research article antiproliferative and apoptosis induction...

Research ArticleAntiproliferative and Apoptosis Induction Potential ofthe Methanolic Leaf Extract of Holarrhena floribunda (G Don)

J A Badmus1 O E Ekpo1 A A Hussein2 M Meyer3 and D C Hiss1

1Department of Medical Biosciences University of the Western Cape New Life Sciences Building Robert Sobukwe RoadPrivate Bag Box X17 Bellville Cape Town 7535 South Africa2Department of Chemistry University of the Western Cape Chemical Sciences Building Robert Sobukwe RoadPrivate Bag Box X17 Bellville Cape Town 7535 South Africa3Department of Biotechnology University of the Western Cape New Life Sciences Building Robert Sobukwe RoadPrivate Bag Box X17 Bellville Cape Town 7535 South Africa

Correspondence should be addressed to D C Hiss dhissuwcacza

Received 24 October 2014 Revised 30 January 2015 Accepted 9 February 2015

Academic Editor Yuping Tang

Copyright copy 2015 J A Badmus et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Natural plant products with potent growth inhibition and apoptosis induction properties are extensively being investigated fortheir cancer chemopreventive potential Holarrhena floribunda (HF) is used in a wide range of traditional medicine practicesThe present study investigated the antiproliferative and apoptosis induction potential of methanolic leaf extracts of HF againstbreast (MCF-7) colorectal (HT-29) and cervical (HeLa) cancer cells relative to normal KMST-6 fibroblasts The MTT assay inconjunction with the trypan blue dye exclusion and clonogenic assays were used to determine the effects of the extracts on the cellsCaspase activities were assayed with Caspase-Glo 37 and Caspase-9 kits Apoptosis induction was monitored by flow cytometryusing the APOPercentage and Annexin V-FITC kits Reactive oxygen species (ROS) was measured using the fluorogenic molecularprobe 5-(and-6)-chloromethyl-2101584071015840-dichlorofluorescein diacetate acetyl ester and cell cycle arrest was detected with propidiumiodide Dose-response analyses of the extract showed greater sensitivity in cancer cell lines than in fibroblast controls Inductionof apoptosis ROS and cell cycle arrest were time- and dose-dependent for the cancer cell lines studied These findings provide abasis for further studies on the isolation characterization and mechanistic evaluation of the bioactive compounds responsible forthe antiproliferative activity of the plant extract

1 Introduction

Cancer remains the leading cause of mortality and morbidityin the world Annually about 10 million newly diagnosedcancer cases are reported of which 6 million result indeaths worldwide [1] In addition about 16 million newcases and 6 hundred thousand deaths have been predictedfor 2013 in the USA alone [2] Chemotherapy is a majormode of treatment for various cancers but its success is con-founded by unwanted toxic side effects and drug resistance[3ndash5] Traditional uses of natural plants to treat differentdiseases including cancer and their scientific isolation andcharacterization for translation into effective drugs againstcancer underscore their importance This viable source oftherapeutic agents from nature might not be unconnected

to the structural diversity inherent in a million species ofplants and microorganisms but still remains beyond thecomprehension of man [6] Drugs currently in use as anti-cancer have over 60 of their origin from natural products[7] Vinblastine (Velban) vincristine (Oncovin) vinorelbine(Navelbine) etoposide (VP-16) teniposide (VM-26) pacli-taxel (Taxol) docetaxel (Taxotere) topotecan (Hycamtin)and irinotecan (Camptosar) are some examples of anticancerdrugs that had passed through different stages of preclinicaland clinical trials and hence were approved for clinical use[8 9] Holarrhena floribunda (G Don) is a tree that belongsto the Apocynaceae family of plants known to be rich inalkaloids Alkaloids are compounds found to be responsiblefor the anticancer activity of the Camptotheca acuminataplant for example from which the currently used anticancer

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 756482 11 pageshttpdxdoiorg1011552015756482

2 Evidence-Based Complementary and Alternative Medicine

drug camptothecin is isolated The leaves of Holarrhenafloribunda are used in folklore medicine as an antimalarialin Ghana [10] In Ivory Coast the bark of this plant is usedas a treatment for diarrhoea and the leaves were used as atreatment for amenorrhea [11] The root is boiled in milk andused to bathe boys attaining puberty and it is also used asa cure for snakebites and venereal disease [12] Extracts ofHolarrhena floribunda have been shown to exhibit significantcytotoxic activity compared to other plants screened [13] Itsantioxidant antimutagenic and lipid peroxidation inhibitionpotential have been reported [14]The present study aimed toevaluate the antiproliferative apoptosis and reactive oxygenspecies inducing activities of the methanolic leaf extractof Holarrhena floribunda in breast cancer (MCF-7) coloncancer (HT-29) and cervical cancer (HeLa) cells relative tonormal fibroblasts (KMST-6)

2 Materials and Methods

21 Plant Material Fresh samples of Holarrhena floribundaleaves were collected during the raining season in IgbajoOsun State Nigeria Messrs E C Chukwuma and O AUgbogu identified the plant at the Federal Research Instituteof Nigeria (FRIN) A voucher specimen (FHI 109764) wasdeposited at the institute herbarium

22 Preparation of Methanolic Leaf Extract of Holarrhenafloribunda The leaves ofHolarrhena floribundawere allowedto air-dry at room temperatureThe dried leaves were soakedin absolutemethanolThemixture was thoroughlymixed andfiltered after 48 husing aBuchner vacuum funnelThe residuewas reconstituted in fresh absolute methanol for 24 h andfiltered again as described aboveThe filtered supernatant wasevaporated to dryness with a rotary evaporator to eliminatemethanol The weight of pulverized extract of the leavesobtained represents a 2178 yield in relation to the weight ofthe leaves used A stock solution of the extract was preparedby dissolving it first in DMSO followed by reconstitutionwithgrowth medium such that the final equivalent concentrationof DMSO in the extract was exactly 01

23 Maintenance of Cell Culture Breast cancer cells (MCF-7) colon cancer cells (HT-29) cervical cancer cells (HeLa)and normal human fibroblasts (KMST-6) were obtained fromthe Department of Biotechnology University of the WesternCape The cell lines were maintained in Dulbeccorsquos ModifiedEaglersquosMedium (DMEM) and supplementedwith 10of fetalbovine serum (FBS) and 1 of penicillin and streptomycin(100UmL penicillin and 100 120583gmL streptomycin) All tissueculture operations were carried out in a model NU-5510ENuAireDHDautoflow automaticCO

2air-jacketed incubator

24 Chemicals 3-(45-Dimethylthiazol-2-yl)-25-diphenyl-tet razolium bromide (MTT) trypan blue dye and crys-tal violet were purchased from Sigma-Aldrich APOP-ercentage dye was obtained from Biocolor Ltd (Car-rickfergus Northern Ireland) 5-(and-6)-chloromethyl-2101584071015840-dichlorofluorescein diacetate acetyl ester (CM-H

2DCFDA)

from Invitrogen (South Africa) Annexin V-FITC apoptosis

kit from BD Pharmingen (USA) and Caspase-Glo 37 andcaspase-9 assay system kits from Promega (USA) All otherchemicals used were of analytical grade

