Rev. bras. farmacogn. vol.25 número6

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w w w. s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Perezone,

from

the

gorgonian

Pseudopterogorgia

rigida

,

induces

oxidative

stress

in

human

leukemia

cells

Paula

A. Abreu

a,b

_,

_Diego

_V.

_Wilke

a

_,

_Ana

_J.

_Araujo

a,c

_,

_José

_Delano

_B.

_{Marinho-Filho}

a,c

_,

Elthon

G. Ferreira

a

_,

_Carlos

_Margo

_R.

_Ribeiro

d

_,

_Leandro

_S.

_Pinheiro

d

_,

_Juliana

_W.

_Amorim

d

_,

Alessandra

L. Valverde

d

_,

_Rosângela

_A.

_Epifanio

d,†

_,

_Letícia

_V.

_Costa-Lotufo

a,e

_,

_Paula

_C.

_Jimenez

a,f,∗

a_Departamento_de_Fisiologia_e_{Farmacologia,}_Universidade_Federal_do_Ceará,_Fortaleza,_CE,_Brazil b_INSERM_U1113,_Strasbourg_University,_Strasbourg,_France

c_Faculdade_de_Medicina,_Universidade_Federal_do_Piauí,_Parnaíba,_PI,_Brazil d_Instituto_de_Química,_Universidade_Federal_Fluminense,_Niterói,_RJ,_Brazil e_Instituto_de_Ciências_Biomédicas,_Universidade_de_São_Paulo,_São_Paulo,_SP,_Brazil f_Departamento_de_Ciências_do_Mar,_Universidade_Federal_de_São_Paulo,_Santos,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1May2015

Accepted20July2015

Availableonline4September2015

Keywords:

Gorgoniancoral

Marinenaturalproducts

Cytotoxicity Quinone ROS

a

b

s

t

r

a

c

t

Fourbisabolanes1–4,includingperezone(1)andtriacetylperezone(2),wereisolatedthrougha bioassay-guidedfractionationoftheextractobtainedfromtheCaribbeangorgoniancoralPseudopterogorgiarigida collectedduringanexpeditioncruisetotheBahamas.Allisolatedcompoundsshowedtobecytotoxic towardpaneloffourhumantumorcelllines,asquantifiedbytheMTTassayafter72hincubation. Pere-zone(1),themostactiveone,wasfurtheranalyzed,showingtobecytotoxic,butnotselective,ina12-cell linepanelcomprisingtumorandnon-tumor,aswellashumanandmurinecells.Additionally,1was assayedforcytotoxicityagainstHL-60leukemiccells.Pre-treatmentwithanacutefreeradicalscavenger (l-NAC)beforeexposureofcellstoperezonevirtuallyeliminatedthegenerationofintracellularROSand lesseneditsseverecytotoxicity.Theprotectiveeffectdeliveredbyl-NACevidencesthatthemechanism ofperezone-inducedcytotoxicityispartiallyassociatedtoproductionofROSandaconsequentinduction ofoxidativestress.

Introduction

Natural or synthetic quinones perform various functions in organisms,as someare tightlyinvolved in the cell’s biochemi-calmechanisms(Asche,2005).Nevertheless,quinoneshavebeen extensively studiedfor their cytotoxicpotential and, currently, areanimportantgroupofanticancerdrugs.Manyquinoid com-pounds,suchastheanthracyclinesdoxorubicinanddaunorubicin and the antibiotic mitomycin, are successfully applied in vari-ouscancerchemotherapyschemes(Asche,2005;Vásquezetal., 2010).

Perezone(1)wasfirstisolatedin 1852fromrootsof Perezia

mangroveplants(renamedAccourtia).Thisquinoneshowed insec-ticideactivityagainstLeptinotarsadecemlineataandMyzuspersicae

∗ Correspondingauthor.

E-mail:pcjimenez@unifesp.br(P.C.Jimenez).

†_in_memorian.

(Burgue ˜no-Tapiaetal.,2008)andseveralpharmacological activ-ities,suchaslaxativeandanti-parasitic(Rubioetal.,1997).The studybyGarcíaetal.(1995)evaluatedtheeffectsof1on intesti-nalcontractilityofsmoothmuscleofratsand showedthat this quinoneseemstoincreasetheavailabilityofintracellularCa+2_. Con-versely,DelaPe ˜naetal.(2001)disclosed1asaninhibitorofplatelet aggregationandsuggestedthatthisfeaturewasassociatedtothe impairmentinCa2+_mobilization_of_internal_stores._Furthermore,

1wasconsideredtobeahitina96druginvitroscreendirected againstneuroblastomacells,atumorthatremainsincurableinover 60%ofcases(Gheeyaetal.,2009).Morerecently,Sánchez-Torres etal.(2010)evaluatedthecytotoxicpropertiesofperezoneonK562 cellsanddemonstratedthattheapoptoticeffectsmaybedependent orindependentofcaspasesactivation,dependingonconcentration. The present study describes the isolation of 1, along with otherknowncompounds2–4,fromtheCaribbeangorgonian

Pseu-dopterogorgia rigida and the respective cytotoxic activity on a

paneloftumorcelllines.Additionally,weusedHL-60cells(acute promyelocyticleukemia) tocharacterizetherespectivemodeof

http://dx.doi.org/10.1016/j.bjp.2015.07.020

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action,whichlargelyinvolvesthegenerationofreactiveoxygen species.

