Protective activity of a novel resveratrol analogue, HS-1793, against DNA damage in 137Cs-irradiated CHO-K1 cells.

1.3 MB·English

by  Jeong, Min Ho

JournalofRadiationResearch,2014,55,464–475
doi:10.1093/jrr/rrt140 AdvanceAccessPublication7January2014
Protective activity of a novel resveratrol analogue, HS-1793, against DNA
damage in 137Cs-irradiated CHO-K1 cells
MinHoJEONG1,KwangMoYANG2,DongHyeokJEONG2,ChangGeunLEE2,SuJungOH2,
SooKyungJEONG2,KiWonLEE3,YoungRaeJO1andWolSoonJO2,*
1DepartmentofMicrobiology,Dong-AUniversityCollegeofMedicine,Daeshingongwon-gil32,Seo-gu,Busan619-953,
RepublicofKorea
2DepartmentofResearchCenter,DongNamInstituteofRadiologicalandMedicalSciences,Jwadong-gil40,Jangan-eup,
Gijang-gun,Busan619-953,RepublicofKorea
3DepartmentofOccupationandEnvironmentalMedicine,Dong-AUniversityCollegeofMedicine,Daeshingongwon-gil32,
Seo-gu,Busan619-953,RepublicofKorea
*Correspondingauthor.DepartmentofResearchCenter,DongNamInstituteofRadiologicalandMedicalSciences,
Jwadong-gil40,Jangan-eup,Gijang-gun,Busan619-953,RepublicofKorea.Tel:[phone];Fax:[phone];
Email:[email protected]
(Received7May2013;revised25September2013;accepted11November2013)
Resveratrolhasreceivedconsiderableattentionasapolyphenolwithanti-oxidant,anti-carcinogenic,andanti-
inflammatory effects. Radiation is an important component of therapy for a wide range of malignant condi-
tions.However,itcausesdamagetonormalcellsand,hence,canresultinadversesideeffects.Thisstudywas
conductedtoexaminewhetherHS-1793,anovelresveratrolanaloguefreefromtherestrictionofmetabolicin-
stability and the high dose requirement of resveratrol, induces a protective effect against radiation-induced
DNA damage. HS-1793 effectively scavenged free radicals and inhibited radiation-induced plasmid DNA
strandbreaksinaninvitroassay.HS-1793significantlydecreasedreactiveoxygenspeciesandcellularDNA
damage in 2Gy-irradiated Chinese hamster ovary (CHO)-K1 cells. In addition, HS-1793 dose-dependently
reducedthelevelsofphosphorylatedH2AXinirradiatedCHO-K1cells.TheseresultsindicatethatHS-1793
haschemicalradioprotectiveactivity.GlutathionelevelsandsuperoxidedismutaseactivityinirradiatedCHO-
K1 cells increased significantly following HS-1793 treatment. The enhanced biological anti-oxidant activity
andchemicalradioprotectiveactivityofHS-1793maintainedsurvivalofirradiatedCHO-K1cellsinaclono-
genic assay. Therefore, HS-1793 may be of value as a radioprotector to protect healthy tissue surrounding
tumorcellsduringradiotherapytoobtainbettertumorcontrolwithahigherdose.
Keywords: resveratrol analogue; HS-1793; free radical scavenging activity; reactive oxygen species (ROS);
DNAstrandbreak;radioprotection
INTRODUCTION reproduce. However, radiation also causes damage to
normal cells, so this can result in adverse side-effects [2].
Theuseofradiationhasbeenincreasingrapidlyfordiagno- The nature and degree of such unwanted side-effects
sis and treatment, and it is now indispensable in virtually dependsonthedoseofionizingradiationandthesensitivity
everybranchofmedicine.Radiationisparticularlyusefulas oftheorgansthatareirradiated.Inaddition,theuseofcom-
acomponent of therapyforawide range of malignant con- bined treatments such as concomitant radiotherapy and
ditions. It is estimated that half of all patients with cancer chemotherapy exacerbates acute damage to normal tissue
will receive radiotherapy during the course of their treat- [3]. The aim of radiotherapy is to destroy cancer cells with
ment [1].Thebasicprincipleofradiotherapyistocausesuf- aslittledamageaspossibletonormalcells,thus,theroleof
ficientdamagetocancercellDNAsuchthatthecancercells radioprotective compounds is very important in clinical
cannot repair their DNA and, therefore, cannot grow or radiotherapy.
