research article antiproliferative effect of the...

Research ArticleIn Vitro Antiproliferative Effect of the Acetone Extract of Rubusfairholmianus Gard Root on Human Colorectal Cancer Cells

Blassan Plackal Adimuriyil George Ivan Mfouo Tynga and Heidi Abrahamse

Laser Research Centre Faculty of Health Sciences University of Johannesburg PO Box 17011 Doornfontein 2028 South Africa

Correspondence should be addressed to Heidi Abrahamse habrahamseujacza

Received 16 February 2015 Accepted 12 May 2015

Academic Editor Anne Hamburger

Copyright copy 2015 Blassan Plackal Adimuriyil George et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Plants and plant derived products exert chemopreventive effects on various cancer cell lines by the induction of cell deathmechanisms The effects of root acetone extract of Rubus fairholmianus (RFRA) on the proliferation of human colorectal cancer(Caco-2) cells have been investigated in this study The extract led to a dose dependent decrease in both viability and proliferationand increased cytotoxicity using trypan blue exclusion adenosine 51015840-triphosphate (ATP) and lactate dehydrogenase (LDH) assayThe morphological features of the treated cells were supportive for the antiproliferative activity The Annexin Vpropidium iodidestaining indicated that R fairholmianus induced toxic effects in Caco-2 cells and the percentages of the early and late apoptoticpopulation significantly increased when compared with control cells Also we studied the apoptosis inducing ability of the extractby analysing caspase 37 activity and the induction of cell death via the effector caspases was confirmed the activity increasedin treated cells compared with control Thus the present findings highlight that the R fairholmianus root acetone extract exhibitsantiproliferative activity on Caco-2 cells by the induction of apoptosis via caspase dependent pathway

1 Introduction

Cancer is a dreaded disease characterized by uncontrolledgrowth and spread of abnormal cells The high mortality rateamongst cancer patients is an indication of limited efficiencyof current therapies [1] Identifying the mechanism of plantderived anticancer agents provides helpful information incancer therapy Natural products have been used for thetreatment of various diseases for centuries Evidences haveshown that active principle compounds from plants mayserve as potent chemotherapeutic agents with less toxicityto normal tissues and at low cost [2] Plants have a longhistory of use in cancer therapy and it is significant that over60 of currently used anticancer agents are from naturalsources and around 80 of people in rural areas dependon plant products for their primary healthcare needs [3]They are associated with induction of apoptosis cell cyclearrest inhibition of various signal transducers and signalingpathways [4 5] Thus it is important to screen the crudeextract of plants or isolated compounds for their apoptoticpotentials The use of alternative medicine is increasing and

many pharmaceutical industries are interested in developingplant derived medicinal compounds [6] The increasing costof conventional treatments and the lack of effective drugsencouraged people to depend more on folk medicine

Berries such as Rubus Fragaria Sorbus Ribes and Vac-cinum are common inWestern dietsThese soft fruits are richin bioactive phytochemicals including several classes of phe-nolic compounds [7] Berries from Rubus genus (cloudberryraspberry and blackberry) primarily contain ellagitanninsand anthocyanins [8] The evidence supports that anticancerproperties of berries are due to the scavenging free radicalsinduction of enzymes involved in xenobiotics metabolismregulation of gene expression alteration of cellular signallingand induction of apoptosis [9 10] The search for bettercytotoxic agents continues to be important in the discoveryof modern anticancer drugs Several bioactive compoundsof plants act as lead compounds in drug discovery due totheir structural diversity bioactivities and the therapeuticpotential of these compounds can be improved by molecularmodifications

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 165037 8 pageshttpdxdoiorg1011552015165037

2 BioMed Research International

Cancers that start in the cells lining inside of the colon andrectum are called colorectal cancers (CRC) CRC are the thirdmost predominant cancers worldwide and are the fourthmost common cause of cancer mortality with approximately94 of global cancer cases irrespective of gender The rate ofincidence of CRC has been observed more in AustraliaNewZealand Europe USA and UK African and Asian coun-tries have a far lower rate of incidence [11] Accordingto epidemiological studies the major risk factors of CRCincidence include obesity smoking alcohol consumptionincreasing age family history and dietary factors [12 13]Several flavonoid-rich foods could display relevant cancer-preventive effects Thus the isolation of potential chemopre-ventive agents from plants has contributed to breakthroughin anticancer studies [14] Carcinoma colon (Caco-2) cellline originates from human colonic adenocarcinoma andshows similarities to small intestinal enterocytes [15] Thesecells develop typical morphology of enterocytes with distinctapical and basolateral membrane domains microvillus andtight junctions [16] The cells can be grown on uncoatedpolycarbonate filters to obtain well-differentiatedmonolayers[17]

The genus Rubus is very diverse with over 750 species in12 subgenera and is found on all continents except Antarctica[18] Due to useful ethnomedicinal and pharmacologicalproperties Rubus species have been used in folk medicine[19] R fairholmianus (R moluccanus L) leaf extract has beenadministrated to reduce headache [20] these leaves possessinsecticidal properties fruits are edible with stimulatingeffect [21] On the basis of the previously reported anti-inflammatory wound healing [22] and in silico anticancerproperties [23] we have selected this plant for this studyEven though this plant has immense ethnomedicinal valuea survey of literature revealed that the in vitro anticancerpotential of this plant has not yet been evaluated Keeping thisin view the aim of the present study was to investigate thein vitro anticancer potential and track the possible cell deathmechanisms of R fairholmianus root acetone extract to putforward a scope to develop an effective drug