Materialsandmethods

Generalprocedures

High-performanceliquidchromatographywascarriedoutusing a semipreparative normal phase (silica gel) Dynamax column (15cmlength,i.d.10mm),aWatersM6000pumpandaWaters R401 differentialrefractometer. NMR spectrawere recorded in CDCl3solutiononaVarianUnity500MHzspectrometer.IRspectra wererecordedonaPerkin-Elmermodel1600(FTIR)spectrometer. MassmeasurementswereobtainedonaHP5989Aspectrometer. Vacuumliquidchromatography(VLC)wasperformedwithsilica gelforthinlayerchromatography.Isolationprocedureswere mon-itoredusingthin-layerchromatography(TLC)onpre-coatedsilica gelplates(Merck,Kieselgel60F-254)throughUVinspectionand H2SO4/heat.

Collectionandidentificationofbiologicalmaterial

Fourteen colonies of thegorgonian werecollected by scuba divingat15–20mdepthat SweatingCays,Bahamas, duringan expeditiononboardtheresearchvesselSewardJohnsonin June 1996. The colonies were labeledand air-driedfor 2h. A small amountofeachcolonywasextractedwithCH2Cl2,andtheextracts comparedbyTLCanalysiswerefurtherclassifiedasasinglespecies,

P.rigida,asidentifiedbyDr.FrederickM.Bayer(NationalMuseum

ofNaturalHistory,SmithsonianInstitution,andavoucher speci-men(ATPH/MO/57)hasbeendepositedattheHerbariumofthe DepartmentofPharmacognosyandChemistryofNaturalProducts, UniversityofAthens.

Extracts,fractionsandpurecompoundsfromP.rigida

Gorgonian tissues (300ml, about 25–30g of dry weight) were cut in to small pieces and extracted with a mixture of MeOH–CH2Cl2,1:1(once),andpureCH2Cl2(twice).Theextracts werecombinedand evaporatedunder reducedpressure afford-ingabrownishgum(12g).Thecrudeextractwasfractionatedby vacuumliquidsilicagelchromatography(VLC),employinga gra-dientrangingfrom0to100% ofEtOAcin (CH3)3CCH2CH(CH3)2 (TMP: 2,2,4-trimethylpentane) and MeOH toyield eleven frac-tions (F1–F11). Fraction 5 (2.67g) was purified by successive re-crystallizations using mixtures of hexane and ethyl acetate, yieldingpureperezone(1)(0.64g)asintenseyellowcrystalwith mp102◦_C._Fraction₇_(1.56_g)_and₁₁₍₃₉_mg)_were_further_purified bynormalphasesemi-preparativeHPLCusinghexaneorTMPand ethylacetatemixturestogive,respectively,compound2(10.7mg) andcompounds3(8mg)and4(9mg).

Compound 1 _(perezone; ₍R

)-3-hydroxy-5-methyl-2-(6-methylhept-5-en-2-yl)cyclohexa-2,5-diene-1,4-dione).Mp102◦_C, 1_H_NMR_(CDCl

3,300MHz):␦1.19(3H,d,J=7.0Hz),1.53(3H,sl), 1.57(1H,m),1.65(3H,d, J=1.8Hz),1.82(2H,m),1.90(1H,m), 2.06(3H,d,J=1.8Hz),3.05(1H,m),5.07(1H,mt,J=7.2Hz),6.48 (1H,q,J=1.8Hz),7.00(1H,s);13_C_NMR_(CDCl

3,75MHz):␦14.6 (CH3), 17.5(CH3), 18.1(CH3),25.6(CH3), 26.6(CH2), 29.2(CH),

34.0(CH2),124.5(CH),124.5(C),131.3(C),135.5(CH),140.4(C), 150.9(C),184.2(C),187.3(C).