25 MTT Assay The viable cells were seeded at a densityof 5 times 104 (100120583Lwell) in 96-well plates and incubatedin a humidified atmosphere of 5 CO

2and 95 air at

37∘C for 24 h to form a cell monolayer After 24 h thesupernatant on the monolayer was aspirated and 100120583L ofmedium and varying log concentrations of extract (01 110 100 and 1000 120583gmL) were added and incubated for24 48 and 72 h time points After the specific times ofexposure to the extract 20 120583L of 5mgmL MTT in PBS wasadded to each well and incubated for 3 h at 37∘C in a 5CO2atmosphere Supernatants were removed and 150120583L of

isopropanol was added and the plates were gently shaken for15min to solubilize the formazan crystals and absorbancewasmeasured at 560 nm using GloMax-Multi Detection System(Promega USA) The percentage inhibition of proliferationwas calculated using the formula below and IC

50values were

calculated from log dose-response curves using GraphPadPrism software version 6 for Windows (GraphPad SoftwareLa Jolla California USA httpwwwgraphpadcom) Con-sider

Inhibition of Proliferation

= 100 minusTest Absorbance at 560 nm

Untreated Control Absorbance at 560 nmtimes100

(1)

26 Trypan Blue Dye Exclusion Assay The viable cells wereseeded at a density of 5 times 104 (1mLwell) in a 12-wellplate and incubated in a humidified atmosphere of 5 CO

2

and 95 air at 37∘C for 24 h to form a cell monolayerAfter 24 h culture medium was gently aspirated and treatedwith 1mL of medium and varying concentrations of extract(100 200 300 400 and 500 120583gmL) for 24 h The adherentcells were removed by trypsinization using 025 EDTAtrypsin (Sigma-Aldrich) The cells were centrifuged usinga Bio-Rad tabletop centrifuge at 2500 rpm for 5min andthe supernatant was discarded to obtain a cell pellet Cellpellets were resuspended in freshmedium fromwhich a 10 120583Laliquot was mixed with an equal volume of 04 trypan bluedye and loaded into Bio-Rad TC20 cell counter Readingswere automatically generated from themachine and recordedbased on the formula below

Inhibition = 100 minus Total Dead Cell CountTotal Cell Count

times 100(2)

27 Clonogenic Assay Theviable cells were plated at a densityof 1 times 103 (1mLwell) in 6-well plates and allowed to attachfor 24 h After 24 h the cells were treated with the methanolicleaf extract ofHolarrhena floribunda (100 200 300 400 and500120583gmL) and incubated further for 24 h in a humidifiedCO2incubatorThemedium containing extract was removed

and replaced with fresh medium and incubated for another 5

Evidence-Based Complementary and Alternative Medicine 3

50

100

150

minus2 minus1 0 1 2 3 4

Log dose

Cell

s pro

lifer

atin

g (

)

24hr exposure48hr exposure72hr exposure

(a)

50

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150

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s pro

lifer

atin

g (

)



Log dose

(b)

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s pro

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atin

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(c)

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s pro

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(d)

Figure 1 The graphs (a)ndash(d) show the log dose cytotoxic effects of methanolic leaf extract of Holarrhena floribunda in HT-29 HeLa MCF-7and KMST-6 cell lines respectively for 24 48 and 72 h treatments MTT assay was employed to assess the cytotoxic effect of the extract andthe graphs were prepared as means plusmn SD of five separate experiments using GraphPad Prism 6 statistical software

days after which the medium was removed and the cells werewashed with PBS Cells were fixed in 500120583L fixative (75methanol and 25acetic acid) for 5min at room temperatureThe fixative was aspirated and the cells were stained with05 crystal violet for 30min at room temperature Cells werethen washed under running tap water to remove the dyeColonies were counted and the survival rate was calculatedas the percentage of treated over untreated

28 APOPercentage Apoptosis Assay Thecells were treated asreported in the trypan blue assay above Following incubationfor 24 h floating cells in each respective treatment weretransferred to 15-mL centrifuge tubes and the adherent cellstrypsinized and added to the respective tubes containingfloating cells Cells were washed with 1 PBS and resus-pended in residual PBS APOPecerntage dye (100120583L) in

complete culture medium (diluted 1 160 vv) was added tothe tubes and allowed to incubate for 30min at 37∘C in ahumidified CO

2incubator After the incubation cells were

washed twice by resuspension in 500120583L of 1 PBS andcentrifugation for 5min at 3000 rpm The cell pellet soobtained was resuspended in PBS and aliquots were analyzedon a BectonDickinson FACScan instrument (BDBiosciencesPharmingen San Diego CA USA) fitted with a 488 nmargon laser A minimum of 10000 cells per sample wereacquired and analyzed using the CellQuest Pro software (BDBiosciences)

29 AnnexinPI Assay The cells (2mL) at a density of 5times104were grown in 40-mm petri dishes and allowed to attach for24 h after which cells were treated with the 200120583gmL extractfor 12 24 and 48 h After the various treatment periods


0

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Figure 2 Methanolic leaf extract effect on the viability of cell lines(HT-29 MCF-7 HeLa and KMST-6) evaluated using the trypanblue exclusion assay Cell viability was assessed using Bio-Rad TC20cell counter The graph represents means plusmn SD of five independentexperiments using GraphPad Prism 6 statistical software while a bc and d represent 119875 lt 05 001 0001 and 00001 respectively

cells were harvested and centrifuged at 335 g for 10min Thesupernatants were washed in 1 PBS and resuspended inAnnexin V binding bufferThe cells were centrifuged at 335 gfor 10min and supernatants were discarded The cell extractswere suspended in 100 120583L Annexin V binding buffer and5 120583L Annexin V Alexa Fluor 488 was added and allowedto incubate in the dark for 15min PI (4 120583L) diluted in 1xAnnexin V binding buffer (1 10) was added and allowed toincubate for 15min in the dark at room temperature AnnexinV binding buffer (500120583L) was added to wash the AnnexinPIstained cells AnnexinPI was evaluated according to previ-ously describedmethod [15] on a BectonDickinson FACScaninstrument (BD Biosciences Pharmingen San Diego CAUSA) fitted with a 488 nm argon laser A minimum of 10000cells per sample were acquired and analyzed using CellQuestPro software

210 Caspases 37 and 9 The evaluation of caspase-37was performed according to the manufacturerrsquos instructionsBriefly 100 120583L of 5 times 104 cellsmL was seeded in white-walled96-well microplates and incubated for 24 h The cells weretreated with the methanolic extract (100 200 300 400 and500120583gmL) for 24 h After treatment an equal volume ofCaspase-Glo 37 reagent was added and agitated for 30 secand the luminescence signal recordedwith theGloMax-MultiDetection System (PromegaUSA) after incubation for 1 hourCaspase-9 was evaluated by treating the cells in the samemanner as described above

