnostics). Following anovernight transfection, the cells were treated with 10 μg/ml of eckol.After an additional incubation for 1 h, the cells were irradiated. After3 h, the cells were thenwashed twicewith PBS and lysedwith reporterlysis buffer (Promega, Madison, Wisconsin, USA). Following thevortex-mixing and centrifugation at 12,000 ×g for 1 min at 4 °C, thesupernatant was stored − 70 °C for the luciferase assay. After mixing20 μl of the cell extract with 100 μl of the luciferase assay reagent atroom temperature, the mixture was placed in an illuminometer tomeasure the light produced.

2.16. Statistical analysis

All measurements were made in triplicate and all values wereexpressed as the means ± standard error of the mean (S.E.M.). Theresults were subjected to an analysis of variance (ANOVA) using theTukey test to analyze the difference. P b 0.05 were consideredsignificantly.

ay irradiation. The V79-4 cells were treated with eckol at 10 μg/ml, followed by γ-rayar reactive oxygen species was detected using (A) a fluorescence spectrophotometer,er. ⁎ Significantly different from 10 Gy irradiated cells (Pb0.05).

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3. Results

3.1. Scavenging effect of eckol on reactive oxygen species generated as aresult of γ-ray irradiation

Wemeasured the radical scavenging effect of eckol on the reactiveoxygen species generated by γ-ray radiation at 24 h and found that thelevel of reactive oxygen species detected with a spectrofluorometer

Fig. 3. Effect of eckol upon the γ-ray radiation-induced cellular DNA damage of V79-4 cells. Than hour later. Next, the cells were incubated for 24 h. (A) Representative image and (B) percdifferent from 10 Gy treated cells (Pb0.05). (C) The cell lysates were electrophoresed and thcontent in cellular DNA was measured by an ELISA kit. ⁎Significantly different from 10 Gy i

decreased in eckol treated irradiated cells, compared to reactiveoxygen species level in irradiated cells (Fig. 2A). As shown in Fig. 2B,the red fluorescence intensity of reactive oxygen species generated bythe γ-ray radiation, using a confocal microscope, was enhanced in theγ-ray irradiated cells at 10 Gy. The eckol reduced the red fluorescenceintensity in γ-ray irradiated cells. This pattern was also confirmed byflow cytometry, showing 489 value of fluorescence intensity whichwas produced from reactive oxygen species stained by DCF-DA

e V79-4 cells were treated with eckol at 10 μg/ml, followed by γ-ray irradiation at 10 Gyentage of cellular DNA damage were detected by an alkaline comet assay. ⁎Significantlye phospho histone H2A.X protein was detected by a specific antibody. (D) The 8-OHdGrradiated cells (Pb0.05).

Fig. 4. Effect of eckol on γ-ray irradiation-induced lipid membrane damage of V79-4 cells.The V79-4 cells were treatedwith eckol at 10 μg/ml, followed by γ-ray irradiation at 10 Gyan hour later. Next, the cells were incubated for 24 h. Lipid peroxidation was assayed bymeasuring the amount of TBARS. The measurement was made in triplicate and values areexpressed as the means±S.E.M. ⁎Significantly different from 10 Gy (Pb0.05).

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fluorescence dye in eckol treated irradiated cells, compared to 739value of fluorescence intensity in irradiated cells (Fig. 2C). The ESRdata showed that the signals of control and eckol were not observed,but in 10 Gy irradiated cells, the single signal was observed. When thecells were treated with 10 μg/ml of eckol before irradiation, the radicalsingle signal was decreased (Fig. 2D). These findings suggest that eckolscavenges reactive oxygen species generated by γ-ray irradiation.

3.2. Effect of eckol on DNA damage and lipid peroxidation induced by γ-ray irradiation

The ability of eckol to inhibit cellular DNA damage and membranelipid peroxidation in γ-ray irradiated cells was investigated after 24 h.The damage to cellular DNA induced by γ-ray radiation was detectedby an alkaline comet assay, by phospho histone-H2A.X expression, andby the amount of 8-OHdG. The exposure of the cells to γ-ray radiationincreased the tail length and percentage of DNA in the tails of the cells.When the cells were exposed to γ-ray radiation, the percentage ofDNA in the tail increased to 35%. In addition, treatment with eckolresulted in a decrease to 14% as shown in Fig. 3A and B. The phos-phorylation of nuclear histone H2A.X, which is a sensitive marker forbreaks of double-stranded DNA (Rogakou et al., 1988), increased in theγ-ray radiation treated cells as shown by western blot (Fig. 3C).However, eckol treatment in γ-ray radiated cells decreased theexpression of phospho H2A.X. In addition, the 8-OHdG adduct ofDNA has been usedmost extensively as a biomarker of oxidative stress(Tope and Panemangalore, 2007). As shown in Fig. 3D, the γ-rayradiation increased the 8-OHdG amount to 10,170 pg/ml compared to3510 pg/ml in the control cells, whereas the eckol treatment decreasedthe 8-OHdG level to 8130 pg/ml, suggesting that eckol providesprotection from γ-ray irradiated DNA damage. In addition, γ-rayirradiation induced damage to the cell membrane is one of the mostimportant lesions responsible for the loss of cell viability. As shown inFig. 4, the V79-4 cells exposed to γ-ray radiation showed an increasein the lipid peroxidation, which was substantiated by the generationof TBARS. However, eckol was found to prevent the γ-ray irradiationinduced peroxidation of lipids.