Compound 2 _(triacetyl _perezone; ₍R

)-6-methyl-3-(6-methylhept-5-en-2-yl)benzene-1,2,4-triyl triacetate). Colorless oil, 1_H_NMR _(CDCl

3,300MHz):1.20(3H,d, J=7.1Hz),1.54(3H, sl),1.59(2H, m),1.64(3H, d,J=1Hz), 1.87(2H,dd,J=15.0Hz; J=7.5Hz),2.14(3H,d, J=0.7Hz),2.29(3H, s),2.29(3H, s),2.28 (3H,s),2.82(1H,ddq,J=7.2Hz;J=7.2Hz;J=7.2Hz),5.04(1H,tdq, J=7.1Hz;J=1.4Hz,J=1.4Hz),6.82(1H,d,0.7Hz);13_C_NMR_(CDCl

3, 75MHz): ␦ 16.0 (CH3), 17.5(CH3), 18.8(CH3), 20.2 (CH3), 20.3 (CH3),21.0 (CH3), 25.6(CH3), 26.2(CH2),30.9 (CH),35.2(CH2), 124.0(CH), 129.5(C),129.9(C),131.7(C),139.2 (C),141.4(C), 146.3(C),167.6(C),167.8(C),168.9(C).EI-MSm/z376(M+).

Compound 3 _(2-((2_R

)-5-hydroxy-6-methylhept-6-en-2-yl)-5-methylbenzene-1,4-diol). Yellowish oil. IV (film, CH2Cl2) ␯max 3339, 2958, 1650, 1423, 1191 e 1006cm−1_; 1_H _NMR _(CDCl

3, 300MHz):␦1.20(3H,d,J=7.0Hz),1.26(1H,m),1.48–1.59(3H,m), 1.66(3H,s),2.16(3H,s),2.76(1H,sl),3.13(1H,m),4.14(1H,m), 4.81(1H,s),4.94(1H,s),6.58(1H,s)e6.60(1H,s);13_C_NMR_(CDCl

3, 75MHz):␦15.5(CH3),17.6(CH3),21.1(CH3),30.8(CH),31.9(CH2), 34.8(CH2),76.8(CH),110.9(CH2),113.0(CH),118.4(CH),122.1(C), 131.5(C),146.8(C),147.6(C)e147.8(C);HR-ESIMS(positivemode) m/z273.1467[M+Na+_]_(calcd_for_C

15H21O3,272.1388).

Compound 4 _(2-((2_R

)-5-hydroxy-6-methylhept-6-en-2-yl)-5-methylbenzene-1,4-diol). Yellowish oil. IV (film, CH2Cl2) ␯max 3334, 2956, 1651, 1442, 12195 e1001cm−1_; 1_H _NMR _(CDCl

3, 300MHz):␦1.15(3H,d,J=7.0Hz),1.26(1H,m),1.42–1.55(3H,m), 1.66(3H,s),2.14(3H,s),2.66(1H,sl),3.03(1H,m),4.05(1H,m),4.82 (1H,s),4.93(1H,s)e6.57(2H,s);13_C_NMR_(CDCl

3,75MHz):␦15.6 (CH3),17.9(CH3),21.2(CH3),31.7(CH),32.1(CH2),33.3(CH2),76.2 (CH),110.8(CH2),113.2(CH),118.1(CH),122.1(C),131.5(C),146.7 (C),147.2(C)e147.8(C);HR-ESIMS(positivemode)m/z273.1467 [M+Na+_]_(calcd_for_C

15H21O3,272.1388).

Cytotoxicityassay

Human tumor cells (HL-60, SF 295, MDA MB435,MDA MB 231andMX1)andthemurinecelllineB16wereobtainedfrom theNationalCancerInstitute,Bethesda,USA.HCT-8celllinewas donatedbytheChildren’sMercyHospital,KansasCity,MO,USA. Non-tumor cells, L929 e J744, were kindly donated by Dr. C.I. Oliveira(CentrodePesquisaGonc¸aloMuniz,Fiocruz,Bahia,Brazil). CellsweremaintainedinRPMI1640mediumsupplementedwith 10%fetalbovineserum,2mMglutamine,100U/mlpenicillin,and 100g/mlstreptomycinat37◦_C_with_5%_CO

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asthepercentageofthecontrolabsorbanceofthereduceddyeat 595nm.Dataarepresentedasmean±S.E.M.TheIC50valuesand their95%confidenceintervals(CI95%)wereobtainedbynon-linear regressionusingtheGraphPadSoftware4.0(IntuitiveSoftwarefor Science).

Phenotypiccharacterizationofperezoneeffects

Flowcytometrywasusedtofurthercharacterizethe cytotox-icity of perezone on the human promyelocitic cell line HL-60. Perezone(1)wastestedat2,4and8␮M.Doxorubicin0.5␮Mwas usedasthepositivecontrolandDMSO1%,thedilutionvehicleof allsamples,servedasthenegativecontrol.

Celldensityandviability

HL-60cells wereseededin a24-wellplate(3×105_cells/ml), culturedfor24htoinitiatecellgrowthandtreated,aspreviously described.Cellswereharvestedafter3,6,12,24,28and72hof exposure, centrifugedand resuspendedin a 5␮g/ml propidium iodide(PI)solution(Sigma–Aldrich).After5minincubationinthe dark,fivethousandeventswereacquiredusingtheGuavaEasyCyte Miniflowcytometer(GuavaTechnologies)andtheGuavaExpress Plussoftwareonagatedregiontoexcludedebrisanddoubletsfrom theanalysis.Concentrationofcellsineachtreatmentand mem-braneintegritywereevaluatedandplottedinanXYgraphusing GraphPadSoftware4.0(IntuitiveSoftwareforScience).