2 Materials and Methods

21 Plant Material Collection Identification and ExtractionThe fresh plant parts of R fairholmianus were collected fromShola forest of Marayoor Idukki District Kerala India dur-ing September 2010 Plant parts were identified and authen-ticated (voucher specimen number BSISRC5232010-11Tech1657) by Botanical Survey of India Southern Cir-cle Coimbatore Tamil Nadu India The shade dried andpowdered roots were extracted in Soxhlet apparatus usingacetone The extractability of phenolic compounds in thepolar solvent like acetone was found to be high Moreoveracetone extract has shown better pharmacological effects inmany previous studies inRubusThe extractwas concentratedto dryness under reduced pressure in a rotary evaporatorto yield dried root acetone extract The dried extract thendissolved in 05 DMSO and was used for further analysisThe preliminary screening of leaves stem and root for totalphenolics tannin flavonoids and antioxidant propertiesshowed that root had the maximum activity [24]

22 Cell Culture The commercially obtained colorectalcancer cells (Caco-2 ATCC HTB-37) were used Caco-2 cellswere cultured in Dulbeccorsquos modified Eaglersquos media (DMEMSigma-Aldrich D 6429) with 12 gL sodium carbonatesupplemented with 10 foetal bovine serum (FBS Gibco30600301) 10mM nonessential amino acids (Gibco 11140)05mMsodiumpyruvate (Gibco 11360) 25mML-glutamine(Gibco 25030) 1 antibiotic (penicillin-streptomycinGibco 15140) and 1 antifungal (amphotericin-B Gibco104813) agents Culture was maintained at 37∘C with 5 CO

2

and 85 humidity Once cells reached 80ndash90 confluencethey were harvested and seeded at a concentration of 5 times104 in 3mL culture media into sterile culture dishes with adiameter of 34 cm Cells were allowed to attach overnight

23 Cellular Morphology-Inverted Microscopy The effect ofthe plant extract on cellmorphologywas determined using aninverted light microscope (Wirsam Olympus CKX41) after24 h of incubationwith different concentrations (5 10 20 and40 120583gmL) of RFRA and 05 DMSO Once digital imageswere recorded cells were trypsinized using 1mL25 cm2 ofTrypLE Express (Invitrogen 12605-028) and resuspended inHankrsquos Balanced Salt Solution (HBSS) to perform furtherassays

24 Cell Viability-Trypan Blue Dye Exclusion Assay Thetrypan blue dye exclusion assay (Sigma-Aldrich T8154) is aquantitative method to determine the percentage viabilityHere the viable cells with an intact cellular membrane do nottake up the blue dye andmaintain a clear appearance whereasthe damaged nonviable cells take up dye and stained blue astheir membrane damages

The cell suspension was carefully mixed with 04 trypanblue reagent in 1 9 ratio and the mixture was transferred to aNeubauer hemocytometer to determine the number of viableand nonviable cells Cells were counted by using automatedcell counter (Countess Automated Cell Counter Invitrogen)

25 Cellular Proliferation-Adenosine Triphosphate (ATP)Luminescent Assay The CellTiter-Glo1 luminescent assay(Promega G7571 Anatech Analytical Technology BellvilleSouth Africa) is a homogeneous method for determinationof cellular proliferation and quantification of ATP present inmetabolically active cells

Fifty microliters of reconstituted reagent was added to anequal volume of cell suspension and then mixed in a shakerfor 2 minutes to induce cell lysis This was then incubatedat room temperature for 10 minutes in dark to stabilize theluminescent signalThe luminescent signalwas read using the1420 Multilabel Counter Victor3 (Perkin-Elmer SeparationScientific)

26 Cytotoxicity-Lactate Dehydrogenase (LDH) Assay Themembrane integrity was assessed by estimating the amount ofLDHpresent in the culturemediaThe cytosolic enzymeLDHwill be released due to membrane damage The CytoTox96X assay (Anatech Promega G 400) was used to measurethe released LDH Fifty microliters of reconstituted reagentwas added to an equal volume of cell culture medium and

BioMed Research International 3

incubated in the dark at room temperature for 30min Thecolorimetric compound was measured spectrophotometri-cally at 490 nm (Perkin-Elmer VICTOR3)

27 Cell Death Analysis-Annexin VPI Staining TheAnnexinV fluorescein isothiocyanate (FITC) apoptosis detection kit(Becton Dickinson 556570 Scientific Group RandburgSouth Africa) was used to detect apoptotic cells by fluo-rescence activated cell sorting (FACS) In apoptotic cellsthe membrane phospholipid phosphatidyl serine which isnormally found in the internal portion of the cell membranebecomes translocated to the outer leaflet of the plasmamembrane thereby exposing phosphatidyl serine to theexternal environment Annexin V is a calcium dependentphospholipid binding protein that has an affinity for phos-phatidylserine and is useful in identifying apoptotic cells

Cells were resuspended in 1x binding buffer at a con-centration of 1 times 106mL One hundred microliters of thecell suspension was transferred to a 5mL falcon tube andstained with 5mL of FITC Annexin and 5mL of propidiumiodide (PI) The cells were gently vortexed and incubatedfor 15min at room temperature and protected from lightAfter the addition of 400mL of 1x binding buffer to eachtube the samples were run in FACSAria flow cytometer(Becton Dickinson) by running 20000 events and analysedwith CellQuest Software (BD Bioscience)

28 Quantification of Caspase 37 Activity The effector cas-pases 3 and 7 play central role in apoptotic process All stepswere performed according to the manufacturerrsquos procedureTumour necrotic factor-alpha (TNF-120572) is able to initiateapoptosis by activating caspase 8 which in turn activatescaspases 3 and 7 Activity of caspases 37 in apoptosis wasdetermined with the Caspase-Glo 37 luminescent assay(Whitehead Scientific Bracken fell South Africa PromegaG8091)

Fifty microliters of treated cells was seeded in a 96-well luminescent plate (Scientific Group Adcock IngramMidrand South Africa BD354651) Fifty microliters ofCaspase-Glo 37 reagent is added and the plate was thenincubated at room temperature for 3 h Luminescence wasread using the Victor3 (Perkin-Elmer Separation ScientificJohannesburg South Africa)

