ResearchArticle - Hindawi Publishing...

Research ArticleCytotoxicity and Proapoptotic Effects ofAllium atroviolaceum Flower Extract by ModulatingCell Cycle Arrest and Caspase-Dependent and p53-IndependentPathway in Breast Cancer Cell Lines

Somayeh Khazaei1 Roslida Abdul Hamid1

Vasudevan Ramachandran2 NorhaizanMohd Esa3 Ashok Kumar Pandurangan4

Fatemeh Danazadeh1 and Patimah Ismail1

1Department of Biomedical Science Faculty of Medicine and Health Sciences Universiti Putra Malaysia Selangor Malaysia2Malaysian Research Institute of Aging Universiti Putra Malaysia Selangor Malaysia3Department of Nutrition and Dietetics Faculty of Medicine and Health Sciences Universiti Putra Malaysia Selangor Malaysia4School of Life Sciences B S Abdur Rahman Crescent University Vandalur Chennai Tamil Nadu 600048 India

Correspondence should be addressed to Patimah Ismail patimahismailgmailcom

Received 1 June 2017 Accepted 12 October 2017 Published 8 November 2017

Academic Editor Jose L Rios

Copyright copy 2017 Somayeh Khazaei et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Breast cancer is the second leading cause of cancer death among women and despite significant advances in therapy it remains acritical health problem worldwide Allium atroviolaceum is an herbaceous plant with limited information about the therapeuticcapability We aimed to study the anticancer effect of flower extract and the mechanisms of action in MCF-7 and MDA-MB-231The extract inhibits the proliferation of the cells in a time- and dose-dependent manner The underlying mechanism involved thestimulation of S and G2M phase arrest in MCF-7 and S phase arrest in MDA-MB-231 associated with decreased level of Cdk1in a p53-independent pathway Furthermore the extract induces apoptosis in both cell lines as indicated by the percentage ofsub-G0 population the morphological changes observed by phase contrast and fluorescent microscopy and increase in Annexin-V-positive cells The apoptosis induction was related to downregulation of Bcl-2 and also likely to be caspase-dependent Moreoverthe combination of the extract and tamoxifen exhibits synergistic effect suggesting that it can complement current chemotherapyLC-MS analysis displayed 17 major compounds in the extract which might be responsible for the observed effects Overall thisstudy demonstrates the potential applications of Allium atroviolaceum extract as an anticancer drug for breast cancer treatment

1 Introduction

After lung cancer breast cancer is the second leading causeof cancer death among women worldwide Among morethan one million new cancer cases breast cancer contains18 of all female cancers globally [1] Despite significantadvances in therapy breast cancer remains a critical healthproblem worldwide Moreover the current breast cancertreatments are expensive not widely available and limitedby side effects and resistance to the treatment Therefore

natural products could be the alternative andnovel anticanceragents [2 3] Natural crude extracts and biologically activecompounds isolated from plant species used in traditionalmedicine can be prolific resources for new drugs [4] Alliumatroviolaceum (A atroviolaceum) an herbaceous perennialbulbous plant is a species in the genus Allium which belongsto the Alliaceae family It is distributed in Crimea Caucasus(Ante-Caucasus Daghestan and Trans-Caucasus) middleAsia (Mountainous Turkmenistan Syr-Darya foothill areas)and Iran [5] A atroviolaceum is used as food (vegetable) and

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 1468957 16 pageshttpsdoiorg10115520171468957

2 Evidence-Based Complementary and Alternative Medicine

a source of vitamins although useful properties of this specieshave not been studied adequately [6] Gas chromatography-mass spectrophotometry study on A atroviolaceum revealedthe presence of various bioactive components in the plantextracts which might be the reason of antioxidant andantibacterial potency and perhaps other biological activ-ities [7] Notably Allium vegetables are rich in flavonolsand organosulfur compounds which have exhibited tumorinhibitory properties in laboratory studies Some compo-nents ofAllium vegetables are reported to block several stagesof carcinogenesis although the underlying mechanisms ofaction are generally unclear [8] The previous studies on Aatroviolaceum bulb extract against breast cervical and livercancers [9] and flower extracts against HepG2 [10] and Hela[11] cells demonstrated potential anticancer activity

