[팬뀔웬괜헝딛핸응
nd Ufe Scien∞
Vol. 6. No. 5. 2009 'Protective effect of dieckol on y-ray radiation-induced
V79-4
lung fibroblast cell damage invol ved in modulation of reacti ve oxygen speciesMei Jing Piao
，
Kyoung Ah Kang，
and Jin Won Hyun Depar1men1이Bi∞
hemis1ry，Jeju Nalional Universily SCh∞
1이Medicine，Jeju，Korea「
Abstract 1----'
lonizing radiation can induce 。치da1ive stress through generation 이reaclive oxygen s야cies (ROS) resulting in cell damage and cell dealh. We have investigated the radioprotective effect 01 dieckol，which was isolated from Ecklonia cava，against。
xidalive stress induced cel1 damage in Chinese hamster lung fibroblast N79-4) cells. Dieckol was found to reduce Ihe in1racellular ROS generated by y-ray radiation. Moreover，dieckol also protected the cell viability damaged by the radiation through inhibltion 이apoptosis. Irradiated cells wi1h dieckol 1rea1ment reduced .1he expression of phospho hislone H2A.X (a marker for DNA slrand breakage) and Ihe activation 01 caspase 9，which were induced by radiation. These resulls suggesl Ihal dieckol protec1ed y-ray radiation induced apoptosis 01 V79-4 lung libroblast cells by inhibiling ROS generation. (J Med Ufe Scl 2009;6:366-372)Key Words : Dieckol，Reaclive oxygen species，Cell damage，Apoplosis
closed-chain trimer of phloroglucino1) ，6，6-bieckol (a hexamer) ，dieckol (Bhexamer) ，phlorofucofuroeckol (a pen1.amerl and triphlorethol-A Lhat are inOuential for biological aetivitieslO. 11. 15)，Among t.hese phlorotannins dieekol is one of 1he major and active compounds. Among these phlorolannins‘dieekol is one of the major and active compounds. Its attributes include antioxidant ae디Vit.y15)‘
antiallergic aetivitylG). i띠libition of human immunodeficieney vlms- 1 reverse tr밍lscnp뻐sel7l
TItis study focused on evaluBting the protective efTect of dieckol on y-ray radiation-indueed V79-4 lung fibroblast cell damage Bnd eell death involved in ROS
1. Reagents
Dieckol (Fig. J) was obtained from Professor Nam Ho Lee
。
f Jeju National University，Korea. 'The purity of dieckol was Bssessed by HPLC and was>
90%. Dieckol was freshly dissolved in dimethyl sulfoxide (DMSO)，yielding a final concentration ，which did not exceed 0.1 %. 2' ，7’-dichlorodihydrofluorescein diacetale (DCF-DA) ， [3-(4.5-dime1hylt1liáwl-2-:피)-2.5-diphenyltetrazoliwn) bromide (MTfl and Hoechst 33342 were pw'이lased from llie Sigma Chemical Company (SI. Louis‘MO. USA). 111e primary casp잉e9and anti-phospho histone H2AX antibodies were pureha<>edfrom Cell Signaling Technology (Beverly. MA. USA)
Introduction
Reactive oxygen species，including the superoxide anion ， hydroxyl radical. single oxygen，and hydrogen peroxide‘are oxygen eontaining molecules with unpaired eleetrons or abstraet elecLrons from other molecules. These reactive
。
xygcn speeies ean lead to functional damage in lipid‘proteins and DNA，which can eventually result in cell death!l，Gamma-ray radiation is known to induce oxidative stress via the generation of reaetive oxygen species in cells2， 3). ln many eases，radiation-induced cell death has been identified as apoptosis"-61
EckJollia cavais 8 brown 81ga (Laminariaceae) that is abundant in the subtidal regions of JEtiu island，Korea. It has been reported that the Ecklonia species e상libits rawcal scavenging activity7-9
’.