CellcycleandDNAfragmentation

HL-60cells wereseededin a24-wellplate(3×105_cells/ml), culturedfor24htoinitiatecellgrowthandtreated,aspreviously described.Afterexposurefor3or24h,cellswereharvested, cen-trifugedandresuspendedinasolutioncontaining5␮g/mlPI,0.1% citrateand0.1%TritonX–100.After30minincubationinthedark, fivethousandeventswereacquiredusingtheGuavaEasyCyteMini flowcytometer(GuavaTechnologies)andtheGuavaExpressPlus softwareonagatedregiontoexcludedebrisanddoubletsfromthe analysis.CellcyclehistogramswereacquiredusingtheFL3channel (excitationatmax488nmandemissionmax620nM)to cap-turetheredfluorescenceemittedfromPI–DNAcomplexes.Thecell cycleprofile(G1/G0–S–G2/M)wasanalyzedinlinearscalewiththe ModFitLT4.0software(VeritySoftwareHouse).Sub-G1cellswere ponderedashavingfragmentedDNA.Thedifferencesbetween neg-ativecontrolandexperimentalgroupsweredeterminedbyanalysis ofvariance (ANOVA)followedbyDunnetpost-testonGraphPad Software4.0(IntuitiveSoftwareforScience).Theminimal signifi-cancelevelwassetatp<0.05.

Analysisofthemechanismsinvolvedinthecytotoxicactivityof

perezone

Furtherexperimentswerecarriedouttoevaluatetheinfluence ofreactiveoxygenspecies(ROS)inthecytotoxicityinducedby1on humanpromyelociticcelllineHL-60.Perezonewastestedat2,4 and8␮M.␤-lapachone2␮Mwasusedasthepositivecontroland DMSO1%,thedilutionvehicleofallsamples,servedasthenegative control.Beforeexposure,cellswerepre-treatedwithl-NAC5␮M during1h.

MorphologicalanalysiswithMay–Grunwald–Giemsastaining

HL-60cellswereseededina24-wellplate(3×105_cells/ml), cul-turedfor24htoinitiatecellgrowthandpre-treatedandtreated, aspreviouslydescribed.Afterexposurefor24htoalltreatment types,cellswereharvested, transferredtocytospinslides,fixed

withmethanolfor10sandstainedwithMay–Grunwald–Giemsa (Bioclin,Brazil).Slideswereexaminedformorphologicalchanges

vialightmicroscopy(Olympus,Tokyo,Japan).

Trypanblueexclusion

HL-60cellswereseededina24-wellplate(3×105_cells/ml), cul-turedfor24htoinitiatecellgrowthandpre-treatedandtreated, aspreviouslydescribed.Afterexposurefor24htoalltreatment types,cellswereharvested,dyedwithTrypanblueand differen-tiallycountedforviable(notdyed)ornon-viable(dyed)counted in a Neubauer chamber. The differences between groups were determinedbyanalysisofvariance(ANOVA)followedbyStudent Newman–Keulspost-testonGraphPadSoftware4.0(Intuitive Soft-wareforScience).Theminimalsignificancelevelwassetatp<0.05.

Measurementofreactiveoxygenspeciesgeneration

HL-60cellswereseededina24-wellplate(3×105_cells/ml), cul-turedfor24htoinitiatecellgrowthandpre-treatedandtreated,as previouslydescribed.Afterexposurefor1htoalltreatmenttypes, cellswereloadedwithH2-DCF-DAandincubatedfor30mininthe darkat37◦_C._Cells_were_then_harvested,_washed,_resuspended_in PBSandimmediatelyanalyzed.Fivethousandeventswereacquired usingtheGuavaEasyCyteMiniflowcytometer(Guava Technolo-gies)andtheGuavaExpressPlussoftwareonagatedregionto excludedebrisanddoubletsfrom theanalysis,usingexcitation andemissionwavelengthsof490and530nm,respectively.The differencesbetweengroupsweredeterminedbyanalysisof vari-ance(ANOVA)followedbyStudentNewman–Keuls post-teston GraphPadSoftware4.0(IntuitiveSoftwareforScience).The mini-malsignificancelevelwassetatp<0.05.