©TheAuthor2014.PublishedbyOxfordUniversityPressonbehalfofTheJapanRadiationResearchSocietyandJapaneseSocietyforRadiationOncology.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/3.0/),which
permitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
RadioprotectiveactivityofHS-1793 465
Radiation damages cells/tissues through both direct and glutathione (GSH) and enzymes such as superoxide dis-
indirect actions [4].The term ‘direct effects’ describes radi- mutase (SOD) and catalase [17–19]. Nevertheless, the bio-
ation that causes direct irreparable damage to critical tar- logical activities of resveratrol require high doses and are
gets within cells, such as DNA. The term ‘indirect effects’ limited by photosensitivity and metabolic instability. In
describes a situation in which radiation interacts with other our previous studies, a resveratrol analogue [4-(6-hydroxy-
molecules in cells that are not critical targets but are close 2-naphthyl)-1,3-benzenediol, HS-1793] was designed to
enough to pass onthisdamage,typicallyin the form offree overcometheseproblems[20–24].Wewereinterestedinex-
radicals. Because mammals are composed of roughly 80% ploringtheuseofHS-1793asameansofamelioratingDNA
water, indirect effects include the production of hydroxyl damage caused by the oxidative stress of radiation. In the
free radicals, which are potent oxidants capable of breaking present study, we demonstrated the protective activity of
chemicalbondsandinitiatinglipidperoxidationinthenano- HS-1793 against radiation-induced DNA damage and anti-
to microsecond timeframe [5]. These free radicals interact oxidantactivityinChinesehamsterovary(CHO)K1cells.
with critical macromolecules leading to DNA damage,
which may be the most important factor in cell death [6].
Althoughcellsandtissuesareequippedwithanendogenous MATERIALSANDMETHODS
anti-oxidant system, it is incapable of protecting cells from
the hazardous effects of free radicals when these reactive Preparationoftheresveratrolanalogue,HS-1793
oxygen species (ROS) increase following exposure to The stilbene double bond present in resveratrol was substi-
irradiation[7,8]. tuted with a naphthalene ring to obtain HS-1793, as
Becauseofthegreatclinicalneedforeffectiveradioprotec- describedpreviously [20,21,24].ThesynthesisofHS-1793
tant agents, many compounds have been tested to develop is summarized in Fig. 1. A 50mM stock solution of
more effective, less toxic, drugs. Initial attempts were HS-1793 was made in absolute ethanol, and working dilu-
focused on synthetic thiol compounds. These agents are tions were prepared directly in saline. The control vehicle
highly effective at reducing the lethality induced by irradi- wasculturemediacontainingamountsofethanolequivalent
ation. However, amifostine isthe only radioprotector in this tothosepresentinHS-1793.
classthathasbeenclinicallyapprovedbytheFoodandDrug
Administration for mitigating side-effects (xerostomia) in FreeradicalscavengingactivityofHS-1793
patients undergoing radiotherapy [9].This drug offers good The following parameters were assayed to determine the
protectionbuthasmanydrawbacksincludinghighcost,side- free radical scavenging activity of HS-1793. DPPH
effects, and toxicity (nausea, vomiting, and hypotension) (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging was
[10]. Several novel approaches have been developed to determined by the method of Gadow et al. [25]with some
identify potent and non-toxic radioprotectors. Use of natu- modifications. Briefly, 10μl of various concentrations of
ral products with antioxidant activity as possible radiopro- HS-1793 (diluted to final concentrations of 1.25, 2.5, 5, 10,
tectors has gained momentum in recent years due to their 20, 40 and 80μM) were mixed with 190μl of DPPH in
lowertoxicity,reducedcost,andotherassociatedadvantages ethanol (final concentration 0.1mM) in wells of a 96-well
[11–13]. plate.Theplatewaskeptinthedarkfor10min,andabsorb-
Resveratrol (trans-3,5,4′-trihydroxy-stilbene) is found in anceofthesolutionwasmeasuredat517nmusingamicro-
a wide variety of plant species, including grapes, pea- platereader(VersaMaxMolecular Devices,Sunnyvale,CA,
nuts, blueberries, bilberries, cranberries, lingonberries, and USA). Superoxide radical scavenging activity was assessed
Polygonum cuspidatum [14],and has received considerable bytheNBT (nitrobluetetrazolium) reduction method ofMc
attention for its reputed health benefits as acardioprotective Cord et al. [26] with some modifications. The reaction
andchemopreventiveagent [15,16].Theantioxidantproper- mixture contained 134μl of buffer (50mM KH PO ,
2 4
tiesofresveratrolaremediatedbyitsabilitytoscavengefree pH 7.4), 2μl of 100mM Na EDTA, 20μl of 3mM hypo-
2
radicals and promote the activities of antioxidants such as xanthine, 2μl of 10mM NBT, and 10μl of various
Fig.1. Synthesisandchemicalstructureoftheresveratrolanalog,HS-1793.
466 M.H.Jeongetal.
™
concentrationsofHS-1793(dilutedtofinalconcentrationsof DAwasmeasuredwithaParadigm DetectionPlatformand
1.25, 2.5, 5, 10, 20, 40 and 80μM). The absorbance of the Multimode Analysis Software version 3.1.0.1 (Beckman
samples was measured immediately after adding 32μl of Coulter, Fullerton, CA, USA). The excitation and emission
xanthine oxidase (1 unit/10ml buffer) at 540nm using the wavelengthswere480nmand530nm,respectively.