3.3. Effect of eckol against apoptosis induced by γ-ray irradiation

The protective effect of eckol on the cell survival of V79-4 cellsexposed to radiation was measured after 48 h. As demonstrated inFig. 5A, the irradiated cells treated with eckol at 10 μg/ml increasedcell survival by 72%, compared to 58% in the irradiated cells at 10 Gy. In

order to study the cytoprotective effect of eckol on radiation-inducedapoptosis, the nuclei of V79-4 cells were stained with Hoechst 33342for visualization by microscopy. The microscopic images in Fig. 5Bdemonstrated that the control cells had intact nuclei, whereas theradiation-exposed cells demonstrated significant nuclear fragmenta-tion, which is characteristic of apoptosis. However, when the cellswere treated with eckol for 1 h prior to radiation, a dramatic decreasein nuclear fragmentation was observed. In addition to the morpho-logical evaluation, the protective effect of eckol against apoptosis wasalso confirmed by apoptotic sub-G1 DNA analysis, ELISA basedquantification of cytoplasmic histone-associated DNA fragmentation,and mitochondrial membrane potential (Δψ) analysis. As shown inFig. 5C, an analysis of the DNA content in 10 Gy irradiated cellsrevealed 23% increase in apoptotic sub-G1 DNA content. However,treatment with 10 μg/ml of eckol decreased the apoptotic sub-G1 DNAcontent to 14%. Moreover, the irradiated cells at 10 Gy increased thelevels of cytoplasmic histone-associated DNA fragmentations com-pared to the control cells; however, treatment with 10 μg/ml of eckolsignificantly decreased the level of DNA fragmentation (Fig. 5D). Toinvestigate the effect of radiation on the pre-treatment and post-treatment of eckol at 10 μg/ml, we determined the cell viability atdifferent times. As shown in Fig. 6A, pre-treatment of eckol protectedcell death against radiation at 10 Gy, however, post-treatment of eckoldid not show the protective effect against radiation. In addition, whenvarious concentrations of pre-treatment of eckol against radiation at10 Gy were compared in terms of cell viability, it did not show atconcentration dependence (Fig. 6B).

3.4. Effect of eckol on the caspases dependent pathway via mitochondriain apoptotic process induced by γ-ray irradiation

To understand the protection mechanism of eckol on the γ-rayirradiation induced apoptotic process at 48 h,we detected the apoptosisrelated protein expression. Beforehand, changes in the expressionof Bcl-2, an anti-apoptotic protein, and the expression of Bax, a pro-apoptoticprotein, were examined. As shown in Fig. 7A, eckol showed an increasein Bcl-2 expression and a decrease of Bax expression in γ-ray irradiatedcells. During the apoptotic process, Bcl-2 prevented the opening of themitochondrial membrane pore, whereas Bax induced the opening ofmembrane pore (Zamzami et al., 1995). The pore opening induces theloss of Δψ, which in turn induces the release of cytochrome c frommitochondria (Zamzami et al., 1996; Cai et al., 1998). The irradiated cellsresulted in the loss ofΔψ, as substantiated by an increase influorescence(FL-1) with the JC-1 dye (Fig. 7B). Eckol treatment at 10 μg/ml blockedthe loss of Δψ in γ-ray irradiation and as shown in Fig. 7A, eckol alsoinhibited the release ofmitochondrial cytochrome c. Next, we examinedthe caspase 9 activity bywestern blot since it is known that this enzymeis activated as a result of mitochondrial membrane disruption (Perkinset al., 2000). Eckol inhibited the γ-ray radiation-induced active form ofcaspase 9 (37 kDa), and caspase 3 (19 and 17 kDa), a target of caspase 9,which is further demonstrated by the cleavage of poly ADP-ribosylpolymerase (PARP) (89 kDa). These results suggest that eckol protectsfrom apoptosis by inhibiting the caspases dependent pathway viamitochondria.