211 Cell Cycle Analysis The cells were seeded at a densityof 2 times 105mL (2mLwell) in 6-well plates and incubated

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Col

ony

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MCF-7

Figure 3 Percentage of colonies formed after the cells were treatedfor 24 h with methanolic leaf extract of Holarrhena floribunda Theparaformaldehyde fixed cells were stained with 05 crystal violetdye and the colonies formed were counted using colony counterColony formation inhibition at each concentration of the extractis expressed in terms of percentage of control and reported as themeansplusmn SD of five independent experiments while a and b represent119875 lt 05 and 00001 respectively

for 24 h at 37∘C in a CO2incubator to form a monolayer

The cells were treated with 200120583gmL of methanolic extractfor 12 and 24 h After treatment the cells were washed with2mL of PBS and cell pellet was resuspended in 1mL (1wv) paraformaldehyde in PBS (pH 74) on ice for 30minThe cell pellets were washed twice in 5mL of PBS Slowly70 of ethanol was added to the cells while vortexing toreduce cell clumping The cells were stored in minus20∘C for 48 hafter which cells were pelleted at 4000 rpm for 10min Thecells were then washed in 2X PBS and 1mL of PI master mixcontaining 100 120583gmL RNase and 40 120583gmL PI in PBS Cellcycle phase distributionwas determinedusing a FACSCaliburflow cytometer (BD Biosciences Franklin Lakes NJ USA)TheDNAcontent of 50000 eventswas determined byModFitsoftware (Verity Software House Topsham ME) whichprovided histograms to evaluate cell cycle distribution

212 Reactive Oxygen Species Detection of reactive oxygenspecies within cells was evaluated using the fluorogenicmolecular probe 5-(and-6)-chloromethyl-2101584071015840-dichloroflu-orescein diacetate acetyl ester (CM-H

2DCFDA Invitrogen)

Briefly cells were cultured in 6-well plates at a densityof 2 times 105mL (2mLwell) The cells were treated with200120583gmL of extract for 12 and 24 h After the treatmentcells were washed with PBS and stained with 75 120583M of (CM-H2DCFDA) prepared in PBS from a DMSO stock solution

and incubated for 30min at 37∘C in a humidified CO2

incubatorThe cells werewashed twice with ice-cold PBS after


HeLa

KMST-6

HT-29

MCF-7

Control 100120583gmL 200120583gmL 300120583gmL 400120583gmL 500120583gmL

Figure 4 Colony formation ability of the cells following treatment with the extract at different concentrations for 24 h The pictures weretaken using ZEISS Primo Vert microscope and the colonies were counted with colony counter

which the cells were acquired and 10000 events analyzed ona Becton Dickinson FACScan instrument (BD BiosciencesPharmingen SanDiego CAUSA) fittedwith a 488 nmargonlaser

213 Statistical Analyses Data are expressed as mean plusmn SDof experiments performed in triplicate The values wereanalyzed by two-way ANOVA followed by Tukeyrsquos multiplecomparison test using GraphPad Prism software version 6for Windows (GraphPad Software La Jolla California USAhttpwwwgraphpadcom)

3 Results

The cytotoxic effect of the methanolic extract of Holarrhenafloribunda was evaluated on HT-29 HeLa MCF-7 andKMST-6 cells using the MTT assay Figure 1 shows the logdose-response curve from which the half-maximal (IC

50)

cytotoxic effects on the extract were estimated by nonlinearregression analysis Table 1 depicts IC

50values for the 24

48 and 72 h treatments The results showed that all the celllines responded to the cytotoxic effects of the plant extractin a dose- and time-dependent manner The HeLa cancercells however were more sensitive to the plant extract asshown by its IC

50values for 24 48 and 72 h (1826 1274 and

1067 120583gmL resp) Moreover the extract exhibited selectivecytotoxicity in normal fibroblast cell KMST-6 with higherIC50

values of 3769 4282 and 3423 in 24 48 and 72 hrespectively Cell viability was evaluated using the trypan blue

Table 1 IC50 values of the methanolic leaf extract of Holarrhenafloribunda effects on HT-29 HeLa MCF-7 and KMST-6 cell linesfor 24 48 and 72 h treatments The IC50 value was obtained fromthe log dose cytotoxic effects of the extract using GraphPad Prism 6statistical software

Time (h) HT-29(120583gmL)

HeLa(120583gmL)

MCF-7(120583gmL)

KMST-6(120583gmL)

24 3492 1826 3576 376948 2175 1274 2443 428272 1594 1067 1267 3423

dye exclusion assay in cell lines exposed to doses of 100ndash500120583gmL of extract The assay further reaffirms the sensi-tivity of the cancer cell lines compared to the noncancerouscell (KMST-6) as presented in Figure 2

The antiproliferative activity of the extract was furtherevaluated using the clonogenic survival assay This assaymeasures the potential of cells to expand into coloniesunrestricted by growth contact inhibitionmdashunlike normalgrowing cells that cease proliferation upon contact inhibitionAs presented in Figures 3 and 4 the colony formation declineswith increasing concentration of the extract RemarkablyMCF-7 and HeLa cell lines exhibited no colonies at extractconcentrations of 400 and 500 120583gmL

The apoptotic effect of the methanolic extract wasexplored by staining the cells with APOPercentage dyeand evaluation by flow cytometry The results showed thatthe induction of cytotoxicity observed occurs through themechanisms associated with apoptosis The extract induced


Table 2The percentage of cell populations in different stages (live apoptotic and necrotic) following the extract treatment and evaluated bydouble staining in Annexin V-FITCpropidium iodide using flow cytometric assay

HeLa MCF-7 HT-2912 h 24 h 48 h 12 h 24 h 48 h 12 h 24 h 48 h

Live () 9241 plusmn 003 8494 plusmn 069 1905 plusmn 051 9522 plusmn 136 9215 plusmn 177 71 plusmn 123 9422 plusmn 223 7845 plusmn 225 8343 plusmn 461Early apoptosis () 341 plusmn 032 626 plusmn 050 341 plusmn 036 469 plusmn 127 611 plusmn 034 015 plusmn 001 456 plusmn 160 1672 plusmn 164 001 plusmn 001Late apoptosis () 353 plusmn 012 653 plusmn 078 3347 plusmn 190 018 plusmn 002 052 plusmn 019 393 plusmn 158 026 plusmn 004 446 plusmn 070 046 plusmn 016Dead () 066 plusmn 023 138 plusmn 120 4360 plusmn 175 003 plusmn 002 016 plusmn 003 8760 plusmn 117 005 plusmn 001 007 plusmn 001 1642 plusmn 173

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(120583gmL)

Figure 5 Apoptotic effect of methanolic leaf extract of Holarrhenafloribunda on cell lines (HT-29 MCF-7 HeLa and KMST-6) Thecells were stained with the APOPercentage dye and evaluated usinga flow cytometer The graph is reported as the means plusmn SD offive independent experiments using GraphPad Prism 6 statisticalsoftwarewhile a b c and d represent119875 lt 05 001 0001 and 00001respectively

apoptosis in a concentration-dependent manner (Figure 5)The HeLa cell line was significantly (119875 lt 00001) sensitiveto the extract when compared with other cell linesThe sensi-tivity of the KMST-6 cells to the apoptosis-inducing potentialof the extract was also low compared to other cell lines Theapoptosis-inducing potential of the extract was further testedin cancer cells using the Annexin-FITCpropidium iodidedouble staining flow cytometric assayThe cells were exposedto 200120583gmL of extract for 12 24 and 48 h Table 2 showsthat after 24 h of exposure HT-29 cells had undergone earlyapoptosis HeLa cells entered a late apoptotic stage after 48 hwhile MCF-7 cells exhibited significant necrotic cell at thistime period