microplatereader.Theplatewaskeptinthedarkfor10min,
and absorbance was measured again at 540nm. Hydroxyl
radical scavenging activity was measured using the Measurementofglutathione(GSH)level
™
OxiSelect HORAC Activity Assay Kit (Cell Biolabs, San andsuperoxidedismutase(SOD)activity
Diego, CA, USA). This assay is based on the oxidation-
The reduced intracellular GSH level was measured using a
mediatedquenchingofafluorescentprobebyhydroxylradi-
glutathione assay kit following the instructions provided by
cals produced by a hydroxyl radical initiator and Fenton
the manufacturer (Bioxytech, OXIS International, Foster
reagent.L-ascorbicacid(1mM)wasusedasthecontrol. City,USA).Inbrief,CHO-K1cells(4×105cells/well)were
seeded in a 100mm culture dish and treated with various
concentrations of HS-1793 at for 24h. The cells were
Gammairradiation
exposedto2Gyof137Csγ-radiation,andtheincubationwas
Gamma irradiation with 137Cs was carried out using
continued for 24h. The cells were then scraped and lysed.
BioBeam 8000 (Gamma-Service Medical GmbH, Bremen,
The lysates were centrifuged at 250g for 10min, and the
Germany)irradiatoratadoserateof1.88Gy/min.
supernatants were used for the assay. This method is based
on the formation of a chromophoric thione, using 4-chloro-
Cellculture 1-methyl-7 trifluoromethylquinolinum methylsulfate as the
CHO-K1 cells were obtained from the American Type chromogen,whoseabsorbancewasmeasuredat420nmona
TissueCollection(Manassas,VA,USA).Thecellswerecul- DU730 spectrophotometer (Beckman Coulter). Total GSH
tured in Ham’s F-12 nutrient mixtures supplemented with activitywascalculatedbasedonthemanufacturer’sformula.
10% fetal bovine serum (Hyclone, Logan, UT, USA). The SODactivitywasmeasuredusingasuperoxidedismutasekit
cells were maintained at 37°C in a humidified atmosphere from Trevigen, Inc. (Gaithersburg, MD, USA). Protein
with5%CO . extract (50μg)wasusedtoassaytotalSODactivity,follow-
2
ingthemanufacturer’sprotocol.Briefly,SODreactionbuffer
was mixed with xanthine solution followed by nitro blue
Cellviabilityassay tetrazoliumsolution.Thesampleproteinswereisolated24h
The number of viable cells was determined by the ability afterirradiation,andtheabsorbancewassettozeroat550nm.
of mitochondria to convert MTT to formazan dye. CHO- Finally, the xanthine oxidase solution was added to each
K1 cells were cultured overnight in 96-well plates, at a sample, and readings were taken at 550nm on a DU730
density of 2 ×104 cells/200μl in each well. The next day, spectrophotometer every 30s for 5min. Total SOD activity
the cells were coincubated with various concentrations of wascalculatedbasedonthemanufacturer’sformula.
HS-1793 for 24h. Following the incubation, the medium
was removed, and the cells were supplemented with 10μl
of 10mg/ml MTT in each well. Following another 4h in-
EstimationofplasmidpSKDNAdamage
cubation at 37°C in a humidified 5% CO atmosphere, the
2
MTT was removed, and the cells were lysed with 150μl A 5.5-kb length of plasmid pSK, was transformed in E.coli
DMSO. The absorbance was measured at 550nm using the and was purified using an Endofree Plasmid Maxi Kit
microplate reader. (QIAGEN, Valencia, CA, USA). ThepSK DNA (0.5μg) in
phosphate buffer (PBS, 0.1 M, pH 7.4) was exposed to
various doses (0–20Gy) of 137Cs γ-radiation. In addition,
DCFHtest the pSK DNA (0.5μg) in PBS was exposed to 5Gy-radi-
Intracellular oxidative stress levels were examined by the ation in the presence and absence of HS-1793 at different
DCFH test [27]. Briefly, CHO-K1 cells were cultured in concentrations. After irradiation, the DNAwas electrophor-
96-well plates, at a density of 3 ×104 cells/200μl in each esed on a 1% agarose gel in 0.08 M Tris borate/0.2mM
well, and were treated with different concentrations of EDTAbuffer(pH8.3).ThebandsofsupercoiledDNA(SC)
HS-1793 at 37°C in a humidified atmosphere with 5% CO and open circular DNA or broken DNA (OC) were visua-
2
for1h.Thecellsweresupplementedwith25μMDCFH-DA lized with SYBR Safe DNA gel staining (Invitrogen,
(2′,7′-dichlorofluorescein, Sigma-Aldrich, St Louis, Mo, Carlsbad,CA,USA)underUVlight,andquantifiedbyscan-
USA) solution and were immediately exposed to 2Gy of ning and densitometric measurement with BIO-1D analysis
137Cs γ-radiation. After irradiation, the cells were incubated software (Vilber Lourmat, France). DNA lesions were
at37°Cfor10minandthefluorescenceintensityofDCFH- expressedasadensityratiooftheOCform.