3.5. Effect of eckol on SEK1-JNK-AP-1 signaling pathway

Because the JNK signal pathway plays such an important role in γ-ray irradiation induced apoptosis (Chen et al., 1996a,b; Verheij et al.,1998; Ruiter et al., 1999), we tested whether eckol regulates thissignaling pathway. As shown in Fig. 8A, the pre-treatment with eckolremarkably inhibited JNK activation in γ-ray irradiated cells at 24 h.Moreover, SEK1 is known to be one of the upstream components inthe JNK signaling pathway (Mann et al., 2006). To investigate whetherthis upstream kinase plays role in γ-ray irradiation induced JNKactivation, the SEK1 phosphorylationwas determined by western blot

Fig. 5. Effect of eckol on γ-ray irradiation-induced cell damage of V79-4 cells. The V79-4 cells were treated with eckol at 10 μg/ml, followed by γ-ray irradiation at 10 Gy an hour later.Next, the cells were incubated for 48 h. (A) The viability of V79-4 cells on the irradiation was determined by MTT assay. The measurements were made in triplicate and values areexpressed as means±S.E.M. ⁎Significantly different from 10 Gy (Pb0.05). (B) Apoptotic body formation was observed under a fluorescence microscope after Hoechst 33342 stainingand apoptotic bodies are indicated by arrows. (C) The apoptotic sub-G1 DNA content was detected by a flow cytometry after propidium iodide staining. (D) DNA fragmentation wasquantified by ELISA kit. The measurements were made in triplicate and values are expressed as means±S.E.M. ⁎Significantly different from 10 Gy irradiated cells (Pb0.05).

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Fig. 6. Effects of pre-treated and post-treated eckol on cell viability of radiation. (A) Pre-treatment of eckol indicates that eckol was treated to the cells, and 1 h later, γ-rayirradiation at 10 Gy was exposed to cells. Post-treatment of eckol indicates that γ-rayirradiation at 10 Gy was exposed to cells, and 1 h later, eckol was treated to the cells.After 48 h, the cell viability was determined by MTT assay. The measurements weremade in triplicate and values are expressed as means±S.E.M. ⁎Significantly differentfrom 10 Gy (Pb0.05). (B) Eckol at 2.5, 5, 10, and 20 μg/ml was treated to the cells and 1 hlater, γ-ray irradiation at 10 Gy was exposed to cells. After 48 h, the cell viability wasdetermined by MTT assay. The measurements were made in triplicate and values areexpressed as means±S.E.M. ⁎Significantly different from eckol untreated cells (Pb0.05).

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analysis at 12 h. As shown in Fig. 8B, the γ-ray irradiated cellsmarkedly increased the SEK1 phosphorylation levels, however, eckoleffectively inhibited γ-ray radiated induced SEK1 phosphorylation.AP-1 is a downstream target of the phospho-JNK pathway, andactivated AP-1 is involved in cell death, including apoptosis (Whit-marshm and Davis,1996). Next, we examined the effect of eckol on theDNA binding activity of AP-1 at 36 h. As shown in Fig. 8C, the γ-rayirradiated cells increased the AP-1 DNA binding activity, whereaseckol inhibited its activity. The transcriptional activity of AP-1was alsoassessed using a promoter construct containing the AP-1 binding DNAconsensus sequences, which are linked to a luciferase reporter gene.As illustrated in Fig. 8D, eckol inhibited the transcriptional activity ofAP-1 induced by γ-ray irradiation. These results suggest that eckolinhibits γ-ray irradiation induced apoptosis via the suppression of theSEK1-JNK-AP-1 pathway.

4. Discussion

Irradiation with γ-ray triggers a diverse array of functionalchanges in cells. The effects of γ-ray irradiation appear due to theability of γ-ray radiation to interact with multiple cell organelles. Todate, considerable evidences have been found that γ-ray radiationgenerates reactive oxygen species, and plays an important role onthe effect of γ-ray irradiation on cells (Bhosle et al., 2005; Choi et al.,2007). We have previously shown that eckol protects cells against