29 Statistical Analysis A colorectal cancer cell line between15 and 20 passages was used Each set of experiments wasrepeated six times (119899 = 6) whereas each assay was performedin duplicates with the results being averaged Untreated cellswere included throughout the course of study and all treatedsamples were compared to those cells by means of one-wayANOVA to determine the statistical difference Statisticalanalysis was performed using SigmaPlot version 120 and themean standard deviation and standard error were obtainedStatistical significances between untreated control cells andtreated cells are shown in the graphs as119875 lt 005 (lowast)119875 lt 001(lowastlowast) and 119875 lt 0001 (lowast lowast lowast) Significant differences wereconsidered at the 95th percentile

3 Results and Discussion

There are many reports available on the increased cancer celldeath via induction of apoptosis or by many other meansusing different plant extracts or the natural compoundsMostof the chemopreventive agents used currently are nonspecificand kill both cancerous and normal cells causing variousside effects But the use of plant derived agents reduces thisrisk and it is more specific to the cancer cells In this studythe antiproliferative activity of R fairholmianus root acetoneextract on Caco-2 cells was examined

The morphological changes of Caco-2 treated cells withvarious concentrations of RFRA extracts were incubatedfor 24 h and compared with the untreated cells shown inFigure 1 Compared to control cells after the incubationperiod morphology of RFRA treated Caco-2 cells signifi-cantly changed The extract treated cells appeared less uni-formwith the loss ofmembrane integrity although still intactat lower concentrations (5 and 10 120583gmL) Whereas at 20and 40 120583gmL concentrations the RFRA treated cells showedremarkable difference with the control groupThe significantchanges such as loss of intact membrane karyopyknosis celldetachment from the plate and change of morphologicalfeatures were evident when compared to untreated cellsThe most identifiable morphological features of apoptosiswere observed by inverted light microscopy in the RFRAtreated cells The treated cells appeared like cells undergoingapoptosis with prominent features such as detaching fromthe culture plate cytoplasmic condensation cell shrinkageand condensation and aggregation of the nuclear chromatinand loss of contact with neighbouring cells [25] However theuntreated cells appeared normal and were confluent

The cell viability of the Caco-2 cells was assessed bytrypan bluemethod Trypan blue is an energy-dependent dyeexclusion viability testing method the dye is being excludedfrom live cells As trypan blue is a weak acid its affinityis increased for basic proteins nuclei uptake is generallyhigher due to the presence of histones yielding marked blueintensity whereas the cytoplasm remains faintly stained [26]Thismethod helped to determine the percentage of viable anddead cells in the RFRA treated Caco-2 cells The results arepresented in Table 1 There was a dose dependent decrease inviability of the treated cells The control cells showed 8383viability whereas RFRA treated cells showed 7133 61505250 and 4100 for 5 10 20 and 40 120583gmL respectivelyThe decrease in percentage of viability becomes significantwith all different concentrations of RFRA (119875 lt 001 for5 120583gmL and 119875 lt 0001 for 10 20 and 40 120583gmL resp) Thenet loss of viability at the higher concentrations in trypanblue assay indicated the cytotoxic effect of RFRA towardsCaco-2 cells No other studies have been published previouslyrevealing the influence of RFRA on viability of Caco-2 celllines Similar results were also observed in R occidentalis(black raspberry) extracts they suppressed the proliferationof colon (HT-29) prostate (LNCaP) oral (KB CAL-27)and breast (MCF-7) tumour cell lines [27 28] and the cellviability decreased in a dose dependent manner The extractsof some Rubus species such as R jamaicensis R rosifoliusR racemosus R acuminatus and R idaeus also exhibited


50120583m50120583m

Control 05 DMSO 5120583gmL RFRA

10120583gmL RFRA 20120583gmL RFRA 40120583gmL RFRA50120583m 50120583m 50120583m

(b)(a) (c)

(d) (e) (f)

50120583m

Figure 1 Morphological features of Caco-2 cells after 24 h treatment with RFRA (R fairholmianus root acetone) extract There were nosignificant visible differences in both control and 05 DMSO treated cells (a and b) The RFRA extract treated cells (c d e and f) after 24 hshowed loss of intact membrane loss of contact with neighbouring cells condensed and detached from the culture plate

Table 1 Trypan blue viability and LDH cytotoxicity assays

Groups Viability percentage LDH cytotoxicityControl 8383 plusmn 128 03490 plusmn 00205 DMSO 8283 plusmn 098 03677 plusmn 0025 120583gmL RFRA 7133 plusmn 275lowastlowast 04580 plusmn 007lowastlowast

10 120583gmL RFRA 6150 plusmn 254lowastlowastlowast 05328 plusmn 006lowastlowastlowast



Values are mean plusmn standard error of six independent assays performed induplicates The significant differences between treated and controls groupsare shown as lowastlowastlowast119875 lt 0001 and lowastlowast119875 lt 001 (RFRA R fairholmianus rootacetone extract LDH lactate dehydrogenase DMSO dimethyl sulfoxide)

great potential to inhibit cancer cell growth inhibiting colonbreast lung and gastric human tumour cells [29]

The LDH leakage assay is a simple reliable and fastcytotoxicity assay based on the measurement of lactatedehydrogenase activity in the extracellular medium LDHa cytoplasmic enzyme released when the cell membranedamages is assessed in cell culture supernatants [30] Thecell membrane damage of Caco-2 cells after the treatmentwith RFRA extract was measured by the release of LDH byfollowingCytoTox961 assayThe control cells and cells treatedwith DMSO showed lesser LDH release when compared

to cells exposed to plant extracts The RFRA treatmentsprovoked a higher release of LDH activity than the controlnontreated cells There was a significant (119875 lt 001 with5 120583gmL and 119875 lt 0001 with 10 20 and 40 120583gmL of RFRA)dose dependent increase in the LDH release observed atincreasing concentrations of RFRA (Table 1)The intracellularLDH release to the medium is a measure of irreversible celldeath due to cell membrane damage whereas Xia et al [31]reported the direct involvement of LDH upregulation andsubsequent induction of apoptosis

