Rev. bras. farmacogn. vol.27 número1

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w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Evaluation

of

cytotoxic

activity

of

protein

extracts

from

the

leaves

of

Morinda

pubescens

on

human

cancer

cell

lines

Anvy

Susan

Thomas,

Rupachandra

Saravanakumar

∗

_,

_Pratiksha

_V.

_Gupta

DepartmentofBiotechnology,SchoolofBioengineering,SRMUniversity,Kattankulathur,Kancheepuram,TamilNadu,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10September2015 Accepted8August2016

Availableonline15September2016

Keywords: Cytotoxicactivity FTIRanalysis Morindapubescens MTTassay Proteinextraction

a

b

s

t

r

a

c

t

Biologicallyactiveproteinsisolatedfromplantspeciescanbeusedintraditionalmedicineasprolific

resourcesfornewdrugsMorindapubescensSm.,Rubiaceae,isapromisingmedicinalplantwhichiswidely

usedinfolkmedicinetotreatfeverduetoprimarycomplex,ulcerandglandularswellings.Inthisstudy,

proteinswereextractedfromtheleavesofM.pubescens,andprecipitatedwithammoniumsulphate

atvarioussaturationconcentrationsrangingfrom20to80%.Theprecipitatedproteinsampleobtained

with80%saturationwasfurtherpurifiedusingultrafiltrationmembrane(<10kDa).SDS-PAGEanalysis

identifiedthepresenceofcrudeandultrafilteredproteinbands.FTIRspectrumoftheultrafilteredprotein

fractionsdepictedthepresenceofhydroxylandcarbonylgroupsofproteins.Theultrafilteredproteins

exhibitedincreasedcytotoxicactivityonA549cellsattheconcentrationsrangingfrom15to100␮g/ml.

About98%cellviabilitywasalsoobservedinVerocellstreatedwiththemaximumconcentrationof

100␮g/mlofultrafilteredproteinextract.DNAfragmentationwasobservedinA549cellstreatedwith

10␮g/mlofultrafilteredproteins,indicatingtheonsetofapoptosis.

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Bioactive proteins and peptides from plant sources exhibit differentactivities,suchasantimicrobial,antioxidant, antithrom-botic, antihypertensive, hypocholesterolemic, hypoglycemic, immunomodulatory,opioid,andantiproliferativeactivities.These activities can affect the condition of major body systems, like cardiovascular,digestive,endocrine,immuneandnervoussystem (Ledesmaetal.,2009).Plantspossessinganticanceractivitywere foundinViolaceae,Rubiaceae,FabaceaeandCucurbitaceae fam-ilies (Gerlach and Mondal,2007).Macrocyclic proteins, suchas circulinsA(32.8kDa)andB(31.5kDa),wereisolatedfromChassalia parvifolia and cyclopsychotride from Psycho trialongipes which belongtoRubiaceaefamily.Thesecyclicproteinsshowcytotoxic activity,antiHIVand hemolyticactivity(Gustafson etal., 1994; Witherupetal.,1994).Anticancerousbyproductsarederivedfrom Morindacitrifolia(noni)fruit(A549humanlungcarcinomacells) (Jang,2012)andbromelain,aproteinfoundinseveralmembers of Rubiaceae family are reported to have anti-tumor activity (MarshallandGolden,2012).

MorindapubescensSm.,commonlyknownasAalisaspeciesof floweringplantofthefamilyRubiaceae,nativetoSouthernAsia.

∗ Correspondingauthor.

E-mail:rupachandra.s@ktr.srmuniv.ac.in(R.Saravanakumar).

ThebarkofM.pubescens,isusefulintreatingeczema,feverdue toprimarycomplex,ulcerandglandularswellings,whileleaves are useful for digestivedisorders and venereal diseases (Nivas etal.,2011).Thepreliminaryphytochemicalstudyofthemethanol extractsofleafandstembarkofM.pubescens,exhibited antimicro-bialandantioxidantproperties(Muruganetal.,2012).Theaimof thisstudyistoisolateandpurifycytotoxicproteinsfromtheleaves ofM.pubescens.

Materialsandmethods

Plantmaterialandreagents

TheleavesofMorindapubescensSm.,Rubiaceae,werecollected andauthentified(PARC/2012/1384)byDr.P.Jayaraman,Directorof PlantAnatomyResearchCentre,Chennai.Allbuffersandchemicals usedwereofanalyticalgrade.HumancancercelllinesuchasA549 (adenocarcinomichumanalveolarbasalepithelialcells)andVero celllines(Africangreenmonkeykidneycells)werepurchasedfrom NCCS,Pune.