Results

Bioassay-guidedfractionationofP.rigida

TheCH2Cl2/MeOHextractofP.rigidaexhibitedastrong cyto-toxicitywithIC50valuesrangingfrom8.8␮g/mlinSF-295cellsto 43.2␮g/mlinMDA-MB-435cells(Table1).Crudeextractwas puri-fiedbyvacuumliquidchromatographyinsilicagelusingagradient ofincreasingpolarityofTMP/EtOAc/MeOH yieldingeleven frac-tions(F1–F11).FractionsF5andF6werethemostactive(Table1) andanalysisbyTLCand1_H_NMR_revealed_the_presence_of_an_almost purecompoundthatwhencrystallizedwithhexane/ethylacetate givepureintenseyellowcrystalswithmp102◦_C._{Spectrometric} andspectroscopicdatarevealedthatthiscompoundisperezone(1). FromF8,themajorcomponentwasisolatedbysemi-preparative normalphaseHPLC(EtOAc/hexane),yieldingtriacethylperezone2

asacolorlessoil.IC50forthiscompoundrangedbetween23.1and 49.8␮Macrossafourhumantumorcelllinepanel(Table2).The mostpolarfraction,F11,wasalsopurifiedbynormalphaseHPLC (EtOAc/TMP),yieldingtwopurebisabolanes3and4.1_H_and13_C NMRdataofthesecompoundsarealmostidentical,andrevealed themtobeepimersatC-10stereochemistry.TheIC50value calcu-latedfor3variedfrom10.8to59.8␮M,while4retainednearlyhalf thecytotoxicitystrengthofhisepimer(Table2).

Cytotoxicityofperezone

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Table1

IC50(CI95%)(␮g/ml)oftotalextractand11fractionsobtainedfromchemicalfractionationofaPseudopterorgiarigidaorganicextractontumorcelllinesinculturedetermined

bytheMTTassayover72hincubation.

Sample HL-60 HCT-8 SF-295 MDA-MB435

P.rigidacrudeextract 14.73 9.87–21.98

9.28 8.46–10.18

8.80 6.45–12.02

43.12 29.76–62.47

P.rigidaF1 11.06

n.d.

11.90 n.d.

13.36 9.87–18.08

12.38 7.93–19.33

P.rigidaF2 >100,0 >100,0 >100,0 >100,0

P.rigidaF3 >100,0 >100,0 >100,0 n.d.

P.rigidaF4 65.41

34.55–123.8

37.32 30.82–45.18

29.62 27.96–31.38

79.83 51.25–124.3

P.rigidaF5 4.81

4.27–5.43

4.85 4.31–5.47

2.09 1.86–2.35

8.31 7.72–8.94

P.rigidaF6 1.75

1.58–1.93

4.12 3.67–4.62

2.03 1.32–3.13

8.91 7.14–11.13

P.rigidaF7 13.10

9.42–18.22

14.49 11.86–17.70

7.12 5.878–8.62

33.49 18.56–60.44

P.rigidaF8 2.28

1.81–2.88

8.43 6.66–10.67

20.35 17.11–24.22

5.96 5.20–6.83

P.rigidaF9 7.53

5.61–10.10

28.25 22.99–34.72

32.63 27.88–38.18

20.89 19.38–22.52

P.rigidaF10 41.37

32.87–52.07

47.20 35.06–63.56

59.38 49.25–71.59

33.86 18.38–62.36

P.rigidaF11 15.91

13.59–18.62

39.39 33.15–46.79

21.00 16.71–26.38

13.98 12.38–15.79

n.d.,notdeterminedornotrated.

Table2

IC50(␮M)and95%confidenceintervalsofcompounds1–4obtainedfromaPseudopterorgiarigidaorganicextractontumorcelllinesinculturedeterminedbytheMTTassay

over72hincubation.TheresultsarepresentedasIC50(␮M)values,with95%confidenceintervals.␤-lapachonewasusedaspositivecontrol.

Celline Histotype IC50(95%CI)␮M

1 2 3 4 ␤-lapachone

HL-60 Promyelocyticleukemia 8.7 7.5–10.2

33.2 n.d.

23.7 4.6–7.7

38.4 30.9–47.8

1.6 1.5–1.8

HCT-8 Colon 17.9

14.5–22.3

23.1 18.8–28.4

59.8 36.5–97.9

89.4 43.1–185.4

0.8 0.7–0.9

SF295 Brain 15.4

13.8–17.1

49.6 34.2–71.7

10.8 8.2–14.2

16.9 12.4–23.2

0.9 0.7–1.1 MDAMB435 Breastcancer 22.0

18.7–25.9

30.6 26.1–35.9

32.4 27.8–37.8

66.9 55.2–81.1

0.2 0.2–0.3 MDAMB231 Breastcancer 13.2

11.3–15.5

n.d. n.d. n.d. n.d.

MX1 Breastcancer 25.2

n.d.

n.d. n.d. n.d. n.d.

B16 Murinemelanoma 24.2

19.1–30.8

n.d. n.d. n.d. n.d.

J774 Murinemacrophage 11.5 9.8–13.5

n.d. n.d. n.d. n.d.

L929 Murinefibroblast 16.32 11.7–22.82

n.d. n.d. n.d. n.d.

n.d.,notdeterminedornotrated.