eytoprotective properties against。
xidat.ive sσ'ess10-14).Phlorotannin components of E. C8V8include phenolic secondary metabolit.es such BS eekol (a
Address lor correspandence : Jin Won Hyun
Departmenl01 Biochemislry，Jeju Nallonal UniversltySCh
。이 。，
Medicine.66 Jejudaehakno，690-756，Jeju，Korea E-mail: jinwonh@jejunu.ac.kr
This research was performed under lhe program 01 Basic Alomic Energy ResearchInslilule (BAERI)whlch is part 01 lhe Nuclear R&D Programsand in part Iromlhe study 011heDNA repa;r regulation with lhe disease program [M1063901]
’
unded by the Mi미stry 01 Science & Technorogy01 Korea (KOSEF)i 、
Protcctiveeπ'ectofdi에mlony-rayradiation-inducedV79-4lungfibroblastcelldamageinvolvedin mod비ationof react.iveoxygenspccics
’
2. Cell culture and irradiation
Chinese hamster lung fibroblasts (V79-4) cells from Ihe American 1γpe C띠ωre Collection (Rock찌lIe. MD. USA) were maintained at 37t in an incubator with a humidified atmosphere of 5% Cα and cultured in Dulbecco’s modified
Eagle’s medium. containing 10% heal-inactivated feta1 ca1f
serum. streptomycin (100 μg/mI) and penicillin 000 unitshnl) 깨e cells were exposed lo y-ray radation at 1.5
Gy/min from a ooCo y-ray source (MDS Nordion C-1B8 standard source. Jeju National University. J잉u.Kor깅a)
3. Intracellular reactive oxygen species (ROS) measurement
The V79-4 cells were
σ
견ated wilh dieckol at 10 μg/mland were exposed ω y-ray radiation an hour later‘111ecells
were incubated for an additional 24 h at 37t. After adding 25 tLM of DCF-DA solution. the nuorescence of 2; . 7'
dichloronuorescein was detected using a Perkin Elmer LS-58 spectronuorometer18)
4. Cell viability
π1e efTect of dieckolon the viabilit;yof the V79-4 cells was determined using Ihe (3-(4. 5-dimelhyllhiazo!-2-yi)-2. 5
이phenyltelrazo!ium] bromide (M'π') assay. which is based on
the reduction of a tetrazolium salt by mitochondrial dehydrogenase in viable cellsl9)‘까1e V79-4 cells were treBlecI
wilh dieckol at 10 I4l/ml and wilh y-ray. Forty ei앙1t hours !ater. 50여
。
f Ihe MTJ‘sωck so!ution (2mg/ml) was added ω each well ω reach a total reaction volume of 200여 After incubating for 4 h. the plate was cenbifuged at 800 X g for 5 Figure 1. Chemical structure of dieckolι
졌
r
띤뱃
값
Z
챔?
min followed by aspiration of the supematants. The fonnazan crystals in each weJl were dissolved in 150여
。
f DMSO and the A.5.oW8Sread on a scanning mtùti-well spectropho1omet:er 5. Nuclear staining with Hoechst 333421꺼e V79-4 cells were b'한.ted wilh diecko! at 10앵Iml 없d
with Y-r8Y radiation at 10 Gy an hour later. Next. the cells were incubated for an additional 48 h at 37'C. 1.5 찌
。
f Hoechst 잃342 (stock 10 mg/ml). which is a DNA-specificf1uorescent dye‘was added 10 each well and incubated for 10 min at 37'C. The stained cells were visualized under a f1uorescent microscopc，equipped with a CoolSNAP-Pro color
이gi어l∞mera 10 examine the de밍-ee of nuclear condensation
6. Western blot analysis
The V79-4 cells were treated with dieckol at 10 μg/ml andwith ‘y-ray raωa
“。
n at 10 Gy. an hour later. Next，the cells were lncubated for 48 h al 37t ，and harvested ，followed by washing twice with PBS. The harvesled. cells were then Iysed on ice for 30 min in 100띠
。