Proteinextraction

LeavesofM.pubescenswerewashedwithdistilledwaterand shadedried.Thedriedleavesweregroundtofinepowder.About 5gofpowderedleafsamplewasextractedwith50mlofextraction

http://dx.doi.org/10.1016/j.bjp.2016.08.003

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buffer(Ribeiroetal.,2007)consistingof10mMNa2HPO4,15mM NaH2PO4,10mMKCl,2mMEDTA(pH7.0)andkeptinconstant stir-ringfor3hat4◦_C._Then_the_contents_were_filtered_and_centrifuged at5000×gfor20min.Thecrudesupernatantwaslyophilizedand

storedforfurtheruse.Thecrudesupernatant(lyophilized)was fur-thertreatedwithammoniumsulphateforprecipitationofproteins withvarioussaturationlimitfrom20to80%.Theconcentrations ofproteinspresentintheprecipitatedsampleswereestimatedby Bradfordassay(Bradford,1976).

Ultrafiltrationofproteinextracts

The precipitated proteins obtained using 80% saturation of ammoniumsulphatefromtheseedsofM.pubescenswere frac-tionatedusingultrafiltrationmembrane(10kDacut-offmembrane, Amicon).Theconcentrated filteredsolutioncontainingproteins smallerthantheporesizewhichislessthan10kDa,waslyophilized andstoredat4◦_C_for_further_use₍_Ketnawa,₂₀₁₁_).

SDS-PAGEanalysis

Sodiumdodecylsulfatepolyacrylamidegelelectrophoresisis awidelyusedtechniquetoseparateproteinsaccordingtotheir electrophoreticmobility(Mahmoodetal.,2012).About40␮lof thecrudesupernatantwasloadedintoLanes1–4,7,8and40␮lof theultrafilteredproteinsamplewasloadedintoLanes9,10.Lanes 5,6representstheproteinmarkerofsizerangingfrom3.5kDato 43kDa.Thegelwaskeptunderelectrophoreticrunfor2hat100V andtheproteinbandswereidentified.

FTIRanalysis

Identificationoffunctionalgroupsoftheultrafilteredproteins isolatedfromtheleavesofM.pubescenswasperformedusing Shi-madzuFourier transforminfraredspectrophotometer (Surewicz andMantsch,1988)Ultrafilteredproteinswerehomogenizedwith potassiumbromidetoobtainapellet.Thepelletwasscannedin theinfraredabsorptionregionbetween400and4000cm−1_with_a resolutionof4cm−1₍_Widjanarko_et_al.,₂₀₁₁_).

Cytotoxicactivityofproteinextracts

The ultrafiltered protein fractions were tested for cytotoxic activity against the selected cancer cell lines using MTT assay (Pascariuetal.,2011).Differentconcentrationsofultrafiltered pro-teinsrangingfrom2to100␮g/mlwereaddedtoeachwellof96 wellplates.Thecellswereculturedin96-wellplates(2×105cells

perwell)inDMEMsupplementedwith10%FBSfor24h.After24h thecellswereobservedunderphasecontrastmicroscopeand mor-phologyofcellswereobserved.Themediumcontainingpositive controlandtestsampleswereremoved.MTT(50␮l)dyewasadded tothewellscontaining200␮loffreshmedium.Thecelllineswere incubatedinCO2incubatorfor4h.After4hofincubation,medium containingdyewasremovedand200␮lofDMSOwasaddedto dis-solvetheformazancrystal.Theabsorbancewasrecordedat570nm andthepercentageofcellviabilitywascalculatedusingthe for-mula:

%ofcellviability=

At Ac×100

whereAt,absorbanceoftreatedcell;andAc,absorbanceofcontrol (untreatedcells).

DNAfragmentationassay

DNAfragmentationanalysis(Kalininaetal.,2002)wascarried outtoevaluatethemechanismofcelldeathinA549cancercell linetreatedwith10␮g/ml, ofultrafiltered proteinextractfrom the leaves of M. pubescens and incubated at −20◦C overnight.

Cells were freezed and thawed three times for detachment of cellsfromtheflasks.Cells(300␮l)fromtheflaskswastakenin aneppendorfand addedwith800␮lofproteinaseKbufferwas added. About4␮l of proteinaseK was added and keptfor 1h incubation at56◦_C _in _a _water _bath._After_incubation, ₇₀₀_␮_l_of phenol:chloroform:isoamylalchohol(25:24:1)and 100␮lof5M sodiumacetatewereaddedtothemixture.Themixturewas cen-trifugedfor15minat5000×g,4◦C.Thesupernatantwasadded

with200␮lofisopropylalcoholandincubatedat−20◦_C_for₁_h. Afterincubation,thecontentswerecentrifugedagainat5000×g,

4◦_C _for₁₅_min._The_supernatant_was_discarded_and ₁_ml_of_70% ethanolwasaddedtothepelletwhichwascentrifugedfor15min at 6000×g, 4◦C. The supernatant was again discarded and air

dried.Nucleasefreewater(20␮l)wasaddedandstoredat−20◦C.