The activation of caspases 37 and 9 was evaluated incancer cells to establish the cell death pathway induced bythe extract Figure 6 indicates that caspase-37 activity inHeLa cells consistently and significantly increased severalfoldsmdashin a concentration-dependent manner between 200and 500 120583gmL of the extractmdashabove those of theMCF-7 andHT-29 cells Caspase-9 activities in all the cell lines decreasein a concentration-dependent manner (Figure 7)

Relat

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in ca

spas

e-3

25

20

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)

Figure 6 Effects ofmethanolic leaf extract ofHolarrhena floribundaon the caspase-3 activation in cell lines (HT-29 HeLa and MCF-7)Caspase-3 activity was evaluated usingCaspase-Glo 37 luminescentassay kit (Promega) The graph is reported as the means plusmn SD ofthree independent experiments using GraphPad Prism 6 statisticalsoftware

Relat

ive f

old

incr

ease

in ca

spas

e-9

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)

Figure 7 Effects ofmethanolic leaf extract ofHolarrhena floribundaon the caspase-9 activation in cell lines (HT-29 HeLa and MCF-7)Caspase-9 activity was evaluated using Caspase-Glo 9 luminescentassay kit (Promega) The graph is reported as the means plusmn SD ofthree independent experiments using GraphPad Prism 6 statisticalsoftware


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Figure 8 Representative histograms of DNA content distribution of cell cycle phases of HeLa cell treated with methanolic leaf extract ofHolarrhena floribunda for 12 and 24 h (a) represents 12 h control cell while (b) (c) and (d) represent 24 h control and 12 h and 24 h cellstreated with the 200 120583gmL extract respectively

The effects on the phases of the cell cycle after 12 and24 h exposure periods of cancer cells to 200120583gmL of extractwere evaluated using flow cytometry Representative cellcycle distribution histograms of HeLa cell are presented inFigure 8 whereas Figure 9 shows the percentage of cells indifferent cell cycle phases for HeLa MCF-7 and HT-29at 12 and 24 h The results show that the extract inducedsignificant accumulation of cells inG

0G1phases and reduced

the number of proliferating cells as shown by reduced S-phase at both 12 and 24 h for all the cells tested Nextthe ability of the extract (200120583gmL) at 12 and 24 h toinduce reactive oxygen species (ROS) was evaluated usingthe cell permeant dye chloromethyl-2101584071015840-dichlorofluorescindiacetate (CM-H

2DCFDA) As shown in Figures 10 and 11

cells treated withHolarrhena floribunda extract (black panel)showed increases in ROS concentration compared to theuntreated control (pink panel) in a time-dependent mannerThe ROS induction effect of the extract was pronounced inHeLa cells at 12 h while in HT-29 at 24 h The inductionof ROS in MCF-7 is significantly low compared with othercancer cell lines

4 Discussion

The use of plants as a source of human therapeutic medicineis as old as recorded history The importance of plantsas agents of therapeutic components is increasingly beingrecognized in line with current advances in technologyGlobally natural plant compounds have attracted attentionas alternative therapeutic strategies in the fight againstdiseases primarily because of their low toxicity and hightherapeutic index [16 17] Many existing and contemporarydrugs in clinical use are derived from the natural plants[7] Holarrhena floribunda leaves are an important source ofdrugs used in traditional medicine to cure different diseasesincluding diabetes malaria cancer and oxidant damagerelated diseases [18 19] The present study evaluated theanticancer activity of the Holarrhena floribunda methanolicleaf extract in breast cancer cell (MCF-7) colon cancer (HT-29) cervical cancer (HeLa) and normal human fibroblastcell (KMST-6) The results of this study show that the extractexhibited cytotoxic effects towards all the cancer cell lines in adose- and time-dependent manner The IC

50values obtained


S0

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HeLa 12h controlHeLa 12h treated


(a)

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c c c

Cell

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ses (

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MCF 12h control MCF 24h controlMCF 24h treatedMCF 12h treated

G0G1 G2M

(b)

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Cell

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HT-29 12h controlHT-29 12h treated


G0G1 G2M

(c)

Figure 9 Percentage of HeLa cell (a) MCF-7 (b) and HT-29 (c) in the G0G1 S and G

2M phases after incubation with the 200120583gmL leaf

extract of Holarrhena floribunda for 12 and 24 h The values are representative of means plusmn SD of five separate experiments using GraphPadPrism 6 statistical software while a b and c represent 119875 lt 05 001 and 0001 respectively

for the various treatment protocols demonstrate that HeLacells are more sensitive to the cytotoxic activity of the plantwhile KMST-6 a normal human fibroblast cell line showedless sensitivity to the extract The potential of the anticanceractivity of the extract to discriminate between normal andcancer cells is an important paradigm in the design anddiscovery of chemotherapeutic agents Consistent with thisconcept trypan blue dye exclusion and colony formationassays confirm the antineoplastic activities of the extractagainst cancer cell lines compared to the normal KMST-6human fibroblast cell lineHolarrhena floribunda is known tobe rich in several phytochemicals like alkaloids flavonoidstannins and cardiac glycosides Some of these phytochem-icals have been reported to possess antineoplastic activities

against different cancer cell lines Lamchouri et al [20] andHoet et al [21] showed that the antiproliferative activities ofPeganum harmala seeds and Cassytha filiformis respectivelywere due to their alkaloid constituents Flavonoid activitiesagainst various cancers have also been reported [22ndash24]

To further elucidate the pathways of the cell deathinduced by the extract phosphatidylserine flipping was eval-uated using the APOPecerntage and AnnexinPI flow cyto-metric assays Exposure of phosphatidylserine on the externalsurface of the cell membrane is generally accepted as one ofthe biomarkers of apoptosis [25] The results demonstratedthe concentration-dependent apoptotic-inducing potential ofthe extract As a necessary corollary of the results of thecytotoxicity assay HeLa cell showed a significant sensitivity


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Cou

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Figure 10 The representative histograms of the cell stained with CM-H2DCFDA dye and evaluated using a flow cytometer The pink

histogram depicts stained control cells while black shows the cells treated with the extract at 12 and 24 h

to the extract compared to the other cell lines tested TheAnnexinPI assay also confirmed the ability of the extract toinduce early and late apoptosis Unlike necrosis apoptosis isan important cell death mechanism that does not trigger aninflammatory response that occasions collateral destructionof normal cells in the surrounding microenvironment [26]Thus apoptosis is a protective mechanism that maintains

tissue homeostasis by removing ailing cells [27] Cancercells however exhibit resistance to apoptosis in order tosustain their uncontrolled proliferation and therefore anyapoptosis modulating compound is desirable as a plausiblechemotherapeutic agent against cancer [28]

Two basic pathways involved in apoptosis areintrinsic (mitochondrial) and extrinsic (death receptor)