Cellular ATP content was assessed to determine the levelof metabolic activity of cells The CellTiter-Glo1 luminescentassay was performed to evaluate the proliferating potential ofcells ATP is a marker for cell viability and proliferation andpresent in all metabolically active cells Some reports suggestthat ATP can induce cell proliferation differentiation andapoptosis mediating different pathophysiological functionsdepending on the target cell [32] The energetic level inCaco-2 cells remained higher which was evident from theincreased ATP level in control and DMSO treated cellsExposure of Caco-2 cells to the RFRA caused a reductionin the intracellular ATP pool (Figure 2) The Caco-2 cellsafter RFRA treatments showed significant changes in cellularproliferation as compared to control cells All the four testedconcentrations of RFRA exhibited significant changes incellular proliferation The cells incubated with increasing


Lum

ines

cenc

e (re

lativ

e lig

ht u

nit)

0

Con

trol

05

DM

SO

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

3e + 5

3e + 5

2e + 5

2e + 5

1e + 5

5e + 4

n = 6

5120583

g RF

RA

10120583

g RF

RA

20120583

g RF

RA

40120583

g RF

RA

Figure 2TheATP luminescent cell proliferation assay was used to determine Caco-2 cell proliferation Control and 05 treated cells showedan increase in ATP level whereas a dose dependent significant (119875 lt 0001) decrease in ATP level was observed in RFRA (R fairholmianusroot acetone) extract treated experimental groups (5 10 20 and 40120583gmL concentrations of RFRA)

concentrations (5 10 20 and 40 120583gmL) of RFRA resultedin a dose dependent highly significant decrease (119875 lt 0001)in cellular proliferation compared with control Caco-2 cellsThe actively dividing cells produce ATP the main productionoccurs in mitochondria and the depletion in ATP correlatedwith the decreased cell proliferation rate [33] The ATPquantity drops soon after cell death and results in a lossof luminescence in assay [34] RFRA extracts decreased thecell proliferation and was manifested from the decline ofluminescence

The Annexin V FITC apoptosis detection kit was usedto distinguish the apoptotic and necrotic cells using flowcytometry To further understand whether the decrease incell viability observed was due to apoptosis we examinedthe behaviour of cells after treatment using Annexin VPIstaining The percentage of apoptotic cells increased withthe increase in concentration of RFRA The control and05 DMSO treated cells showed no significant changesin percentage of cell population most of the cells linedin the live cells range during flow cytometry analysis TheRFRA treated cells showed an increase in the percentages ofearly and late apoptotic cells The nonapoptotic or necroticcells concentration in the treated extracts was found to bevery low compared with the control group Figure 3 showsthe results of Annexin V-PI staining after the treatmentwith RFRA extract to Caco-2 cells In the control groupsthe Annexin V+PI+ population was less (33) and wascompared with experimental groups The RFRA treatedgroups such as 5 10 20 and 40 120583gmL showed 247 312396 and 558 percentages of late apoptotic cells populationWhen Caco-2 cells were treated with the RFRA the early(Annexin V+PIminus) and late (Annexin V+PI+) apoptoticpopulation increased concurrently and induced a decrease inthe viable cell (Annexin VminusPIminus) and necrotic or dead cell(Annexin VminusP+) population Although both late apoptoticand necrotic cells are AnnexinV and PI positive the presence

of these cells with early apoptotic cells suggests that suchdead cells resulted from the apoptosis rather than necrosis[35] These findings confirmed that apoptosis may be thepossible mechanism by which the RFRA triggers cell deathin Caco-2 cellsThe absence or significantly low percentage ofcell population in quadrant (Annexin VminusPI+) of the RFRAtreated Caco-2 cells ruled out necrosis [36]

To evaluate the inhibition of cell growth release of LDHthe increase in early and late apoptotic cell population inresponse to RFRA treatments was due to caspase dependentpathway the caspase 37 activity was measured Caspasesare a family of cysteine-aspartic proteases known as the vitalmediators of apoptotic signalling [37] Caspases 3 and 7 arewell established as the major executioner (effector) caspasesand their activation eventually leads to cell death [38] Themeasure of caspase 37 activity in the cells is a direct meanfor the determination of caspase dependent apoptosis Therewas a significant dose dependent increase in the caspase 37activities (119875 lt 001 for 5 120583gmL and 119875 lt 0001 for 10 20and 40 120583gmL) observed in treated cells with the increase inconcentrations of RFRA (Figure 4) Low levels of caspase 37activity were detected in the control and 05 DMSO treatedcells which was presumably due to the small amount ofapoptotic cells present in the growing cell population Amongthe effector caspases caspases 3 and 7 play a vital role inthe execution phase of intrinsic and extrinsic pathways ofapoptosis by cleavingmany crucial cellular proteins [39]Thiscleavage facilitates disassembly of cell which is evident fromthemorphological changes such as cell shrinkage chromatincondensation membrane blebbing and DNA fragmentation[40] These morphological changes were also observed fromthe RFRA treated cells and strongly suggested the caspasemediated apoptotic cell death in Caco-2 cells Kim andcoworkers [41] reported the caspase 37 activity and apoptosisinducing ability of R coreanuma closely related species of Rfairholmianus on HT-29 human colon cancer cells


101

102

103

104

105

106

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101

102

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102

103

104

105

106

107

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102

103

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105

106

107

101

102

103

104

105

106

107

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102

103

104

105

106

107

101

102

103

104

105

106

107

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102

103

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106

107

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103

104

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102

103

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105

106

107

101

102

103

104

105

106

107

10120583gmL RFRA 20120583gmL RFRA 40120583gmL RFRA

5120583gmL RFRAControl

Annexin V FITCAnnexin V FITCAnnexin V FITC

Annexin V FITC Annexin V FITCAnnexin V FITC

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

05 DMSO

(a) (b) (c)