B).Precisely,duringthefirst24h,culturesexposedto1suffered a rapid decline in cell number and membrane integrity, espe-ciallyunderhigherconcentrations.Alterationsinthecellcycleof perezone-treatedcellswith4and8␮Mweredetectableafter24h, andshowedasignificantandratherstrikingincreaseoftheG2/M

phase,accompaniedbyadecreaseintheSphase(Fig.1C). Inter-nucleosomalfragmentation,however,wasdetectedonlyincells treatedwith1at8␮M(Fig.1D).

InvolvementofROSgenerationincytotoxicityinducedby

perezone

Interestingly, cytotoxic activity of 1 on HL-60 cells proved stronger aftera shorter exposuretime, as IC50 valueincreased a3.5-foldbetween24 and72h. Nevertheless,whenHL-60cells werepre-incubated withl-NAC,those became lesssensitiveto perezone-induced cytotoxicity,as IC50 increased about8 times

after 24htreatment and, after 72h, toxicity was undetectable (Table3).

SubstantialmorphologicalchangeswereobservedinHL-60cells treatedwith1or␤-lapachone,thelaterconsidered,herein,asthe positivecontrol(Fig.2A).In␤-lapachone-treatedcells,vacuolesin thecytoplasmcouldbeobserved,along withfurther character-isticsofapoptosis,suchasreducedvolume,membraneblebbing and nuclearfragmentation.Additionally, aconsiderablenumber cellsresemblingnecrosis,werealsoobservedafter24htreatment. Perezone-treatedcellsshowedvolumereductionandsomecellular aggregation.LittleDNAfragmentationandsomemembrane rup-tureweredetected.Moreover,picnoticcellswerewidespreadin slides.Suchcharacteristicsseamedtoretainconcentration depen-dency.Cellspre-treatedwithl_-NAC_prior_to_exposure_to₁_showed fewersignsoftoxicity,especiallythoserelatedtoapoptosis,atall testedconcentrations.

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8 4 2 (+) (–) 0 10 20 30 40 50

a

a b

Perezone (µM)

DNA fragmentation (%)

3 h 24 h

47%

45% 8%

(–)

44%

43% 13%

Perezone 2 µM

(+)

* 80% * 14%

* 6%

Perezone 8 µM

* 21% *

48% * 31% 49%

Perezone 4 µM

* 30%

* 21%

G0/G1

G2/M

S

D

72 48

24 0

0 20 40 60 80 100

Hours

Membrane integrity (%)

Doxorubicin 0.5 µM Control

Perezone 2.0 µM Perezone 4.0 µM Perezone 8.0 µM

B

72 48

24 0

0 10 20 30

Hours

Cell density

(X10

5cells/ml)

A

C

Doxorubicin 0.5 µM Control

Perezone 2.0 µM Perezone 4.0 µM Perezone 8.0 µM

Fig.1.Characterizationofperezone(1)cytotoxicityonHL-60cells.(A)Depictstheeffectsoncellgrowthafter3,6,12,24,48and72hexposure;(B)theeffectsonmembrane

integrityafter3,6,12,24,48and72h;(C)theeffectsoncellcycleafter24hexposure;and(D)theeffectsoninternucleossomalfragmentationafter3and24hexposure.

CellswereeithertreatedwithDMSO1%(−),0.5␮Mdoxorubicin(+),orwith1at2,4or8␮M,andanalyzedbyflowcytometryafterstainingwithPI.Dataarepresentedas

meanvalues±standarderrorofthemean(S.E.M.)fromthreeindependentexperiments,eachconductedintriplicate.Fivethousandeventswereacquiredforeachreplicate

andcomparedwiththerespectivecontrolsbyANOVAandfollowedbyDunnett’scomparisontest.In(C),*indicatesstatisticallysignificantlydifferentfrom(−)(p<0.05).In

(D),a,indicatesstatisticallysignificantlydifferentfrom(−)at24h(p<0.05);andb,indicatesstatisticallysignificantlydifferentfrom(−)at3h(p<0.05).

Table3

CytotoxicityofperezoneevaluatedbytheMTTassayinHL-60cellsafter24or

72hexposure,pre-treatedornotpre-treatedfor1hwithl-NAC.Theresultsare

presentedasIC50(␮M)values,withconfidenceinterval95%.

Treatment 24h 72h

Perezone 2.45 2.01–2.98 8.747.46–10.18 Perezone+NAC 20.3313.29–30.6 >25

increased the number of non-viable ones in a concentration-dependentmanner(Fig.2B). Pre-incubationwithl-NACfor 1h waseffectiveinlesseningperezone-inducedcytotoxicity,butnot sufficient to reverse the effects prompted especially by higher concentrationsonneitherviable andnon-viablecellcount.It is noteworthythatthepresentdatashowedareductioninthe num-berof viable cellsin control groups whenincubated onlywith l-NAC,whichindicatesthatthisantioxidantitselfhasamild cyto-toxicity.