RadioprotectiveactivityofHS-1793 467
Cometassay Cellsurvivalandcolony-formingefficiency
CHO-K1 cells were cultured overnight in 6-well plates, at a Thecellsurvivalassayisbasedontheclonogenicityofcells
density of 2 ×105 cells/3ml in each well. The next day, the to divide indefinitelyand form colonies [27]. To investigate
cells were treated with different concentrations of HS-1793 cellsurvivalcurvesafterexposuretovariousdoses(0–14Gy)
for 15min, exposed to 2Gy of 137Cs γ-radiation, and incu- of 137Cs γ-radiation, CHO-K1 cells were harvested and sus-
bated at 37°C in a humidified atmosphere with 5% CO for pended in medium, and diluted cells were inoculated into
2
15min.Thecellswerecollectedandmixedwithlowmelting 100-mm dishes for colony formation. Clonogenic cell sur-
pointagaroseat37°C.Thismixturewasplacedonthetopof vival assays were performed with CHO-K1 cells exposed
thepreviouslayerof0.5%normalmeltingpointagaroseona to radiation (0–14Gy), and the experimental doses were
slide covered with a coverslip, and returned to 4°C until determined for inducing radiation-induced DNA damage.
solid. The coverslip was gently removed and some NMP CHO-K1 cells were pre-treated with HS-1793 at different
agarose was added to the slide. The slide was then covered concentrations for 24h prior to exposure to a fixed dose of
again with a coverslip and placed at 4°C until the mixture radiation (2Gy), orat aconcentration of 20μM prior to ex-
wassolid.Theslidewasplacedinchilledlysisbuffer(100mM posure to various doses of radiation (2, 4 and 6Gy). After
EDTA,2.5Msodiumchloride,10mMTrizmabaseand1% roughly 14 d, the colonies were fixed, stained with 1%
N-lauroylsarcosinate, adjusted to pH 10.0, with 1% Triton crystalviolet,andcounted.Platingefficiency(PE)wascalcu-
X-100) and unwinding buffer (1mM EDTA and 300mM latedasfollows:
sodium hydroxide, pH>13), respectively, and subjected to
PE¼ðno. of colonies counted/no. of cells platedÞ(cid:2)100:
electrophoresis. Thereafter, the slides were gently washed
with 0.4 M Tris buffer, stained with Gel green DNA
Thesurvivingfraction(SF)wascalculatedas:
dye(Biotium,Inc.,Hayward,CA,USA),andanalyzedundera
fluorescence microscope (Carl Zeiss, Oberkochen, Germany). SF¼PET/PEC;
Theimageswerecaptured,andaminimumof100cometsper
where PET isthe plating efficiencyof the treated group and
slide,intriplicatesforagroup,wereanalyzedusingMetafer4
PECisthevalueofthecontrol.
software (MetaSystems, Carl Zeiss) which gives % DNA in
the tail, tail length, tail moment (TM), and olive moment
(OM)directly.TheparameterTMistheproductoftaillength
Statisticalanalysis
and%DNAinthetail,andtheolivemomentistheproductof
the distance between the center of the head and the center of Alldataareexpressedasmeanstandarddeviation.Statistical
thetailand%DNAinthetail[28,29]. significance was tested using the Statistical Package for the
Social Sciences statistical software for Windows, Ver. 18.0
(SPSSInc.,Chicago,IL,USA).Dataweretestedfornormal-
Immunofluorescentstainingofphoshphorylated ityusingtheKolmogorov–Smirnovtestandforhomogeneity
H2AX of variance using Levene’s test, prior to any statistical
CHO-K1cellswereculturedovernightin6-wellplates,ata analysis. The data were normally distributed, and the var-
densityof3×105cells/3mlineachwell.Thenextday,the ianceswerehomogeneous.Therefore,significantdifferences
cells were treated with various concentrations of HS-1793 between two groups were evaluated by Student’s t-test and
for 15min, exposed to 2Gyof 137Cs γ-radiation and incu- significant differences among more than two groups were
batedat37°Cinahumidifiedatmospherewith5%CO for evaluated by one-way analysis of variance with Dunnett’s
2
45min. The cells were cytocentrifuged on slides, fixed posthoctestformultiplecomparisons.Adifferencewascon-
with4%formaldehyde(Biosesang,Seoul,Korea),permea- sideredtobesignificantatP<0.05.
bilizedfor10minonicein0.2%TritonX-100inPBS,and
washed thoroughly with PBS. The slides were treated with
the primary antibody (anti-phosphorylated histone H2AX
atserine139antibody;Abcam,Cambridge,MA,USA)and RESULTS
Texas Red Goat anti-mouse IgG secondary antibody
(Vector Laboratories, Burlingame, CA, USA). The slides FreeradicalscavengingactivityofHS-1793
were then incubated with 4μg/ml Hoechst 33342 (4′,6- To investigate the free radical scavenging activity of
diamidino-2-phenylindole;Invitrogen)atroomtemperature HS-1793,theDPPHassay,NBT/XOassayandtheoxidation
for 15min. All slides were mounted with 0.05ml PBS con- ofthefluorescentprobebyhydroxylradicalswerepeformed.