Agarosegelelectrophoresis(0.8%)wasperformedat100V.

Statisticalanalysis

The experiments werecarried out in triplicates. The results werecalculatedasmeanalongwithstandarderrorvalues. Statisti-calsignificancewascalculatedusingone-wayanalysisofvariance (ANOVA).Avalueofp<0.05wasconsideredasstatistically signifi-cant.

Resultsanddiscussion

Plant-basedproductsincludingproteinsand smallmolecular compoundshavebeensuggestedasthefavorabledrugsfor can-certreatmentinregardtomanyadverseeffectsexertedbycurrent cancertreatments,namelychemotherapyand radiationtherapy (Ledesmaetal.,2005).Theconcentrationofproteinspresentinthe crudesupernatant(1mg/ml),80%precipitatedsample(0.8mg/ml) and ultrafiltrate (0.5mg/ml) wereestimated by Bradfordassay. Similarstudieshavebeenperformedbyextractingbioactive pro-teinsfromvariousplantsources(Mauryaetal.,2011;Kumarand Santhi,2012).SDS-PAGEgel(12%)showedthepresenceofprotein

Lane1Lane2Lane3 Lane4 Lane5 Lane6Lane7 Lane8Lane9Lane10

43kda 29kda

14kda

9kda

3.5kda

Lane 1: Crude supernatant Lane 2: Crude supernatant Lane 3: Crude supernatant Lane 4: Crude supernatant Lane 5: Protein marker

Lane 6: Protein marker Lane 7: Crude supernatant Lane 8: Crude supernatant Lane 9: Ultra filtered protein Lane 10: Ultra filtered protein

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54.6

52

50

48

46

44

42

40

38

36

34

32

30

27.5

4000.0 3000

MP

MP 001 7201 4000.00 400.00 27.52 53.30 4.00 %T 4 1.00

REF 4000 34.94 2000 41.90 600

3353.97 27.52 2359.95 30.64 2341.43 32.94 1631.26 37.19 1403.00 39.55 1295.63 39.36 1036.60 34.36 909.81 45.45 668.03 41.80 597.22 43.68

pK

3353.97 2359.95 2341.43

1631.26 1403.00

1295.63

1036.60 668.03 909.81

597.22

2000 1500

cm-1

%T

1000 400.0

Fig.2. FTIRspectralanalysisofultrafilteredproteinextractofMorindapubescens.

bandsofultrafilteredprotein(lanes:9,10)andcrudesupernatant (lanes:1–4,7,8).Lanes5,6depictsthebandsofproteinmarker rangingfromthesizeof3.5–43kDa(Fig.1).

TheFTIRspectrumoftheultrafilteredproteinfractions(Fig.2) detectedthepeaksat 1631.26cm−1 _and_3353.97_cm−1 _showing the presence of carbonyl (C O) and hydroxyl (OH) stretching vibrations. Further FTIR analysis of ultrafiltered protein frac-tionsalsodepictedIRabsorptionbandsat2359.95,2341.43and

Fig.3.Photomicrographs(40×)ofuntreatedVerocells(Control).

1403.00cm−1 _indicating _the _presence _of _aminoacids, _peptides and proteinscontainingN H bonds.Characteristicbandsfound intheinfraredspectraofproteinsandpolypeptidesinclude the Amide I and Amide II. These arise from the amidebonds that linktheaminoacids.TheabsorptionassociatedwiththeAmideI bandleadstostretchingvibrationsoftheC Obondoftheamide, absorptionassociatedwiththeAmideIIbandleadsprimarilyto

bending vibrations of the N H bond. Since both the C O and

theN Hbondsareinvolvedinthehydrogenbondingthattakes placebetweenthedifferentelementsofsecondarystructure,the locationsofboththeAmideIandAmideIIbandsaresensitiveto thesecondarystructurecontentofaprotein(SusiandByler,1983; BylerandSusi,1986).Similarstudiesweredoneonsoya,wheat, cornetc.,whereproteinwereisolatedwhichwerefoundoutusing FTIRstudies(Thanietal.,2010).

Fig.4.Photomicrographs(40×)ofVerocellstreatedwithdifferentconcentrationsofultrafilteredproteinextractrangingfrom2to100␮g/ml,isolatedfromtheleavesof

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120

100

80

60

40

20

0

0 2 5 10 15

Concentration of ultrafiltrate protein extract (µg/ml) Vero cell lines

Vero cell lines

% cell viability

20 40 60 100

Fig.5. PercentageofcellviabilityofVerocelllinestreatedwithdifferent concen-trationsofultrafilteredproteinextractrangingfrom2to100␮g/ml,isolatedfrom theleavesofMorindapubescens.