f a Iysis buffer [120 mM NaCI. 40 mM 1뇌s (pH 8). 0.1% NP 40] and centrifuged at 13.α)()X g for 15 min. The supernatants were collected from the Iysates 밍1d the protein concentrationswere detennined. Aliquots of the lysates (40 pg of protein) were boiled for 5 min and elecU'ophoresed in 10% SDS-polyacrylamide gel. 111e blots in the gels were transferred onto nilrocellulose membranes (Bio-Rad. Hercules，CA. USA)，and subsequently incubated with anti-primary antibodies. The membranes were further incubated with secondary antiimmunoglobulin-G-horseradish peroxidase conjugates (Pierce，Rockford，IL. USA). followed by exposure lo X-ray film. The proæin bands were detected using 뻐
enhanced chemiluminescence westem blotting detection kit (Amersham. Li띠e Chalfonι Buckinghamshire. UK)
7. Statistical analysis
All measurements were made in triplicale and all values were exþl-esscd as the means :!::standard error of the mean (S.E.MJ. 1'he results were subjected to an analysis of variance (ANOVA) using the Tukey test ω analyze lhe difference. P
<
0.05 was considered significantJy.Results
1. Protective effect 01 dieckol on r-ray radiation induced ROS generation
MeiJing Piao. KyoungAh K어19.Jin WonHyun
damage. To detennine whether the radioprotective effect of dieckol on V79-4 lung fibroblast cells involve in ROS， intracellular ROS were detected by spectrof1uorometer 만，e levels of intracellular ROS increased markedly in V79-4 cells exposure to different dose of irradiation from 0 to 20 Gy (data not shown). We measured the radical scavenging effect of dieckol on the ROS generated by y-ray radiation at 24 h and found that the level of ROS produced by racliation is increased to 137% compared to control and in dieckoltreated irrad1ated cells the ROS level is decreased to 119%，suggesting that dieckol scavenged 상1e ROS generated by irradiation (Fig. 2)
Figure 2. Effect of dieckol on scavenging intraee!l비ar reaetive oxygen specîes generated by y-ray irradiation. The V79-4 cells were treated with dieckol at 10 명1m!，followed by y-ray lπadiation at 20 Gy an hour later. Next，the cells were incubated for 24 h，the intrac마l버앙 reactive oxygen species
、
Nas detected using f1uorescence spectrophotomerer after DCF-DA staining’
Signillcantlydifferent from 20 Gy따-adiatedcells (P<
0.05) 2QO2. Protective eftecl 01 dieckol on y-ray radiation ind uced cell death
돼e viabilities of V79-4 lung fibroblast cells exposure to
며fferent dose of iπadiation were detected to figure out the destruetive effect of irradiation. It shown that irradiation reduced cell viabilities in a dose-dependent marmer from 0 to 20 Gy (data not shown). The protective effect of dieckol
。
n cell survival in iπadîated' y-raycells was measured. Cells were treated、”다
1 dieekol at 10 g/ml for 1 h，prior to the exposed to radiEi.tion.Cell viability was detennined 48 h later by the M'tr assay. As shown in Fig. 3，treatment with dieckôl increased the cell surviva1 by 76% as comparèd to 66% of ìrradiated y-ray at 20 Gy3. Protective effecl 01 dieckol on y-ray radiation induced apoptosis
To evaluate a cytoprotective effect of dieckolon apoptosis induced by y-ray radiation，the nuclei of V79-4 eells were stained with Hoechst 33342 and assessed by microscopy The microscopic pict니res in Fig. 4A showed that the control
cells had intact nuclei，while Ìrradiated y-ray cellsshowed signilicant nuclear fragmentation. which is characteristic of apoptosis. However，when the cells were treated with dieckol for 1 h prior to radiation，a drama디c decrease in nuclear fragmentation was observed. In addition ，the phosphorγlation of the nuclear histone H2A.X，a sensitive
'"
'"
'# 80 þ:0
6ρ1 연 〉꽁애
Control Dieckol 10 Gy Dieckol+10Gy
Activecaspase 9 ß-Actin Phospho H2A.X
B
A
멘헬灣렐|
Figure 4. Effect of diecko] upon the y-ray radiution-induced ccllular damage of V79-4 cells. The V79-4 cells were treated