taining 10% glycerol (Wako, Osaka, Japan) and were exam- The stable free radical DPPH with characteristic absorption
ined using a Zeiss fluorescence microscope.The red intensity at 517nm decreased significantly following HS-1793 treat-
of the phospho-H2AX signal on digitized images was ana- ment (P<0.05), and 50% DPPH reduction was observed at
lyzedusingAxioVisionRel.4.8software(CarlZeiss). 5μM HS-1793 (Fig. 2A). HS-1793 inhibited the generation
468 M.H.Jeongetal.
ofsuperoxideradicalsinaconcentration-dependentmanner, EffectofHS-1793oncellularROSproductionand
and a 50% reduction of superoxide generation occurred at anti-oxidantactivityin137Csγ-radiation-exposed
10μM HS-1793 (Fig. 2A). Moreover, HS-1793 suppressed CHO-K1cells
hydroxyl radical production in a dose-dependent manner ToinvestigatetheeffectofHS-1793oncellularROSproduc-
(Fig. 2B), suggesting that HS-1793 may prevent hydroxyl tion,radiation-exposedCHO-K1cellswereused.Theexperi-
radical-induced damage. The degree of free radical scaven- mental doses of HS-1793 and 137Cs γ-radiation were
gingactivityofHS-1793wascomparedwiththatoftheposi- determined as follows (Fig. 3). CHO-K1 cell viability was
tive control, ascorbic acid, at the maximum concentration measuredusingtheMTTassayinthe presence ofHS-1793,
tested(80μM). and HS-1793 induced little toxicity in CHO-K1 cells up to
a concentration of 80μM (Fig. 3A). The survivability of
137Cs γ-irradiated CHO-K1 cells was measured using the
clonogenic assay, and radiation-induced cell damage was
Fig. 2. Free radical scavenging activity of HS-1793. (A) DPPH
radical scavenging activity of HS-1793 was determined by the
reduction of DPPH when cells were incubated with the indicated Fig. 3. CHO-K1 cell viability in response to HS-1793 and the
concentrations of HS-1793. Superoxide radical scavenging ability survivability of 137Cs γ-irradiated CHO-K1 cells. (A) The cells
of HS-1793 was measured by the level of nitroblue tetrazolium were treated with the indicated concentrations of HS-1793 (1.25–
(NBT) reduction. The percent inhibition of the DPPH free radical 80μM)for24h.CellviabilitywasdeterminedbytheMTTassay.
and superoxide radicals was calculated as a measure of the Eachpercentagevalueintreatedcellswascalculatedwithrespectto
radical scavenging activity of HS-1793. (B) The hydroxyl radical that in the untreated control. All samples were performed in
scavenging activity of HS-1793 was measured using an HORAC triplicate and experiments were repeated three times. Results are
Activity Assay kit. The antioxidant capacity of HS-1793 was expressedaspercentagesofcontrol,anddataaremeans±standard
calculated based on the area under the fluorescence decay curve deviations of three independent experiments. (B) CHO-K1 cells
comparedwiththeantioxidantstandardcurveobtainedwithgallic were exposed to 0.5–14Gy irradiation to generate clonogenic cell
acid (for HORAC). Ascorbic acid (AA, 1mM) was used as the survival curves. After exposure, the cells were trypsinized and
positivecontrol.Allsampleswererunintriplicateandexperiments platedatmultipledensities.Afterabout14days,thecolonieswere
wererepeatedthreetimes.Dataaremeans±standarddeviationsof fixed, stained with crystal violet, and counted. Surviving cell
three independent experiments. Significant differences between fractions were normalized to non-irradiated controls. All samples
more than two groups were assessed by one-way analysis of were run in triplicate and experiments were repeated three times.
variancefollowedbyDunnett’s-test.*,**and#P<0.05compared Each plot presents average data from three independent
withtheuntreatedcontrol. experiments.
RadioprotectiveactivityofHS-1793 469
prominentat2Gy(logarithmicsurvivingfraction[SF]=0.51, preventedthedecreaseintheintensityoftheSCformandan
Fig. 3B). When the cells were irradiated with 2Gyof 137Cs, increaseintheintensityofOCform(Fig.6Cand6D).
the ROS production, determined by DCFH-DA assay, was
~2.7 times higher than that in non-irradiated cells. HS-1793
EffectofHS-1793oncellularDNAdamagein137Cs
significantlyreducedROSproductioninirradiatedcells,and
γ-radiation-exposedCHO-K1cells
little difference was observed in the production of ROS
To demonstrate the protective effect of HS-1793 against
at HS-1793 concentrations >20μM in non-irradiated cells
DNAsingle-strandbreaks(SSBs)inirradiatedcells,alkaline
(Fig.4).
single-cell gel electrophoresis (the comet assay) was per-
To identify cellular antioxidant activity resulting from
formed. As shown Fig. 7A, CHO-K1 cells irradiated with
HS-1793pretreatment,theSODactivityandGSHlevelwere
2Gy increased SSBs (percentage DNA in the tail: 27.32±
measured in CHO-K1 cells following 137Cs γ-irradiation.