12 240

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Fold

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FI

HeLaHT-29MCF-7

Figure 11 The graph shows fold increase in mean fluorescenceintensity (MFI) of cells undergoing induction of ROS due to thetreatment with the methanolic extract at 12 and 24 h The resultsare means plusmn SD of three separate experiments evaluated using flowcytometry

pathways [29] Caspase-37 is one of the effector caspasesthat is involved in the final execution of dying cells whilecaspase-9 is an initiator caspase that is involved in theintrinsic pathway [26 27] To understand the mechanismof action induced by the extract caspase-37 and caspase-9activities were evaluated The results showed that the extractinduced concentration-dependent increases in caspase-3activity in HeLa cell lines while in contrast a concentration-dependent decrease in such activity was observed in MCF-7and HT-29 cells Caspase-9 results showed a similar trend ofdecrease in activities in all the cell lines The possible reasonfor these observed results can be explained in two waysThe first is that the increase in caspase-3 activity observedin HeLa cells suggests that the extract induced apoptosis isa caspase-dependent manner while the decrease in caspaseactivities in MCF-7 and HT-29 cells presumably involvesdegradation of the protease although the mechanisms ofapoptosis induction need to be clarified

However in agreement with antiproliferative activity ofthe extract the results of the cell cycle evaluation showthat the extract arrests cell cycle progression by significantlyrestricting cells in G

0G1phase This implies that the extract

perturbs the protein synthesis that is important to cellprogression from G

1to S-phase It is known that p53 and

MDM2 proteins are important to the progression of the cellcycle at G

0G1[30 31] It may be possible that the extract

plays a role in the disturbance of these proteins but this aspectwas not investigated in this study The effect of the extracton cell cycle progression may be due to its phytochemicalconstituents such as flavonoids and alkaloids

Cells are known to thrive in low levels of reactive oxygenspecies (ROS) but a relative increase in ROS induces cellcycle arrest and apoptosis [24] ROS-modulating drugs are

however being proposed as therapeutic strategies to selec-tively target the destruction of cancer cells [32]The results ofthis study indicate that the extract induced a time-dependentincrease in ROS production ROS production due to extract(200120583gmL) treatment for 12 and 24 h is more evident inHeLa cells which may explain why this cell line is moresensitive to the extract with regard to its antiproliferativeapoptotic and cell cycle arrest effects

5 Conclusion

Taken together the results of this study clearly show thatthe extract was able to induce growth inhibition apoptosiscell cycle arrest and induction of ROS in cancer cells Thecompelling result shows that the extract contains possibleanticancer bioactive compounds that require isolation andfurther characterization

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] D Maxwell Parkin F Bray J Ferlay and P Pisani ldquoEstimatingthe world cancer burden Globocan 2000rdquo International Journalof Cancer vol 94 no 2 pp 153ndash156 2001

[2] R Siegel DNaishadham andA Jemal ldquoCancer statistics 2013rdquoCA Cancer Journal for Clinicians vol 63 no 1 pp 11ndash30 2013

[3] N S Yaacob N Hamzah N N Nik Mohamed Kamal etal ldquoAnticancer activity of a sub-fraction of dichloromethaneextract of Strobilanthes crispus on human breast and prostatecancer cells in vitrordquo BMC Complementary and AlternativeMedicine vol 10 article 42 2010

[4] R C F Leonard S Williams A Tulpule A M Levine andS Oliveros ldquoImproving the therapeutic index of anthracyclinechemotherapy focus on liposomal doxorubicin (Myocet)rdquoBreast vol 18 no 4 pp 218ndash224 2009

[5] K YWonders and B S Reigle ldquoTrastuzumab and doxorubicin-related cardiotoxicity and the cardioprotective role of exerciserdquoIntegrative Cancer Therapies vol 8 no 1 pp 17ndash21 2009

[6] A B da Rocha R M Lopes and G Schwartsmann ldquoNaturalproducts in anticancer therapyrdquo Current Opinion in Pharmacol-ogy vol 1 no 4 pp 364ndash369 2001

[7] G M Cragg P G Grothaus and D J Newman ldquoImpactof natural products on developing new anti-cancer agentsrdquoChemical Reviews vol 109 no 7 pp 3012ndash3043 2009

[8] H-K Wang S L Morris-Natschke and K-H Lee ldquoRecentadvances in the discovery and development of topoisomeraseinhibitors as antitumor agentsrdquoMedicinal Research Reviews vol17 no 4 pp 367ndash425 1997

[9] K-H Lee ldquoNovel antitumor agents from higher plantsrdquoMedic-inal Research Reviews vol 19 no 6 pp 569ndash596 1999

[10] A Bouquet and M Debray ldquoPlantes medicinalis de Cote drsquoIvoirerdquo Travaux et Documents de lrsquoORSTOM ORSTOM ParisFrance 1974

[11] J Kerharo and J G Adam Pharmacopee Senegalaise Tradi-tionelle Plantes Medicinales et Toxiques Vigot Freres ParisFrance 1974


[12] M M Iwu Handbook of African Medicinal Plants CRC PressBoca Raton Fla USA 2nd edition 2014

[13] P M Abreu E S Martins O Kayser et al ldquoAntimicrobialantitumor and antileishmania screening of medicinal plantsfrom Guinea-Bissaurdquo Phytomedicine vol 6 no 3 pp 187ndash1951999

[14] J A Badmus O A Odunola E M Obuotor and O OOyedapo ldquoPhytochemicals and in vitro antioxidant potentialsof defattedmethanolic extract of Holarrhena floribunda leavesrdquoAfrican Journal of Biotechnology vol 9 no 3 pp 340ndash346 2010

[15] A M Rieger K L Nelson J D Konowalchuk and D RBarreda ldquoModified annexin Vpropidium iodide apoptosisassay for accurate assessment of cell deathrdquo Journal of VisualizedExperiments no 50 Article ID e2597 2011

[16] P D Sanchez-Gonzalez F J Lopez-Hernandez J M Lopez-Novoa and A I Morales ldquoAn integrative view of the patho-physiological events leading to cisplatin nephrotoxicityrdquoCriticalReviews in Toxicology vol 41 no 10 pp 803ndash821 2011

[17] I L Jung ldquoSoluble extract from Moringa oleifera leaves witha new anticancer activityrdquo PLoS ONE vol 9 no 4 Article IDe95492 2014

[18] J A Badmus O A Odunola T A Yekeen et al ldquoEvaluationof antioxidant antimutagenic and lipid peroxidation inhibitoryactivities of selected fractions of Holarrhena floribunda (GDon) leavesrdquo Acta Biochimica Polonica vol 60 no 3 pp 435ndash442 2013

[19] J Fotie D S Bohle M L Leimanis E Georges G Rukungaand A E Nkengfack ldquoLupeol long-chain fatty acid esters withantimalarial activity from Holarrhena floribundardquo Journal ofNatural Products vol 69 no 1 pp 62ndash67 2006

[20] F Lamchouri M Zemzami A Jossang A Settaf Z H IsrailiandB Lyoussi ldquoCytotoxicity of alkaloids isolated fromPeganumharmala seedsrdquoPakistan Journal of Pharmaceutical Sciences vol26 no 4 pp 699ndash706 2013

[21] S Hoet C Stevigny S Block et al ldquoAlkaloids from Cassythafiliformis and related aporphines antitrypanosomal activitycytotoxicity and interaction with DNA and topoisomerasesrdquoPlanta Medica vol 70 no 5 pp 407ndash413 2004