(d) (e) (f)

GroupsQ5-UL nonapoptotic

or necrotic cells ()

LL live cells () LR earlyapoptotic cells ()

UR lateapoptotic dead

cells ()Control 05 DMSORFRA (5120583gmL)RFRA (10120583gmL)RFRA (20120583gmL)RFRA (40120583gmL)

62 plusmn 098 33 plusmn 078

39 plusmn 089

681 plusmn 232 223 plusmn 057271 plusmn 059654 plusmn 22136 plusmn 081

05 plusmn 038lowastlowastlowast 245 plusmn 122

lowastlowast 503 plusmn 052 lowastlowastlowast 247 plusmn 103 lowastlowast

312 plusmn 154 lowastlowastlowast





395 plusmn 200 lowastlowast

74 plusmn 103lowastlowastlowast



11 plusmn 049lowastlowast



Values are mean plusmn standard error of three independent assays performed in duplicates The significant differences between treated and controls groups are shown aslowastlowastlowastP lt 0001 andlowastlowastP lt 001 (RFRA R fairholmianus root acetone extract)

Figure 3 Annexin V FITCPI staining used to assess the mode of cell death RFRA (R fairholmianus root acetone) treated Caco-2 cellsshowed an increased percentage of apoptotic population after 24 h incubation The population of early and late apoptotic cells in controlgroup found to be lower (223 and 33) compared with experimental groups the attached table shows the statistical data of flow cytometricanalysis

Many plant extracts and isolated compounds have beenreported for the antiproliferative activities and some of themshowed their phototoxic effects on different cancer cell linesby inducing the cell death Hypericin a second generationphotosensitizer isolated from Hypericum perforatum hasbeen shown to act as a potent light-activated photosensitizerin numerous in vitro and in vivo systems [42] The furtherresearch is warranted to find out the photosensitising abilityof R fairholmianus since it has great antiproliferative activ-ities The irradiation of the extracts and isolated bioactivecompounds may be helpful in killing cancer cells via genera-tion of reactive oxygen species

4 Conclusions

The present study demonstrated a dose dependent antitumoreffect of R fairholmianus root acetone extract on humancolorectal cancer cell (Caco-2) A significant decrease in thecell viability proliferation and increase in cytotoxicity andcaspase 37 activity were observed after the treatment withRFRA The Annexin VPI staining supports the apoptosisinduced cell death Moreover the morphological alterationsin the Caco-2 cells exposed to RFRA extract were suggestivefor the apoptotic activities and this is described for thefirst time The data demonstrate that this plant exhibits

BioMed Research International 7Lu

min

esce

nce (

read

ing

light

uni

ts RL

U)

0

5000

10000

15000

20000

25000n = 6

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con

trol

05

DM

SO

5120583

gm

L RF

RA

10120583

gm

L RF

RA

20120583

gm

L RF

RA

40120583

gm

L RF

RA

Figure 4 Caspase 37 activity determined as a function of caspasedependent apoptosis in Caco-2 cells after the treatment with RFRA(R fairholmianus root acetone) extract There was a significant dosedependent increase in caspase 37 activity after RFRA treatments(119875 lt 001 and 119875 lt 0001) compared to control and 05 DMSOtreated cells

an inhibitory effect and seems to indicate the caspase 37mediated apoptotic cell death triggered in Caco-2 cellsThe outcome of this study recommends that the substantialantiproliferative activity of R fairholmianus may be due tothe caspase dependent apoptosis and the compounds of thisextractmay have promising use as a cancer chemotherapeuticagent

Conflict of Interests

The authors declare that there is no conflict of interests

References

[1] J Xu X S Liu S-F Zhou and M Q Wei ldquoCombinationof immunotherapy with anaerobic bacteria for immunogenetherapy of solid tumoursrdquoGeneTherapy andMolecular Biologyvol 13 no 1 pp 36ndash52 2009

[2] W-S Jeong I-W Kim R Hu and A-N T Kong ldquoModu-latory properties of various natural chemopreventive agentson the activation of NF-120581B signaling pathwayrdquo PharmaceuticalResearch vol 21 no 4 pp 661ndash670 2004

[3] D M Sakarkar and V N Deshmukh ldquoEthnopharmacologicalreview of traditional medicinal plants for anticancer activityrdquoInternational Journal of PharmTech Research vol 3 no 1 pp298ndash308 2011

[4] Y-S KeumW-S Jeong and A N Tony Kong ldquoChemopreven-tion by isothiocyanates and their underlying molecular signal-ing mechanismsrdquo Mutation ResearchFundamental and Molec-ular Mechanisms of Mutagenesis vol 555 no 1-2 pp 191ndash2022004

[5] C Chen and A N Kong ldquoDietary cancer-chemopreventivecompounds from signaling and gene expression to pharmaco-logical effectsrdquo Trends in Pharmacological Sciences vol 26 no6 pp 318ndash326 2005

[6] Y Zhang X-M Xu M Zhang et al ldquoEffects of tubeimoside-1 on the proliferation and apoptosis of BGC823 gastric cancercells in vitrordquo Oncology Letters vol 5 no 3 pp 801ndash804 2013

[7] E M Brown C I R Gill G J McDougall and D StewartldquoMechanisms underlying the anti-proliferative effects of berrycomponents in in vitro models of colon cancerrdquo CurrentPharmaceutical Biotechnology vol 13 no 1 pp 200ndash209 2012

[8] M P Kahkonen A I Hopia and M Heinonen ldquoBerryphenolics and their antioxidant activityrdquo Journal of Agriculturaland Food Chemistry vol 49 no 8 pp 4076ndash4082 2001

[9] G D Stoner ldquoFoodstuffs for preventing cancer the preclin-ical and clinical development of berriesrdquo Cancer PreventionResearch vol 2 no 3 pp 187ndash194 2009