Intracellular reactive oxygen species (ROS) accumulation was monitored using 2,7-dichlorodihydrofluorescein diacetate (H2-DCF-DA),whichisconvertedtothehighlyfluorescent dichlo-rofluorescein(DCF)inthepresenceofintracellularROS.Perezone (1)showedtobeastrongproducerofintracellularROS,andthis effectcouldbeperceivedaftermerely1hexposure(Fig.2C). Pre-incubationofcellswithl-NAC,asexpected,weakenedmostofthis effect.

Discussion

Marinesecondarymetabolitescontaininginteresting biomedi-calpropertieshavebeenisolatedfrommanyorganisms,including

bacteria, algae, sponges, bryozoans, mollusks, ascidians, and cnidarians(Costa-Lotufoetal.,2009).Fromachemicalstandpoint, withinthe cnidarians, thegreatest attentions in this groupare sessileand softbody organisms,suchascorals,gorgonians and zoanthids.MarineGorgonaceaOctocoralsareknowntobearich sourceofbiologicallyactiveterpenoids,whichareanimportant partinecologicalrelationships(MartinsandEpifanio,1998). Sev-eralstudiescarriedoutwithpseudopterosin,aditerpeneglycoside isolatedfromPseudopterogorgiaelisabethaespecies,demonstrated apotentanti-inflammatoryactivity(Ataetal.,2003;Mayeretal., 1998).

TheCaribbeangorgonianP.rigida(Bielschowsky,1929)belongs tothephylumCnidaria,classAnthozoaanditisknownfor produc-inganumberofsesquiterpenes,ofwhichmostarequinonesand hydroquinones(McEnroeand Fenical,1978;Freyer etal.,1997; D’Armaset al., 2000).Some of thesecompounds seem to play importantecologicalrolesinthehostorganism,suchasdefense against predation. In fact, curcuphenol and curcuhydroquinone proved to be responsible for the feeding deterrent activity of thecrudeextractobtainedfromP.rigidaagainstgeneralistfishes (Harvelletal.,1988).Inplants,bisabolanesarealsoallelopatic com-poundswithherbicidalandantifeedantactivities,hencerecruiting agrochemicalinterests(Burgue ˜no-Tapiaetal.,2008).

Perezone(1)hasbeenpreviouslyreportedinextractsofP.rigida

(Georganteaetal.,2013),besidesbeingisolatedfromthedryroots

ofPereziaspp.(Enrıquezetal.,1980).Itsspectraldataarealso

(6)

L-NAC pre-treatment

(+) (–)

–

+

Perezone 2 µM Perezone 4 µM Perezone 8 µM

8 4 2 (+) (–) 0 20 40 60 80

c

Perezone (µM)

Viable cell count (x10

4/ml)

c

a a

a b

b b

b

8 4 2 (+) (–) 0 5 10 15 20

a

a b

b

b b

a

Perezone (µM)

Non-viable cell count (x10

4/ml)

c c

8 4 2 (+) (–) 0 20 40 60 80

Perezone (µM)

c

b

a b b b

c c c

ROS generation (% of cells)

B

_C

A

20 µM

No pre-treatment L-NAC pre-treatment

Fig.2.Influenceofreactiveoxygenspecies(ROS)onthecytotoxicityinducedbyperezone(1)onHL-60cellswithorwithout1hpre-treatmentwith5␮Ml-NAC5.(A)

DepictsrepresentativestructuraleffectsoncellmorphologybyMay–Grümwald–Giemsastainingafter24hexposure;(B)theeffectsoncellviabilitybyexclusionoftrypan

bluedyeafter24hexposure;and(C)theeffectsonROSgenerationbyH2-DCF-DAstainingandflowcytometryanalysisafter1hexposure.Cellswereeithertreatedwith

DMSO1%(−),2␮M␤-lapachone(+),orwith1at2,4or8␮M.Dataarepresentedasmeanvalues±standarderrorofthemean(S.E.M.)fromthreeindependentexperiments,

eachconductedintriplicate,andcomparedbyANOVAfollowedbytheStudentNewman–Keulstest.a,indicatesstatisticallysignificantlydifferentfrom(−)withl-NAC

pre-treatment(p<0.05);b,indicatesstatisticallysignificantlydifferentfrom(−)withoutl-NACpre-treatment(p<0.05);andc,indicatesstatisticallysignificantlydifferent

fromtheirrespectivetreatmentcounterpart(p<0.05).

fromthisspecieandtheNMRdataarealsoaccordingtothe litera-ture(Georganteaetal.,2014).