[22] S Yadegarynia A Pham A Ng et al ldquoProfiling flavonoidcytotoxicity in human breast cancer cell lines determination ofstructure-function relationshipsrdquoNatural Product Communica-tions vol 7 no 10 pp 1295ndash1304 2012

[23] M Matsuo N Sasaki K Saga and T Kaneko ldquoCytotoxicity offlavonoids toward cultured normal human cellsrdquo Biological andPharmaceutical Bulletin vol 28 no 2 pp 253ndash259 2005

[24] H Li J Chen C Xiong HWei C Yin and J Ruan ldquoApoptosisinduction by the total flavonoids from Arachniodes exilis inHepG2 cells through reactive oxygen species-mediated mito-chondrial dysfunction involving MAPK activationrdquo Evidence-Based Complementary and Alternative Medicine vol 2014Article ID 906941 11 pages 2014

[25] V A Fadok D R Voelker P A Campbell J J Cohen D LBratton and P M Henson ldquoExposure of phosphatidylserine onthe surface of apoptotic lymphocytes triggers specific recogni-tion and removal by macrophagesrdquo Journal of Immunology vol148 no 7 pp 2207ndash2216 1992

[26] S Elmore ldquoApoptosis a review of programmed cell deathrdquoToxicologic Pathology vol 35 no 4 pp 495ndash516 2007

[27] T-J Fan L-H Han R-S Cong and J Liang ldquoCaspase familyproteases and apoptosisrdquo Acta Biochimica et Biophysica Sinicavol 37 no 11 pp 719ndash727 2005

[28] Y S Tor L S Yazan J B Foo et al ldquoInduction of apoptosisthrough oxidative stress-related pathways in MCF-7 humanbreast cancer cells by ethyl acetate extract of Dillenia suffruti-cosardquo BMC Complementary and Alternative Medicine vol 14article 55 2014

[29] Y L Hsu C C Chia P J Chen S E Huang S C Huangand P L Kuo ldquoShallot and licorice constituent isoliquiritigeninarrests cell cycle progression and induces apoptosis throughthe induction of ATMp53 and initiation of the mitochondrialsystem in human cervical carcinoma HeLa cellsrdquo MolecularNutrition and Food Research vol 53 no 7 pp 826ndash835 2009

[30] C Plasencia R Dayam QWang et al ldquoDiscovery and preclin-ical evaluation of a novel class of small-molecule compoundsin hormone-dependent and -independent cancer cell linesrdquoMolecular CancerTherapeutics vol 4 no 7 pp 1105ndash1113 2005

[31] Y-Q Tang I B Jaganath and S D Sekaran ldquoPhyllanthus sppinduces selective growth inhibition of PC-3 and mewo humancancer cells through modulation of cell cycle and induction ofapoptosisrdquo PLoS ONE vol 5 no 9 Article ID e12644 2010

[32] N Lampiasi A Azzolina N DrsquoAlessandro et al ldquoAntitumoreffects of dehydroxymethylepoxyquinomicin a novel nuclearfactor-120581B inhibitor in human liver cancer cells are mediatedthrough a reactive oxygen species-dependent mechanismrdquoMolecular Pharmacology vol 76 no 2 pp 290ndash300 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


drug camptothecin is isolated The leaves of Holarrhenafloribunda are used in folklore medicine as an antimalarialin Ghana [10] In Ivory Coast the bark of this plant is usedas a treatment for diarrhoea and the leaves were used as atreatment for amenorrhea [11] The root is boiled in milk andused to bathe boys attaining puberty and it is also used asa cure for snakebites and venereal disease [12] Extracts ofHolarrhena floribunda have been shown to exhibit significantcytotoxic activity compared to other plants screened [13] Itsantioxidant antimutagenic and lipid peroxidation inhibitionpotential have been reported [14]The present study aimed toevaluate the antiproliferative apoptosis and reactive oxygenspecies inducing activities of the methanolic leaf extractof Holarrhena floribunda in breast cancer (MCF-7) coloncancer (HT-29) and cervical cancer (HeLa) cells relative tonormal fibroblasts (KMST-6)

2 Materials and Methods

21 Plant Material Fresh samples of Holarrhena floribundaleaves were collected during the raining season in IgbajoOsun State Nigeria Messrs E C Chukwuma and O AUgbogu identified the plant at the Federal Research Instituteof Nigeria (FRIN) A voucher specimen (FHI 109764) wasdeposited at the institute herbarium

22 Preparation of Methanolic Leaf Extract of Holarrhenafloribunda The leaves ofHolarrhena floribundawere allowedto air-dry at room temperatureThe dried leaves were soakedin absolutemethanolThemixture was thoroughlymixed andfiltered after 48 husing aBuchner vacuum funnelThe residuewas reconstituted in fresh absolute methanol for 24 h andfiltered again as described aboveThe filtered supernatant wasevaporated to dryness with a rotary evaporator to eliminatemethanol The weight of pulverized extract of the leavesobtained represents a 2178 yield in relation to the weight ofthe leaves used A stock solution of the extract was preparedby dissolving it first in DMSO followed by reconstitutionwithgrowth medium such that the final equivalent concentrationof DMSO in the extract was exactly 01

23 Maintenance of Cell Culture Breast cancer cells (MCF-7) colon cancer cells (HT-29) cervical cancer cells (HeLa)and normal human fibroblasts (KMST-6) were obtained fromthe Department of Biotechnology University of the WesternCape The cell lines were maintained in Dulbeccorsquos ModifiedEaglersquosMedium (DMEM) and supplementedwith 10of fetalbovine serum (FBS) and 1 of penicillin and streptomycin(100UmL penicillin and 100 120583gmL streptomycin) All tissueculture operations were carried out in a model NU-5510ENuAireDHDautoflow automaticCO

2air-jacketed incubator

24 Chemicals 3-(45-Dimethylthiazol-2-yl)-25-diphenyl-tet razolium bromide (MTT) trypan blue dye and crys-tal violet were purchased from Sigma-Aldrich APOP-ercentage dye was obtained from Biocolor Ltd (Car-rickfergus Northern Ireland) 5-(and-6)-chloromethyl-2101584071015840-dichlorofluorescein diacetate acetyl ester (CM-H

2DCFDA)

from Invitrogen (South Africa) Annexin V-FITC apoptosis

kit from BD Pharmingen (USA) and Caspase-Glo 37 andcaspase-9 assay system kits from Promega (USA) All otherchemicals used were of analytical grade

25 MTT Assay The viable cells were seeded at a densityof 5 times 104 (100120583Lwell) in 96-well plates and incubatedin a humidified atmosphere of 5 CO

2and 95 air at

37∘C for 24 h to form a cell monolayer After 24 h thesupernatant on the monolayer was aspirated and 100120583L ofmedium and varying log concentrations of extract (01 110 100 and 1000 120583gmL) were added and incubated for24 48 and 72 h time points After the specific times ofexposure to the extract 20 120583L of 5mgmL MTT in PBS wasadded to each well and incubated for 3 h at 37∘C in a 5CO2atmosphere Supernatants were removed and 150120583L of

isopropanol was added and the plates were gently shaken for15min to solubilize the formazan crystals and absorbancewasmeasured at 560 nm using GloMax-Multi Detection System(Promega USA) The percentage inhibition of proliferationwas calculated using the formula below and IC