[10] A Bishayee ldquoCancer prevention and treatment with resvera-trol from rodent studies to clinical trialsrdquo Cancer PreventionResearch vol 2 no 5 pp 409ndash418 2009

[11] J Ferlay H R Shin F Bray D Forman C Mathers and D MParkin GLOBOCAN 2008 v12 Cancer Incidence and MortalityWorldwide IARC Cancer Base No 10 International Agency forResearch on Cancer Lyon France 2010

[12] M M Center A Jemal and E Ward ldquoInternational trendsin colorectal cancer incidence ratesrdquo Cancer EpidemiologyBiomarkers and Prevention vol 18 no 6 pp 1688ndash1694 2009

[13] A Jemal F Bray M M Center J Ferlay E Ward and DForman ldquoGlobal cancer statisticsrdquo CA A Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011

[14] D Cooke W P Steward A J Gescher and T MarczyloldquoAnthocyans from fruits and vegetables does bright coloursignal cancer chemopreventive activityrdquo European Journal ofCancer vol 41 no 13 pp 1931ndash1940 2005

[15] F Delie and W Rubas ldquoA human colonic cell line sharingsimilarities with enterocytes as amodel to examine oral absorp-tion advantages and limitations of the Caco-2 modelrdquo CriticalReviews in Therapeutic Drug Carrier Systems vol 14 no 3 pp221ndash286 1997

[16] M Pinto S Robine Leon and M D Appay ldquoEnterocyte-likedifferentiation and polarization of the human colon carcinomacell line Caco-2 in culturerdquo Biology of the Cell vol 47 no 3 pp323ndash330 1983

[17] P Artursson ldquoEpithelial transport of drugs in cell culture I amodel for studying the passive diffusion of drugs over intestinalabsorptive (Caco-2) cellsrdquo Journal of Pharmaceutical Sciencesvol 79 no 6 pp 476ndash482 1990

[18] C E Finn ldquoRubus spp blackberryrdquo in The Encyclopedia ofFruits and Nuts J Janick and R E Paull Eds pp 348ndash351CABI Cambridge Mass USA 2008

[19] A V Patel J Rojas-Vera and C G Dacke ldquoTherapeuticconstituents and actions of Rubus speciesrdquo Current MedicinalChemistry vol 11 no 11 pp 1501ndash1512 2004

[20] N J Das S P Saikia S Sarkar and K Devi ldquoMedicinal plantsof north kamrup district of Assam used in primary health caresystemrdquo International Journal of Traditional Knowledge vol 5no 22 pp 489ndash493 2006

[21] J Barukial and JN Sarmah ldquoEthnomedicinal plants used by thepeople of Golaghat district Assam Indiardquo International Journalof Medicinal and Aromatic Plants vol 1 no 3 pp 203ndash211 2011


[22] B P George T Parimelazhagan and R Chandran ldquoAnti-inflammatory and wound healing properties of Rubusfairholmianus Gard Root-An in vivo studyrdquo Industrial Cropsand Products vol 54 pp 216ndash225 2014

[23] B Plackal George P Thangaraj C Sulaiman S Piramanay-agam and S K Ramaswamy ldquoBioassay directed isolationand biological evaluation of compounds isolated from Rubusfairholmianus Gardrdquo BioMed Research International vol 2014Article ID 204340 15 pages 2014

[24] B P George T Parimelazhagan and R Chandran ldquoEvaluationof total phenolic content antioxidant and analgesic potential ofRubus fairholmianus gardrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 5 no 3 pp 484ndash488 2013

[25] J Monga S Pandit C S Chauhan andM Sharma ldquoCytotoxic-ity and apoptosis induction in human breast adenocarcinomaMCF-7 cells by (+)-cyanidan-3-olrdquo Experimental and Toxico-logic Pathology vol 65 no 7-8 pp 1091ndash1100 2013

[26] K Grankvist A Lernmark and I B Taljedal ldquoAlloxan cyto-toxicity in vitro Microscope photometric analyses of TrypanBlue uptake by pancreatic islet cells in suspensionrdquo BiochemicalJournal vol 162 no 1 pp 19ndash24 1977

[27] J-H Jeong H Jung S-R Lee H-J Lee K T Hwang and T-Y Kim ldquoAnti-oxidant anti-proliferative and anti-inflammatoryactivities of the extracts from black raspberry fruits and winerdquoFood Chemistry vol 123 no 2 pp 338ndash344 2010

[28] N P Seeram L S Adams Y Zhang et al ldquoBlackberry blackraspberry blueberry cranberry red raspberry and strawberryextracts inhibit growth and stimulate apoptosis of humancancer cells in vitrordquo Journal of Agricultural and FoodChemistryvol 54 no 25 pp 9329ndash9339 2006

[29] C S Bowen-Forbes Y Zhang and M G Nair ldquoAnthocyanincontent antioxidant anti-inflammatory and anticancer prop-erties of blackberry and raspberry fruitsrdquo Journal of FoodComposition and Analysis vol 23 no 6 pp 554ndash560 2010

[30] B Saad S Dakwar O Said et al ldquoEvaluation of medicinal planthepatotoxicity in co-cultures of hepatocytes and monocytesrdquoEvidence-Based Complementary and Alternative Medicine vol3 no 1 pp 93ndash98 2006

[31] G-H Xia B-A Chen Z-Y Shao H-X Lu D Konstanzeand D Hartmut ldquoMechanism of 2-methoxyestradiol-inducedapoptosis in myelodysplastic syndrome MUTZ-1 cell linerdquoZhongguo Shi Yan Xue Ye Xue Za Zhi vol 15 no 2 pp 296ndash301 2007

[32] N White and G Burnstock ldquoP2 receptors and cancerrdquo Trendsin Pharmacological Sciences vol 27 no 4 pp 211ndash217 2006