AnaturalproductscreeningcarriedoutbytheNationalCancer Institute(NCI)hasidentifiedthatquinonesareanimportant phar-macological elementwithcytotoxic activity(Pérez-Sacauet al., 2007).Accordingly, 1 showed tobe cytotoxicin a four-human tumorcelllinepanel.Furthermore,bisabolanes2–4alsoshowed thesamekindofactivity,howevertoalesserpotency(Table2). Perezone(1)wasnotselectivebetweentumorandnon-tumorcells, and itscytotoxicityseemedtoberelated toapoptosisinducing propertiesinleukemiacells.Thesefindingsareinagreementwith apreviousstudyfromSánchez-Torresetal.(2010)showingthat

1iscytotoxictoK562leukemiccellsandcancausemitochondrial depolarizationinaconcentrationdependentmanner.

Quinoidalstructuresarewidespreadinnatureandarehighly redox active molecules. Such reactivity leads to the produc-tion of ROS (reactive oxygen species), a normal mitochondrial metabolicby-product, which, in excess, triggers cellular oxida-tivestressthroughtheoxidationofmacromolecules(Boltonetal., 2000).These proprieties, nonetheless, can be explored for the developmentofnewdrugsincancertherapy.ProductionofROS is involved in induction of apoptosis and this death pathway is considered the most appropriate when it comes to provok-ing tumorcells death (Bironaite et al., 2004).One ofthe main features responsible for quinone cytotoxicity is the alkylation of cellular nucleophiles, suchas DNA, lipids and proteins, and therefore,theirabilitytogenerateROS.Theoneelectron reduc-tionfromaquinonecompoundproducesasemi-quinoneradical, which,inturn,generatesROSwhilealteringmitochondrial trans-membrane potential. This alteration can interfere in some cell signsassociatedwithdeathpathways(Tudoretal.,2003). Pere-zone(1)hasbeenshowntobeboth, anelectron-donor andan

electron-acceptor, and theseoxido-reductionproprietiesinhibit mitochondrialelectrontransportat50␮M(CarabezandSandoval, 1988).

Glutathioneisanacutefreeradicalneutralizerduetoitsrole asasubstratetointracellularROSscavengingenzymes(Circuand Aw,2008).l-NACisaprecursorofglutathioneand,therefore,an importantantioxidant.Thus,l-NAChasbeenwidelyusedastoolfor investigatingtheinvolvementofROSincytotoxicity(Sun,2010). Perezone (1) is a potent inducer of intracellular ROS in HL-60 cells,andthiseffectcanbeobservedafteraslittleas1hexposure (Fig.2C).Pre-treatment withl-NAC,ontheotherhand,showed competencetovirtuallyeliminatemeasurableROSinductionby1

and␤-lapachone.Actually,l_-NAC_desensitized_HL-60_to perezone-inducedcytotoxicity,andIC50increasedatleast8-foldafter24h exposure(Table3).Theprotective effect deliveredby l-NACis evidencethatthemechanismofperezone-inducedcytotoxicityis partiallyassociatedtogenerationofROSandaconsequent induc-tionofoxidativestress.

(7)

Otherquinones,suchasthatisolatedfromtherootsofCordia

leucocephala,alsoinduceapoptosisandnecrosisbyamechanism

involvingoxidativestress.Thisnaphthoquinone,rapidlyinduced ROS-relateddeathinHL-60cellsafteronly3hexposure( Marinho-Filho et al., 2010). Studies also suggest that several cytotoxic agentsappliedclinicallyincancerchemotherapy,likedoxorubicin, bleomycinandcisplatin,inducestressthroughROSgenerationin tumorcells(Tiligadaetal.,2002).

Conclusion

Perezone(1),isolatedhereinfromthebisabolane-richtropical gorgonianP.rigida,isacytotoxicquinonewithlowselectivitythat inducescellsdeathtroughamechanismthatisassociated,though incompletely,withtheexcessivegenerationofintracellular reac-tiveoxygenspeciesandconsequentinductionofoxidativestress.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Authorcontribution

CMRR,LSP,JWA,ALVandRAEconductedtheextract fraction-ation,naturalproductisolationandstructureelucidationefforts, includingtheMS andNMR studies.PAA, PCJ,DVW,JDBMF, AJA andEGFconductedbiologicalassayswithfractionsandpure com-pounds, including cytotoxicity,flow cytometry and microscopy analysis.PCJ,ALV,CMRR,LVCLandRAEdevelopedtheprojectand interpretedtheresults.Allauthorscontributedtothewriting.

Acknowledgements

WethankDr.FrederickBayer,NationalMuseumofNatural His-torySmithsonianInstitution,forthegorgonianidentification,and thestaffattheMassSpectrometryCenter,Universityof California-Riverside,forprovidingHRMS.WealsothankDr.WilliamFenical, fromScrippsInstitutionofOceanography,UniversityofCalifornia SanDiego,forstructuralsupportduringthecollectionexpedition andplentifulscientificdiscussions.Thisworkwassupportedby grantsfromtheUnitedStatesNationalScienceFoundation(NSF) andtheBrazilianfundingagencyConselhoNacionalde Desenvolvi-mentoCientíficoeTecnológico(CNPq).

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