ThecytotoxicactivityofultrafilteredproteinsfromleavesofM. pubescensSm.,onnormalVerocellsandA549cancercellswere measuredby MTT colorimetric assay.Untreated Verocell lines were used as control (Fig.3).Vero cell lines treated with dif-ferentconcentrationsofultrafilteredproteinsrangingfrom2to 100␮g/ml,showedincreasedcellviabilityof98%(Figs.4and5). AnincreasedpercentageofcellviabilityofA549cellswasnoted attheminimumconcentrationsof2–5␮g/mlofultrafiltered pro-teinextract(Figs.6–8).Cellviability(50%)ofA549cells treated with10␮g/mlofultrafilteredproteinextractwasalsoexamined (Figs. 6–8).Furtherit wasobservedthatthere wasadecreased percentageofcellviabilityofA549cells treatedwithincreasing

Fig.6.Photomicrographs(40×)ofuntreatedA549cells(Control).

concentrationsofultrafilteredproteinextractrangingfrom15to 100␮g/ml (Figs. 6–8).Previous studieswere reported on anti-cancerpropertyof proteinsextracted fromGynuraprocumbens, Asteraceae,onbreastcancercellline, MDA-MB-231,atanEC50 valueof3.8␮g/ml(Ngetal.,1992).Similarstudieswerecarriedout fortheanticancerpropertyofBidensalbaprotein-extractagainst humancolorectalcancer(SW480)cells whichdepictedmarked DNAdamagesandapoptosis-relatedcellularmorphologies(Hew etal.,2013).

FurtherDNAfragmentationassaywasperformedbyagarose gel electrophoresis. A DNA ladder characteristic of apoptosis was observed in A549 cells treated with 10␮g/ml of ultrafil-teredproteins(Lane2,Fig.9).However,theDNAextractedfrom theuntreatedVerocells,appearedasdistinctbandshowingthe

Fig.7. Photomicrographs(40×)ofA549cellstreatedwithdifferentconcentrationsofultrafilteredproteinextractrangingfrom2to100␮g/ml,isolatedfromtheleavesof

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120 100 80 60 40 20 0

0 2 5 10 15

Concentration of ultrafiltrate protein extract (µg/ml) A549 cell lines

A549 cell lines

% cell viability

20 40 60 100

Fig.8.PercentageofcellviabilityofA549celllinestreatedwithdifferent concen-trationsofultrafilteredproteinextractrangingfrom2to100␮g/ml,isolatedfrom theleavesofMorindapubescens.

Fig.9.AgarosegeldepictingDNAfragmentationofA549cellsshowing,Lane1: Marker(M)–1kbpladder;Lane2:A549cellstreatedwith10␮g/mlofultrafiltered proteinextract;Lane4:UntreatedVerocelllines.

viabilityofVerocells(Lane4,Fig.9).Apoptosis,akindofcellular deathischaracterizedbytheearlyactivationofendogenous pro-teases,cellshrinkageandDNAfragmentation(Nagata,2000).The nuclearDNAofapoptoticcellsshowsacharacteristicladdering pat-ternofoligonucleosomalfragments(Nagataetal.,2003).Similar observationswerenotedwiththehumancolorectalcancerSW480 cellsand monocyticleukemiaTHP-1cells treatedwiththe pro-teinextractsofCalvatialilacina,PleurotusostreatusandVolvariella volvacea.TheviabilitiesofTHP-1andSW480cellsdecreasedina concentration-dependentmannerafter24hoftreatmentwiththe proteinextractsoftheselectedplants.Apoptoticanalysisrevealed thatthepercentageofSW480cellsintheSubG1phase(amarkerof apoptosis)wasincreasedupontreatmentwithprotein-extractsof PleurotusostreatusandVolvariellavolvacea,indicatingthat oligonu-cleosomalDNAfragmentationexistedconcomitantlywithcellular death(Ongetal.,2008;Jin-Yietal.,2011).

Inconclusiontheultrafilteredproteinsextractedfromtheleaves ofM. pubescensexhibitedsignificantcytotoxicactivityonA549 cellsattheIC50concentrationof10␮g/ml.Besides,theultrafiltered proteinfractiondidnotshowanyinhibitoryeffectsonthe pro-liferationofVerocells.Further,DNAfragmentationwasobserved inA549cellstreatedwithultrafilteredproteins,thereby indicat-ingtheonsetofapoptotic celldeath. Thus,theresultsobtained in this study suggest that the ultrafiltered proteins (10kDa) with apoptosis-inducing activity, isolated from the leaves of

M. pubescens can act as potential anticancer agents in cancer chemotherapy.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

ASTcontributedtobiologicalstudiesandlaboratorywork.SR andPVGcontributedtocriticalreadingofthemanuscript.PVG con-tributedtoplantcollection.SRdesignedthestudyandsupervised thelaboratorywork.Alltheauthorshavereadthefinalmanuscript andapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

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