[33] C Vidau J L Brunet A Badiou and L P BelzuncesldquoPhenylpyrazole insecticides induce cytotoxicity by alteringmechanisms involved in cellular energy supply in the humanepithelial cell model Caco-2rdquo Toxicology in Vitro vol 23 no 4pp 589ndash597 2009

[34] F Fan and K V Wood ldquoBioluminescent assays for high-throughput screeningrdquo Assay and Drug Development Technolo-gies vol 5 no 1 pp 127ndash136 2007

[35] P Hansakul K Aree S Tanuchit and A Itharat ldquoGrowth arrestand apoptosis via caspase activation of dioscoreanone in humannon-small-cell lung cancer A549 cellsrdquo BMC Complementaryand Alternative Medicine vol 14 article 413 2014

[36] F Naselli L Tesoriere F Caradonna et al ldquoAnti-proliferativeand pro-apoptotic activity ofwhole extract and isolated indicax-anthin from Opuntia ficus-indica associated with re-activationof the onco-suppressor p161198681198731198704119886 gene in human colorectal

carcinoma (Caco-2) cellsrdquoBiochemical and Biophysical ResearchCommunications vol 450 no 1 pp 652ndash658 2014

[37] W C Earnshaw L M Martins and S H Kaufmann ldquoMam-malian caspases structure activation substrates and functionsduring apoptosisrdquo Annual Review of Biochemistry vol 68 pp383ndash424 1999

[38] E A Slee C Adrain and S J Martin ldquoExecutioner caspase-3 -6 and -7 perform distinct non-redundant roles during thedemolition phase of apoptosisrdquo Journal of Biological Chemistryvol 276 no 10 pp 7320ndash7326 2001

[39] A G Porter and R U Janicke ldquoEmerging roles of caspase-3 inapoptosisrdquo Cell Death and Differentiation vol 6 no 2 pp 99ndash104 1999

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

[41] E J Kim Y-J Lee H-K Shin and J H Y Park ldquoInduction ofapoptosis by the aqueous extract of Rubus coreanum in HT-29human colon cancer cellsrdquo Nutrition vol 21 no 11-12 pp 1141ndash1148 2005

[42] L A Schmitt Y Liu P A Murphy J W Petrich P M Dixonand D F Birt ldquoReduction in hypericin-induced phototoxicityby Hypericum perforatum extracts and pure compoundsrdquoJournal of Photochemistry and Photobiology B Biology vol 85no 2 pp 118ndash130 2006

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


Cancers that start in the cells lining inside of the colon andrectum are called colorectal cancers (CRC) CRC are the thirdmost predominant cancers worldwide and are the fourthmost common cause of cancer mortality with approximately94 of global cancer cases irrespective of gender The rate ofincidence of CRC has been observed more in AustraliaNewZealand Europe USA and UK African and Asian coun-tries have a far lower rate of incidence [11] Accordingto epidemiological studies the major risk factors of CRCincidence include obesity smoking alcohol consumptionincreasing age family history and dietary factors [12 13]Several flavonoid-rich foods could display relevant cancer-preventive effects Thus the isolation of potential chemopre-ventive agents from plants has contributed to breakthroughin anticancer studies [14] Carcinoma colon (Caco-2) cellline originates from human colonic adenocarcinoma andshows similarities to small intestinal enterocytes [15] Thesecells develop typical morphology of enterocytes with distinctapical and basolateral membrane domains microvillus andtight junctions [16] The cells can be grown on uncoatedpolycarbonate filters to obtain well-differentiatedmonolayers[17]

The genus Rubus is very diverse with over 750 species in12 subgenera and is found on all continents except Antarctica[18] Due to useful ethnomedicinal and pharmacologicalproperties Rubus species have been used in folk medicine[19] R fairholmianus (R moluccanus L) leaf extract has beenadministrated to reduce headache [20] these leaves possessinsecticidal properties fruits are edible with stimulatingeffect [21] On the basis of the previously reported anti-inflammatory wound healing [22] and in silico anticancerproperties [23] we have selected this plant for this studyEven though this plant has immense ethnomedicinal valuea survey of literature revealed that the in vitro anticancerpotential of this plant has not yet been evaluated Keeping thisin view the aim of the present study was to investigate thein vitro anticancer potential and track the possible cell deathmechanisms of R fairholmianus root acetone extract to putforward a scope to develop an effective drug

2 Materials and Methods

21 Plant Material Collection Identification and ExtractionThe fresh plant parts of R fairholmianus were collected fromShola forest of Marayoor Idukki District Kerala India dur-ing September 2010 Plant parts were identified and authen-ticated (voucher specimen number BSISRC5232010-11Tech1657) by Botanical Survey of India Southern Cir-cle Coimbatore Tamil Nadu India The shade dried andpowdered roots were extracted in Soxhlet apparatus usingacetone The extractability of phenolic compounds in thepolar solvent like acetone was found to be high Moreoveracetone extract has shown better pharmacological effects inmany previous studies inRubusThe extractwas concentratedto dryness under reduced pressure in a rotary evaporatorto yield dried root acetone extract The dried extract thendissolved in 05 DMSO and was used for further analysisThe preliminary screening of leaves stem and root for totalphenolics tannin flavonoids and antioxidant propertiesshowed that root had the maximum activity [24]

22 Cell Culture The commercially obtained colorectalcancer cells (Caco-2 ATCC HTB-37) were used Caco-2 cellswere cultured in Dulbeccorsquos modified Eaglersquos media (DMEMSigma-Aldrich D 6429) with 12 gL sodium carbonatesupplemented with 10 foetal bovine serum (FBS Gibco30600301) 10mM nonessential amino acids (Gibco 11140)05mMsodiumpyruvate (Gibco 11360) 25mML-glutamine(Gibco 25030) 1 antibiotic (penicillin-streptomycinGibco 15140) and 1 antifungal (amphotericin-B Gibco104813) agents Culture was maintained at 37∘C with 5 CO