2.70%, tail length: 45.80±4.23μm, TM: 14.80±1.30μm,
Ourdatashowedthat2Gyγ-irradiationcausedasignificant
and OM: 8.40±0.90μm), whereas 20μM HS-1793
decrease in the SOD activityand the GSH level when com-
pared with non-irradiated cells, however, HS-1793 pretreat-
mentsignificantlyrecoveredthecells(P<0.05,Fig.5).
ProtectiveeffectofHS-1793against137Cs
γ-radiation-inducedDNAdamageinplasmidpSK
ExposureofplasmidpSKDNAto137Csγ-radiationresulted
in the production of strand breaks in which the supercoiled,
covalentlyclosed,circular(SC)formofDNAwasconverted
to open circular or linear forms (OC) in a radiation dose-
dependent manner (Fig. 6B). Hence, this plasmid DNA re-
laxation assay with pSK DNA was useful for studying the
radioprotective efficacy of HS-1793 against direct DNA
damage. DNA strand breaks in pSK DNAwere sufficiently
induced at 5Gy γ-irradiation, however, HS-1793 effectively
Fig. 5. Effect of HS-1793 on glutathione (GSH) level and
superoxide dismutase (SOD) activity in 137Cs γ-radiation-exposed
CHO-K1 cells. The cells were treated with the indicated
Fig. 4. Effect of reactive oxygen species (ROS) production by
HS-1793 in 137Cs γ-radiation exposed CHO-K1 cells. CHO-K1 concentrations of HS-1793 for 24h and were exposed to 2Gy
137Csγ-radiation.Proteinswereextractedfrom24hirradiatedcells
cellsweretreatedwiththeindicatedconcentrationsofHS-1793for
1h and then 25μM DCFH-DA was added. The cells were aftertreatmentwithHS-1793.(A)SODactivitywasassayedusing
immediately exposed to 2Gy 137Cs γ-radiation and incubated for the superoxide dismutase kit by mixing SOD reaction buffer,
xanthine, and nitro blue tetrazolium solution with 50μg protein.
10min.ROSproductionwasmeasuredbytheDCFHassay.Results
(B)GSHlevelwasmeasuredusingaglutathioneassaykitwith50μg
areexpressedasintensityofDCFHfluorescence.Allsampleswere
totalprotein.Allsampleswererunintriplicateandexperimentswere
runintriplicateandexperimentswererepeatedthreetimes.Dataare
repeated three times. SOD and GSH results are means±standard
means±standard deviations of three independent experiments.
deviationsofthreeindependentexperiments.Significantdifferences
Significant differences between two groups were evaluated by
Student’s t-test and those between more than two groups were between two groups were evaluated by Student’s t-test and those
amongmorethantwogroupswereexaminedbyone-wayanalysisof
examined by one-way analysis of variance followed by
Dunnett’s-test.#P<0.05comparedwithnon-irradiatedcontroland variance followed by Dunnett’s-test. #P<0.05 compared with
non-irradiatedcontroland*P<0.05comparedwithirradiationalone.
*P<0.05comparedwithirradiationalone.
470 M.H.Jeongetal.
Fig.6. EffectofHS-1793on137Csγ-radiation-inducedstrandbreaksinplasmidpSKDNA.(A)AplasmidpSKmapwasconstructed.(B)
pSK DNA (500ng) in phosphate buffer (0.1 M, pH 7.4) was exposed to the indicated doses of γ-radiation. (C) The plasmid DNAwas
exposed to 5Gy γ-radiation in the presence of different concentrations (0–20μM) of HS-1793. After irradiation the DNA was
electrophoresedona1%agarose gelandDNA damagewasanalyzedusingagel documentationsystem.The agarosegelelectrophoresis
patternofpSKDNAexposedto5Gy(lane1:controlpSKDNA;lane2:irradiationalone(RT,5Gy);lane3:RT(5Gy)+HS-1793(1.25
μM);lane4:RT(5Gy)+HS-1793(2.5μM);lane5:RT(5Gy)+HS-1793(5μM);lane6:RT(5Gy)+HS-1793(10μM);lane7:RT(5
Gy)+HS-1793 (20μM)). (D) Quantification of plasmid DNA breaks were expressed by the density ratio (irradiated pSK DNA/
non-irradiated pSK DNA) of the OC form of pSK DNA. Data are means with standard deviations calculated from the densitometric
measurements of the OC plasmid DNA in three independent experiments. Significant differences among more than two groups were
assessedbyone-wayanalysisofvariancefollowedbyDunnett’s-test.*P<0.05comparedwithirradiationalone.