H2O2-induced cell damage via cellular antioxidant activation, and theERK-NFκB activation pathway (Kang et al., 2005b). Eckol is a polymerof phloroglucinol with a polyphenol structure. The existence of aphenolic group with an aromatic conjugation of the structure of eckolcontributes to the quenching of reactive oxygen species generated byirradiation. In our system, the DCF-DA method was used for detectingreactive oxygen species generated by irradiation. A major problem ofDCF-DA probe exists in detecting the reactive oxygen species; namely,the superoxide anion generation itself, especially in the presence ofcytochrome c (Mikkelsen and Wardman, 2003). Despite this problem,DCF-DA probe is still considered a suitable probe for detecting ofnonspecific free radical oxidants. In addition, the eckol inducedactivity of catalase, which converts hydrogen peroxide to molecularoxygen and water, was found to play a role in cytoprotection againstoxidative stress induced cell damages (Kang et al., 2005b). Conse-quently, the effect of eckol on reactive oxygen species scavenging mayinvolve the direct action on reactive oxygen species scavenging andthe indirect action of the induction of an antioxidant enzyme.Radiation-induced reactive oxygen species attack at vital cellularsites such as DNA and cell membranes, often resulting in lethal cellulardamage. Moreover, the DNA and cellular membrane are the two maintargets of radiation-induced lethality. Eckol was found to inhibit DNAtail length, expression of phospho-H2AX, and the amount of 8-OHdGinduced by γ-ray irradiation. These results confirm the protection ofcellular DNA by eckol. In addition, the formation of lipid peroxidationin cells exposed to γ-ray irradiation, is one of the importantmarkers ofmembrane damage. Eckol was found to protect cell membrane lipidsfrom peroxidation damage induced by radiation. These inhibitoryeffects of eckol against lipid and DNA damage resulted in theprotective effects against radiation-induced cell death. In manycases, the γ-ray irradiation-induced cell death has resulted inapoptosis (Kim et al., 2007; Lee et al., 2007). Eckol inhibited the γ-ray irradiation induced caspase-dependent apoptotic biochemicalchanges. Mitochondria act as an important apparatus for signalsduring apoptosis, and the loss of mitochondrial integrity can beprompted or inhibited bymany regulators of apoptosis (Kroemer et al.,1997; Green and Reed, 1998). In many cases, oxidative stress inducedcaspase activation through cytochrome c release from the mitochon-drial inter-membrane space into the cytosol (Green and Reed, 1998;Liu et al., 1996). To further elucidate the mechanisms on inhibition ofγ-ray radiation-induced apoptosis by eckol treatment, we examinedthe Δψ and mitochondrial release of cytochrome c. We found thateckol inhibited γ-ray irradiation induced the loss of Δψ and themitochondrial release of cytochrome c. During the apoptotic process,Bcl-2 prevents the opening of the mitochondrial membrane pores,whereas Bax induces the opening of membrane pores (Zamzami et al.,1995). Therefore, the blocked loss of Δψ by eckol may be a result of theup-regulation of Bcl-2, and the down-regulation of Bax. Variousstudies have suggested the possible mechanisms for the JNK pathwayrelated to mitochondrial depolarization and apoptosis induction. Fanet al. (2000) reported that JNK translocates to mitochondria and thenphosphorylates Bcl-2 and Bcl-XL, antiapoptotic members of Bcl-2family, and presumably inactivate them. Malhi et al. (2006) reportedthat JNK induces themitochondrial pathway of apoptosis by activatingBim and Bax, proapoptotic members of the Bcl-2 family. One of thesignaling pathways that has mediated γ-ray radiation-inducedapoptosis is the JNK cascade (Chen et al., 1996a; Ruiter et al., 1999).The activation of this pathway appeared to be essential in transducingapoptosis signals since the disruption of the JNK pathway by thedominant negative mutant abrogated radiation-induced apoptosis(Chen et al., 1996b; Bowen et al., 2002). The eckol blocked γ-rayradiation induced activation of the SEK1-JNK-AP-1 signaling pathway,which in turn resulted in the protection from γ-ray irradiation-induced apoptosis. Taken together, the cytoprotective effect of eckolagainst γ-ray radiation-induced apoptosis was exerted via reactiveoxygen species scavenging activity, inhibition of the JNK pathway, and

Fig. 7. Effects of eckol on apoptosis regulatory proteins and mitochondrial function. V79-4 cells were treated with eckol at 10 μg/ml, followed by γ-ray irradiation at 10 Gy an hourlater. Next, the cells were incubated for 48 h. (A) The cell lysates were electrophoresed and Bcl-2, Bax, cytochrome c, active caspase 9, active caspase 3, and cleaved PARP proteins weredetected by their specific antibodies. (B) The mitochondrial membrane potential (Δψ) was analyzed with flow cytometer after staining cells with JC-1.

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the inhibition of mitochondria involved caspase-dependent apoptosis(Fig. 9).

Acknowledgements

This research was performed under the program of Basic AtomicEnergy Research Institute (BAERI) which is part of the Nuclear R&DPrograms and in part from the study of the DNA repair regulationwiththe disease program [M1063901] funded by the Ministry of Science &Technology of Korea (KOSEF).

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