50values were

calculated from log dose-response curves using GraphPadPrism software version 6 for Windows (GraphPad SoftwareLa Jolla California USA httpwwwgraphpadcom) Con-sider

Inhibition of Proliferation

= 100 minusTest Absorbance at 560 nm

Untreated Control Absorbance at 560 nmtimes100

(1)

26 Trypan Blue Dye Exclusion Assay The viable cells wereseeded at a density of 5 times 104 (1mLwell) in a 12-wellplate and incubated in a humidified atmosphere of 5 CO

2

and 95 air at 37∘C for 24 h to form a cell monolayerAfter 24 h culture medium was gently aspirated and treatedwith 1mL of medium and varying concentrations of extract(100 200 300 400 and 500 120583gmL) for 24 h The adherentcells were removed by trypsinization using 025 EDTAtrypsin (Sigma-Aldrich) The cells were centrifuged usinga Bio-Rad tabletop centrifuge at 2500 rpm for 5min andthe supernatant was discarded to obtain a cell pellet Cellpellets were resuspended in freshmedium fromwhich a 10 120583Laliquot was mixed with an equal volume of 04 trypan bluedye and loaded into Bio-Rad TC20 cell counter Readingswere automatically generated from themachine and recordedbased on the formula below

Inhibition = 100 minus Total Dead Cell CountTotal Cell Count

times 100(2)

27 Clonogenic Assay Theviable cells were plated at a densityof 1 times 103 (1mLwell) in 6-well plates and allowed to attachfor 24 h After 24 h the cells were treated with the methanolicleaf extract ofHolarrhena floribunda (100 200 300 400 and500120583gmL) and incubated further for 24 h in a humidifiedCO2incubatorThemedium containing extract was removed

and replaced with fresh medium and incubated for another 5


50

100

150


Log dose

Cell

s pro

lifer

atin

g (

)


(a)

50

100

150

Cell

s pro

lifer

atin

g (

)



Log dose

(b)

50

100

150


Log dose

Cell

s pro

lifer

atin

g (

)


(c)

50

100

150

Cell

s pro

lifer

atin

g (

)


Log dose


(d)









0

50

100

150

b

b

c

a

d

dd

ddd

Viab

ility

()

HT-29MCF-7

HeLaKMST-6

Con

trol

100120583

gm

L

200120583

gm

L

300

120583g

mL

400

120583g

mL

500

120583g

mL





0

50

100

150

HeLa

a

a

ab

b

bb

b

HT-29KMST-6

100 200 300 400 500

(120583gmL)

Col

ony

form

ed re

lativ

e to

cont

rol (

)

MCF-7





2DCFDA Invitrogen)





HeLa

KMST-6

HT-29

MCF-7





3 Results


50)


50values for the 24







HeLa(120583gmL)

MCF-7(120583gmL)

KMST-6(120583gmL)

24 3492 1826 3576 376948 2175 1274 2443 428272 1594 1067 1267 3423








0

50

100

150

a

bb

cc

dd

d

HeLaHT-29

KMST-6MCF-7

Cell

s in

apop

tosis

()

100 200 300 400 500

(120583gmL)




Relat

ive f

old

incr

ease

in ca

spas

e-3

25

20

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)


Relat

ive f

old

incr

ease

in ca

spas

e-9

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)



800

600

400

200

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(a)

800

600

400

200

0

Num

ber

0 30 60 90 120 150

Channels (FL2-A)

Dip G1Dip G2

Dip S

(b)

160

120

80

40

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(c)

1200

900

600

300

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(d)







4 Discussion


50values obtained


S0

20

40

60

80

a

c c c cC

ell cy

cle p

hase

s (

)

G0G1 G2M



(a)

0

20

40

60

80

c b

c c c

Cell

cycle

pha

ses (

)

S


G0G1 G2M

(b)

0

20

40

60

c c c c

c

Cell

cycle

pha

ses (

)

S



G0G1 G2M

(c)








0

100

200

300

400

101 102 103 104 105 106 1072

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

100

200

300

400

Cou

nt24

h

FL1-H

MCF

12h

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt

FL1-H

HT-29

101 102 103 104 105 106 1072

1600

1000

500

0

Cou

nt24

h

FL1-H

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

200

400

600

800

Cou

nt24

h

FL1-H

HeLa







12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















50

100

150


Log dose

Cell

s pro

lifer

atin

g (

)


(a)

50

100

150

Cell

s pro

lifer

atin

g (

)



Log dose

(b)

50

100

150


Log dose

Cell

s pro

lifer

atin

g (

)


(c)

50

100

150

Cell

s pro

lifer

atin

g (

)


Log dose


(d)









0

50

100

150

b

b

c

a

d

dd

ddd

Viab

ility

()

HT-29MCF-7

HeLaKMST-6

Con

trol

100120583

gm

L

200120583

gm

L

300

120583g

mL

400

120583g

mL

500

120583g

mL





0

50

100

150

HeLa

a

a

ab

b

bb

b

HT-29KMST-6

100 200 300 400 500

(120583gmL)

Col

ony

form

ed re

lativ

e to

cont

rol (

)

MCF-7





2DCFDA Invitrogen)





HeLa

KMST-6

HT-29

MCF-7





3 Results


50)


50values for the 24







HeLa(120583gmL)

MCF-7(120583gmL)

KMST-6(120583gmL)

24 3492 1826 3576 376948 2175 1274 2443 428272 1594 1067 1267 3423








0

50

100

150

a

bb

cc

dd

d

HeLaHT-29

KMST-6MCF-7

Cell

s in

apop

tosis

()

100 200 300 400 500

(120583gmL)




Relat

ive f

old

incr

ease

in ca

spas

e-3

25

20

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)


Relat

ive f

old

incr

ease

in ca

spas

e-9

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)



800

600

400

200

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(a)

800

600

400

200

0

Num

ber

0 30 60 90 120 150

Channels (FL2-A)

Dip G1Dip G2

Dip S

(b)

160

120

80

40

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(c)

1200

900

600

300

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(d)







4 Discussion


50values obtained


S0

20

40

60

80

a

c c c cC

ell cy

cle p

hase

s (

)

G0G1 G2M



(a)

0

20

40

60

80

c b

c c c

Cell

cycle

pha

ses (

)

S


G0G1 G2M

(b)

0

20

40

60

c c c c

c

Cell

cycle

pha

ses (

)

S



G0G1 G2M

(c)








0

100

200

300

400

101 102 103 104 105 106 1072

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

100

200

300

400

Cou

nt24

h

FL1-H

MCF

12h

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt

FL1-H

HT-29

101 102 103 104 105 106 1072

1600

1000

500

0

Cou

nt24

h

FL1-H

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

200

400

600

800

Cou

nt24

h

FL1-H

HeLa







12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

b

b

c

a

d

dd

ddd

Viab

ility

()

HT-29MCF-7

HeLaKMST-6

Con

trol

100120583

gm

L

200120583

gm

L

300

120583g

mL

400

120583g

mL

500

120583g

mL





0

50

100

150

HeLa

a

a

ab

b

bb

b

HT-29KMST-6

100 200 300 400 500

(120583gmL)

Col

ony

form

ed re

lativ

e to

cont

rol (

)

MCF-7





2DCFDA Invitrogen)