decreased the production of radiation-induced SSBs in a significantly in a dose-dependent manner in HS-1793 pre-
dosedependentmanner(percentageDNAinthetail:10.14± treated cells (Fig. 9A). Pretreament with 20μM HS-1793
1.70%, tail length: 16.30±1.70μm, TM: 1.80±0.30μm showed a higher radioprotective effect on 2-Gy γ-irradiated
andOM:3.20±0.40μm). CHO-K1 cells(SF=0.90)thanthatinirradiatedcontrolcells
TodemonstratetheprotectiveeffectofHS-1793againstcel- (SF=0.51). In addition, pretreatment with 20μM HS-1793
lular DNA double-strand breaks (DSBs) after irradiation, the also significantly increased the surviving fractions of
level of γ-H2AX foci was measured. As shown in Fig. 8, cellsirradiatedwithhigherdosesofγ-radiationsuchas4and
higher levels of red phosphorylated H2AX foci were clearly 6Gy(P<0.05,Fig.9B).Asexpectedwiththeresultsofcyto-
observed in nuclei after irradiation compared with those in toxicity of HS-1793, there was no change in surviving frac-
non-irradiated cells. HS-1793 reduced the numberof positive tionsofCHO-K1cellstreatedwith20μMHS-1793alone.
cellswithγ-H2AXfociinadose-dependentmanner(Fig.8B).
DISCUSSION
RadioprotectiveeffectofHS-1793onCHO-K1cells
The radioprotective effects of HS-1793 were verified using Radiation protection is an area of great significance due to
the clonogenic cell survival assay in 2Gy γ-irradiated its possible applications in planned radiotherapy as well
CHO-K1 cells pretreated with varying concentrations as in unplanned radiation exposure [11, 30]. Ionizing
(1.25–20μM) of HS-1793. Colony formations increased radiation-induced damage to cellular DNA is mainly due to
RadioprotectiveactivityofHS-1793 471
Fig. 7. Effect of HS-1793 on DNA damage in irradiated CHO-K1 cells. The cells were pretreated with the indicated concentrations
of HS-1793 for 15min before irradiation (RT) with 2Gy. Then, the comet assay was performed after 15min of irradiation.
(A) Photomicrographs of comet length, and (B) representative comet parameters (% DNA in tail, tail length, tail moment, and olive tail
moment) presented foreach condition. A minimum of 100 cells were analyzed using Metafer 4 software, and data are means±standard
deviations of three independent experiments. Significant differences between two groups were evaluated by Student’s t-test, and
comparisonsamongmorethantwogroupswereassessedbyone-wayanalysisofvariancefollowedbyDunnett’s-test.#P<0.05compared
withnon-irradiatedcontroland*P<0.05comparedwithirradiationalone.
strandbreaks,whichmayleadtolossofnormalcellviability. secondary superoxide radicals that lead to serious cell
In addition, waterdecomposes afterexposure toionizing ra- damage from DNA breaks [31]. Hence, compounds that
diation and generates primary hydroxyl radicals ((cid:129)OH) and protect DNA against ionizing radiation have considerable
472 M.H.Jeongetal.
Fig.8. ReducedlevelofDNAdouble-strandbreaksbyHS-1793in137Csγ-radiation-exposedCHO-K1cells.(A)Fluorescenceimagesof
phosphorylatedH2AX(red)orwithHoechst33342stainingnuclei(blue)andfluorescentintensitiesofphosphorylatedH2AXinirradiated
cells45minafterirradiation(RT)intheabsenceorpresenceofHS-1793.(B)Thenumberofpositivecellswithγ-H2AXfociwasobtained
fromthreeindependentexperiments,and200cellswereanalyzed.Dataaremeans±standarddeviations.Significantdifferencesbetweentwo
groupswereevaluatedbyStudent’st-test,andcomparisonsamongmorethan twogroupswereassessed byone-wayanalysisofvariance
followedbyDunnett’s-test.#P<0.05comparedwithnon-irradiatedcontroland*P<0.05comparedwithirradiationalone.
potential as radioprotectors. Recent studies on the develop- oxidase (XXO) system has been observed at a resveratrol
ment of radioprotectors have focused on screening avariety concentration of 245μM [35]. Resveratrol also inhibits the
of chemical and biological compounds. Various natural or generation of O(cid:129)− in blood platelets at 22.3–360μM [36].
2
syntheticdrugs,i.e.antioxidantcytoprotectiveagents,immu- Our results demonstrate that the new resveratrol analogue
nomodulators, vitamins, and DNA binding molecules, have HS-1793canalsoeffectivelyscavengeinvitrohydroxylradi-
beenextensivelyevaluatedfortheirradioprotectivepotential cals.TheEC ofDPPHradicalandsuperoxideanionswere
50
inbothinvitroandinvivomodels[11,32,33]. 5μM and 10μM, respectively. Furthermore, a protective
Resveratrolwasreportedpreviouslytobeaneffectiveanti- effect of HS-1793 against plasmid DNA damage after
oxidant in different in vitro assays including: total antioxi- irradiation (suchas strandbreaks) was demonstrated invitro
dant activity, reducing power, DPPH(cid:129), ABTS(cid:129)+, DMPD(cid:129)+ by quantifying the amount of DNA in both nicked-circular
(cid:129)−
and O radical scavenging, hydrogen peroxide scavenging and supercoiled form. These results provide reasonable evi-
2
andmetalchelatingactivities[34].A50%scavenging(EC ) dence for a chemical radioprotective action of HS-1793 at
50
of superoxide anion produced in the xanthine/xanthine themolecularlevel.