~“
1 diecko] at 10ttglml. followed by y-ray irradiation at 10 Gy an hour later. Next. the cells were incubated for 24 h. (A) Apoptotic bo여 forma디on was obseπed under a f1uoreseenee mieroscope after Hoechst 33342 sæining and apoptotic bodies are indieated by arrows. (B) The cell lysates were electrophoresed，phospho histone H2A.Xprotein and caspase 9 were detected by a specifie antibody20Gv 미~k이+20Gv 20Gy Dleckol+20Gy
c，"띠 미ecl\ol Cootr이 Dleckol
"
Figure 3. Effect of dieckol on y-ray irradiatîon-jnduced cell death of V79-4 cells. The V79-4 cells were treated with dieekol at 10탱1m]，followed by y-ray irradiation at 20 Gy an hour later. Next，the ceils were incubated for 48 h. The viability of V79-4 cells on the irradiation was determined by MTT assay. The measurement.s were made in triplicate and values are expressed as means ::t S.E.M. *Sîgnificantly different from 20 Gy îrradiated cells (P
<
0.05)Protκtiveeffectofdicckol00y-ray
’
-adiation-indu∞
d \179-4lungfibroblastcelldwnagcinvolvedin modulationof reactiveoxygensp떠csmarker for breaks of double stranded DNA20l，increased in the ÎlTadiated y-ray cells. as shown by western blot (Fig 48). However. dieckol treatment in irradiated y-ray cells decreased the expression of phosphor H2A.X. NexL. we examined the activity of caspase."g by wesrern blot. since caspase g. i.5 known as an 때tiaωr of apoptosis21l. Dieckol
inhibited the y-ray radiation-induced 8ctive fonn of caspase 9 (37 kDa) (I'ig. 4B). These resuJts suggest that dieck이
protects ceU viability by inhibiling the damage of cellular components and apoptosis induced by y-ray radiation
1) HaJJiwell B. Gutteridge JM. RoJe of free radicals and catalytic metal ions in human disease: an overγlew Methods Enzymol 1990;186:1-85.
2) Ewing D. Jones SR. Superoxide removal a띠d radiation
protection in bacteria. A떠1 Biochem Biophys 1987 Apr; 254(1):53-62.
3) Mikke1sen .RB. Wardman P. Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms. Oncogene 2003 Sep 1;22(37):5734-54
Exposure of cells to ionizing radiation can lead t
。
increased generation of ROS，including hydroxyl radicals ((HO.). superoxide anions(αn
，sing1et oxygen (lÛ2) and hydrogen pero잉de (H，
o，).
which 앙e major deterrninants of‘cellular damage. Therefore‘ROS-scavenging agents are
considered as radioprotectors22. 23). Dieckol is a polyrner of ph1oroglucino1with a polyphenol strucωre. The existence of a pheno1ic group with an aromatic conjugation of the struclure of dieckol contributes ω the quenching of reactive
。
xygen species generated by irradiation. Our data demonstrate that dieckol produces a radioprotective effect。
n V79-4 lung fibrob1ast cells. which are known ω be sensitive ω irradiation π113protective mechanism of dieckol invo1ves its ability to scavenge ROS. Jn màl1Ycases‘they-ray radiation-induced cell death has resulted in apopωsis6 24). Diecko1 increased cell survival vîa inhibition of )'-ray radiation-induced apoptosis. This cytoprotective effect induced by dieckol was associated with the inhibited expression of phospho-H2A.X and caspase 9 activity. Taken together. the radioprotective efTect of diecko1 against y-ray radiation-induced cell damage was exerted via ROS scavenging acti찌ty
亡
Discussion
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![Figure 4. Effect of diecko] upon the y-ray radiution-induced ccllular damage of V79-4 cells](https://thumb-ap.123doks.com/thumbv2/123dokinfo/4963294.51528/3.889.175.791.804.1080/figure-effect-diecko-radiution-induced-ccllular-damage-cells.webp)