HeLa

KMST-6

HT-29

MCF-7





3 Results


50)


50values for the 24







HeLa(120583gmL)

MCF-7(120583gmL)

KMST-6(120583gmL)

24 3492 1826 3576 376948 2175 1274 2443 428272 1594 1067 1267 3423








0

50

100

150

a

bb

cc

dd

d

HeLaHT-29

KMST-6MCF-7

Cell

s in

apop

tosis

()

100 200 300 400 500

(120583gmL)




Relat

ive f

old

incr

ease

in ca

spas

e-3

25

20

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)


Relat

ive f

old

incr

ease

in ca

spas

e-9

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)



800

600

400

200

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(a)

800

600

400

200

0

Num

ber

0 30 60 90 120 150

Channels (FL2-A)

Dip G1Dip G2

Dip S

(b)

160

120

80

40

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(c)

1200

900

600

300

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(d)







4 Discussion


50values obtained


S0

20

40

60

80

a

c c c cC

ell cy

cle p

hase

s (

)

G0G1 G2M



(a)

0

20

40

60

80

c b

c c c

Cell

cycle

pha

ses (

)

S


G0G1 G2M

(b)

0

20

40

60

c c c c

c

Cell

cycle

pha

ses (

)

S



G0G1 G2M

(c)








0

100

200

300

400

101 102 103 104 105 106 1072

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

100

200

300

400

Cou

nt24

h

FL1-H

MCF

12h

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt

FL1-H

HT-29

101 102 103 104 105 106 1072

1600

1000

500

0

Cou

nt24

h

FL1-H

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

200

400

600

800

Cou

nt24

h

FL1-H

HeLa







12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HeLa

KMST-6

HT-29

MCF-7





3 Results


50)


50values for the 24







HeLa(120583gmL)

MCF-7(120583gmL)

KMST-6(120583gmL)

24 3492 1826 3576 376948 2175 1274 2443 428272 1594 1067 1267 3423








0

50

100

150

a

bb

cc

dd

d

HeLaHT-29

KMST-6MCF-7

Cell

s in

apop

tosis

()

100 200 300 400 500

(120583gmL)




Relat

ive f

old

incr

ease

in ca

spas

e-3

25

20

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)


Relat

ive f

old

incr

ease

in ca

spas

e-9

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)



800

600

400

200

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(a)

800

600

400

200

0

Num

ber

0 30 60 90 120 150

Channels (FL2-A)

Dip G1Dip G2

Dip S

(b)

160

120

80

40

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(c)

1200

900

600

300

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(d)







4 Discussion


50values obtained


S0

20

40

60

80

a

c c c cC

ell cy

cle p

hase

s (

)

G0G1 G2M



(a)

0

20

40

60

80

c b

c c c

Cell

cycle

pha

ses (

)

S


G0G1 G2M

(b)

0

20

40

60

c c c c

c

Cell

cycle

pha

ses (

)

S



G0G1 G2M

(c)








0

100

200

300

400

101 102 103 104 105 106 1072

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

100

200

300

400

Cou

nt24

h

FL1-H

MCF

12h

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt

FL1-H

HT-29

101 102 103 104 105 106 1072

1600

1000

500

0

Cou

nt24

h

FL1-H

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

200

400

600

800

Cou

nt24

h

FL1-H

HeLa







12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















0

50

100

150

a

bb

cc

dd

d

HeLaHT-29

KMST-6MCF-7

Cell

s in

apop

tosis

()

100 200 300 400 500

(120583gmL)




Relat

ive f

old

incr

ease

in ca

spas

e-3

25

20

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)


Relat

ive f

old

incr

ease

in ca

spas

e-9

15

10

05

00

HeLaHT-29 MCF-7

100 200 300 400 500

(120583gmL)



800

600

400

200

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(a)

800

600

400

200

0

Num

ber

0 30 60 90 120 150

Channels (FL2-A)

Dip G1Dip G2

Dip S

(b)

160

120

80

40

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(c)

1200

900

600

300

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(d)







4 Discussion


50values obtained


S0

20

40

60

80

a

c c c cC

ell cy

cle p

hase

s (

)

G0G1 G2M



(a)

0

20

40

60

80

c b

c c c

Cell

cycle

pha

ses (

)

S


G0G1 G2M

(b)

0

20

40

60

c c c c

c

Cell

cycle

pha

ses (

)

S



G0G1 G2M

(c)








0

100

200

300

400

101 102 103 104 105 106 1072

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

100

200

300

400

Cou

nt24

h

FL1-H

MCF

12h

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt

FL1-H

HT-29

101 102 103 104 105 106 1072

1600

1000

500

0

Cou

nt24

h

FL1-H

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

200

400

600

800

Cou

nt24

h

FL1-H

HeLa







12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















800

600

400

200

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(a)

800

600

400

200

0

Num

ber

0 30 60 90 120 150

Channels (FL2-A)

Dip G1Dip G2

Dip S

(b)

160

120

80

40

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(c)

1200

900

600

300

0

Num

ber

0 20 40 60 80 100 120

Channels (FL2-A)

Dip G1Dip G2

Dip S

(d)







4 Discussion


50values obtained


S0

20

40

60

80

a

c c c cC

ell cy

cle p

hase

s (

)

G0G1 G2M



(a)

0

20

40

60

80

c b

c c c

Cell

cycle

pha

ses (

)

S


G0G1 G2M

(b)

0

20

40

60

c c c c

c

Cell

cycle

pha

ses (

)

S



G0G1 G2M

(c)








0

100

200

300

400

101 102 103 104 105 106 1072

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

100

200

300

400

Cou

nt24

h

FL1-H

MCF

12h

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt

FL1-H

HT-29

101 102 103 104 105 106 1072

1600

1000

500

0

Cou

nt24

h

FL1-H

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

200

400

600

800

Cou

nt24

h

FL1-H

HeLa







12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















S0

20

40

60

80

a

c c c cC

ell cy

cle p

hase

s (

)

G0G1 G2M



(a)

0

20

40

60

80

c b

c c c

Cell

cycle

pha

ses (

)

S


G0G1 G2M

(b)

0

20

40

60

c c c c

c

Cell

cycle

pha

ses (

)

S



G0G1 G2M

(c)








0

100

200

300

400

101 102 103 104 105 106 1072

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

100

200

300

400

Cou

nt24

h

FL1-H

MCF

12h

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt

FL1-H

HT-29

101 102 103 104 105 106 1072

1600

1000

500

0

Cou

nt24

h

FL1-H

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

200

400

600

800

Cou

nt24

h

FL1-H

HeLa







12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

100

200

300

400

101 102 103 104 105 106 1072

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

100

200

300

400

Cou

nt24

h

FL1-H

MCF

12h

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt

FL1-H

HT-29

101 102 103 104 105 106 1072

1600

1000

500

0

Cou

nt24

h

FL1-H

101 102 103 104 105 106 1072

0

100

200

300

Cou

nt12

h

FL1-H101 102 103 104 105 106 1072

0

200

400

600

800

Cou

nt24

h

FL1-H

HeLa







12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















12 240

2

4

6

8

10

Time (h)

Fold

incr

ease

in M

FI

HeLaHT-29MCF-7












5 Conclusion




References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