RadioprotectiveactivityofHS-1793 473
misrepair of these lesions [38]. The increased parameters
induced by radiation were effectively prevented by a short-
termincubationwithHS-1793beforeirradiation.Wefurther
investigated DSBs in gamma radiation-exposed CHO-K1
cells. DNA DSBs are potentially damaging events in cells,
which are highly mutagenic when misrepaired and lethal if
leftunrepaired.FollowingtheinductionofDSBs,phosphor-
ylation mediated either by ataxia telangiectasia-mutated,
ataxia telangiectasia-related, or DNA-dependent protein
kinase,occursonSer-139attheC-terminusofH2AXmole-
cules flanking the DSBs in chromatin [39, 40]. The phos-
phorylated form of H2AX is called γ-H2AX [41]. The
appearance of γ-H2AX in chromatin in the form of discrete
nuclear foci, each focus representing a single DSB, can be
detected immunocytochemically shortly after induction of
DSBs [42]. The increased γ-H2AX foci number induced by
radiationinCHO-K1cellswasalsoeffectivelypreventedby
short-term incubation with HS-1793 before irradiation.
These results provide reasonable evidence for a chemical
radioprotectiveactionofHS-1793atthecellularlevel.
Cells have developed antioxidant defense systems against
ROS,includinglow-molecular-weightantioxidantmolecules
such as GSH, melatonin, and various antioxidant enzymes
[43].SOD,afirstlineofdefenseagainstoxygen-derivedfree
Fig. 9. Effects of HS-1793 on CHO-K1 cell surviving fractions radicals, catalyzes the dismutation of the superoxide anion
after 137Cs γ-radiation. (A) Clonogenic CHO-K1 cell survival (O(cid:129)−)intoH O .Thelevelsofenzymaticandnon-enzymatic
2 2 2
curvesaftertreatmentwiththeindicatedconcentrationsofHS-1793 antioxidants decrease after irradiation [44]. The decreased
for24hprior toexposureto2Gyirradiation.(B)Clonogeniccell
GSH level in gamma-irradiated cells may be due to its util-
survival curvesof CHO-K1cellstreated with 20μMHS-1793for
ization byenhanced ROS levels [45].Adecreasein enzyme
24hpriortoradiationexposurewith2,4or6Gy.Afterirradiation,
levels could also be due to feedback inhibition or oxidative
the cells were trypsinized and plated at multiple densities. After
inactivationofanenzymecausedbyROSgeneration,which
about14days,thecolonieswerefixed,stainedwithcrystalviolet,
in turn impairs the antioxidant defense mechanism, leading
and counted. Surviving cell fractions were normalized to those of
thenon-irradiatedcontrol.Dataaremeans±standarddeviationsof toincreasedDNAdamageandmembranelipidperoxidation
threeindependentexperiments.Significantdifferencesbetweentwo [46]. The protective effects of resveratrol against oxidative
groupswereevaluatedbyStudent’st-test,andcomparisonsamong injuryarelikelyattributedtotheupregulationofendogenous
more than two groups were assessed by one-way analysis of cellular antioxidant systems rather than to the direct ROS
variance followed by Dunnett’s-test. #P<0.05 compared with scavenging activity of the compound. Indeed, long-term
non-irradiatedcontroland*P<0.05comparedtoirradiationalone. treatmentofH9C2cellswithresveratrolincreasestheactivity
**P<0.05 compared with irradiation alone at 2, 4 and 6Gy,
of cellular antioxidant systems including SOD, catalase,
respectively.
glutathione peroxidase and GSH [47]. As shown in our
results, the decreased SOD activity and GSH level induced
by radiation in CHO-K1 cells were effectively recovered by
Resveratrol is a potent inhibitor of cellular ROS produc- relatively long-term incubation with HS-1793 before irradi-
tion in unopsonized zymosan-stimulated RAW 264.7 cells ation. We further investigated the biological radioprotective
(EC , 17μM), human monocytes (EC , 18μM) and neu- action of HS-1793 on CHO-K1 cells using a clonogenic
50 50
trophils(EC ,23μM)[37].OurresultsrevealthatHS-1793 survival assay. When CHO-K1 cells were preincubated
50
is a strong inhibitor of cellular ROS production in 2Gy of with HS-1793 for 1h, the surviving fractions of 137Cs
137Cs radiation-exposed CHO-K1 cells (EC 5μM). We γ-radiation-exposed CHO-K1 cells did not change signifi-
50,
also investigated the comet parameters of gamma radiation- cantly(datanotshown).However,long-term(24h)incubation
exposed CHO-K1 cells, in which most of the strand with HS-1793 effectively recovered the surviving fractions of
breaks measured by the alkaline comet assay were SSBs. 2Gy or higher doses of 137Cs γ-radiation-exposed CHO-K1
Consideringthat~1000SSBsareproducedper40DSBsper cells. These results suggest that relatively long-term incuba-
cell per gray, the alkaline comet assay is not sensitive tion with HS-1793 upregulatedendogenouscellularantioxi-
enough to measure variations in DSB levels or possible dant systems needed before irradiation to achieve biological