2

and 85 humidity Once cells reached 80ndash90 confluencethey were harvested and seeded at a concentration of 5 times104 in 3mL culture media into sterile culture dishes with adiameter of 34 cm Cells were allowed to attach overnight

23 Cellular Morphology-Inverted Microscopy The effect ofthe plant extract on cellmorphologywas determined using aninverted light microscope (Wirsam Olympus CKX41) after24 h of incubationwith different concentrations (5 10 20 and40 120583gmL) of RFRA and 05 DMSO Once digital imageswere recorded cells were trypsinized using 1mL25 cm2 ofTrypLE Express (Invitrogen 12605-028) and resuspended inHankrsquos Balanced Salt Solution (HBSS) to perform furtherassays

24 Cell Viability-Trypan Blue Dye Exclusion Assay Thetrypan blue dye exclusion assay (Sigma-Aldrich T8154) is aquantitative method to determine the percentage viabilityHere the viable cells with an intact cellular membrane do nottake up the blue dye andmaintain a clear appearance whereasthe damaged nonviable cells take up dye and stained blue astheir membrane damages

The cell suspension was carefully mixed with 04 trypanblue reagent in 1 9 ratio and the mixture was transferred to aNeubauer hemocytometer to determine the number of viableand nonviable cells Cells were counted by using automatedcell counter (Countess Automated Cell Counter Invitrogen)

25 Cellular Proliferation-Adenosine Triphosphate (ATP)Luminescent Assay The CellTiter-Glo1 luminescent assay(Promega G7571 Anatech Analytical Technology BellvilleSouth Africa) is a homogeneous method for determinationof cellular proliferation and quantification of ATP present inmetabolically active cells

Fifty microliters of reconstituted reagent was added to anequal volume of cell suspension and then mixed in a shakerfor 2 minutes to induce cell lysis This was then incubatedat room temperature for 10 minutes in dark to stabilize theluminescent signalThe luminescent signalwas read using the1420 Multilabel Counter Victor3 (Perkin-Elmer SeparationScientific)

26 Cytotoxicity-Lactate Dehydrogenase (LDH) Assay Themembrane integrity was assessed by estimating the amount ofLDHpresent in the culturemediaThe cytosolic enzymeLDHwill be released due to membrane damage The CytoTox96X assay (Anatech Promega G 400) was used to measurethe released LDH Fifty microliters of reconstituted reagentwas added to an equal volume of cell culture medium and













50120583m50120583m



(b)(a) (c)

(d) (e) (f)

50120583m













Lum

ines

cenc

e (re

lativ

e lig

ht u

nit)

0

Con

trol

05

DM

SO


lowastlowastlowast

lowastlowastlowast

3e + 5

3e + 5

2e + 5

2e + 5

1e + 5

5e + 4

n = 6

5120583

g RF

RA

10120583

g RF

RA

20120583

g RF

RA

40120583

g RF

RA







101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107





Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

05 DMSO

(a) (b) (c)

(d) (e) (f)







39 plusmn 089



















4 Conclusions



min

esce

nce (

read

ing

light

uni

ts RL

U)

0

5000

10000

15000

20000

25000n = 6

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con

trol

05

DM

SO

5120583

gm

L RF

RA

10120583

gm

L RF

RA

20120583

gm

L RF

RA

40120583

gm

L RF

RA





References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























50120583m50120583m



(b)(a) (c)

(d) (e) (f)

50120583m













Lum

ines

cenc

e (re

lativ

e lig

ht u

nit)

0

Con

trol

05

DM

SO


lowastlowastlowast

lowastlowastlowast

3e + 5

3e + 5

2e + 5

2e + 5

1e + 5

5e + 4

n = 6

5120583

g RF

RA

10120583

g RF

RA

20120583

g RF

RA

40120583

g RF

RA







101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107





Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

05 DMSO

(a) (b) (c)

(d) (e) (f)







39 plusmn 089



















4 Conclusions



min

esce

nce (

read

ing

light

uni

ts RL

U)

0

5000

10000

15000

20000

25000n = 6

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con

trol

05

DM

SO

5120583

gm

L RF

RA

10120583

gm

L RF

RA

20120583

gm

L RF

RA

40120583

gm

L RF

RA





References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Lum

ines

cenc

e (re

lativ

e lig

ht u

nit)

0

Con

trol

05

DM

SO


lowastlowastlowast

lowastlowastlowast

3e + 5

3e + 5

2e + 5

2e + 5

1e + 5

5e + 4

n = 6

5120583

g RF

RA

10120583

g RF

RA

20120583

g RF

RA

40120583

g RF

RA







101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107





Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

05 DMSO

(a) (b) (c)

(d) (e) (f)







39 plusmn 089



















4 Conclusions



min

esce

nce (

read

ing

light

uni

ts RL

U)

0

5000

10000

15000

20000

25000n = 6

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con

trol

05

DM

SO

5120583

gm

L RF

RA

10120583

gm

L RF

RA

20120583

gm

L RF

RA

40120583

gm

L RF

RA





References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107

101

102

103

104

105

106

107





Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

Prop

idiu

m io

dide

05 DMSO

(a) (b) (c)

(d) (e) (f)







39 plusmn 089



















4 Conclusions



min

esce

nce (

read

ing

light

uni

ts RL

U)

0

5000

10000

15000

20000

25000n = 6

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con

trol

05

DM

SO

5120583

gm

L RF

RA

10120583

gm

L RF

RA

20120583

gm

L RF

RA

40120583

gm

L RF

RA





References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















min

esce

nce (

read

ing

light

uni

ts RL

U)

0

5000

10000

15000

20000

25000n = 6

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

Con

trol

05

DM

SO

5120583

gm

L RF

RA

10120583

gm

L RF

RA

20120583

gm

L RF

RA

40120583

gm

L RF

RA





References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article antiproliferative effect of the...

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