In current study methanol extract from flower of Aatroviolaceum (FAA) has been used in scientific research toreveal the therapeutic properties of this plant against twohuman breast cancer cell lines MCF-7 and MDA-MB-231We hypothesized that FAA extract induces anticancer effectsthrough the induction of cell cycle arrest and apoptosis inhuman breast cancer cells Based on this hypothesis the studyaimed to investigate the possible mechanisms of action inMCF-7 and MDA-MB-231 cells

2 Materials and Methods

21 Preparation of Plant Extract The process of extractpreparation was described previously [10] Fresh flowers ofA atroviolaceum were collected in July of 2013 from KojurIran (latitude = 363825 longitude = 517106 Lat = 36 degrees230 minutes north Long = 51 degrees 426 minutes east)The plant was compared with the voucher specimen number720ndash722 deposited at the Faculty of BiologyHerbarium AzadUniversity of Ghaemshahr Iran Harvested fresh flowerswere rinsed and air dried at room temperature The driedmaterial was homogenized to obtain a coarse powder andextracted by 70 methanol (CheMarCo Inc SC USA) for6 h using Soxhlet apparatus (Electrothermal Eng RochfordUK) The extract was concentrated in a rotary evaporator(Buchi Labortechnik AG Flawil Switzerland) The resultantcondensate was subjected to freeze-drying (VirTis Bench-Top K freeze dryer NY USA)The stock solution of extractwas prepared by dimethyl sulfoxide (DMSO) (Sigma-AldrichMO USA) prior to usage

22 Cell Lines The human breast cancer cell lines MCF-7 (human hormone-dependent breast cancer cell lineATCC HTB-22) and MDA-MB-231 (human non-hormone-dependent breast cancer cell line ATCC HTB-26) andthe normal 3T3 cell line (mouse embryo fibroblast ATCCCRL-1658) were purchased from American Type CultureCollection (VA USA) Cell lines were grown in tissue cultureflasks at 37∘C 5 CO2 and 90 humidity (IR censoredCO2 incubator) in RPMI-1640 medium (Sigma-AldrichSteinheim Germany) containing 10 fetal bovine serum(Sigma-Aldrich Steinheim Germany) and 100 IUml peni-cillin streptomycin (Sigma-Aldrich Steinheim Germany)The cells were grown confluence which could be observed

under an inverted microscope and subcultured at three tofour days interval

23 MTT Cell Proliferation Test The antiproliferative effectsof FAA on MCF-7 and MDA-MB-231 cells were examinedusing the MTT colorimetric assay Cells were seeded in 96-well plates (TPP Switzerland) at a density of 106 cellsml for24 h before exposure to the indicated concentrations of FAAfor 24 48 and 72 hMedia without FAA extract were used as anegative control After removal of the medium 20 120583l of MTTreagent (Sigma-Aldrich MO USA) was added to each welland followed by 4 h incubationThe purple formazan crystalswere dissolved in 100 120583l of DMSO The optical density (OD)of each well was measured at 570 nm on FLUOstar Omegamicroplate reader (BMG Labtech Ortenberg Germany) Anexperiment was done in triplicate and cell viability has beencalculated by following equation

Cytotoxicity = OD of treated cellsOD of negative control

times 100 (1)

24 Microscopic Examination To determine the effects ofFAA on the cell morphology the cells were seeded in 6-well plates (106ml) After 24 h 5ml of media containingIC50 concentrations of the FAA was added while the controlcontained untreated media After 24 48 and 72 h the cellswere then washed with PBS and observed by OlympusCulture Microscope model CK40 (Olympus CorporationTokyo Japan)

Moreover acridine orange (AO) and propidium iodide(PI) double staining was applied to observe the apoptoticfeature of the cells by fluorescent microscope MCF-7 andMDA-MB-231 cells were seeded at density of 5 times 106 cellswelland incubated for 24 h then the medium was replaced withIC50 concentration of FAA dissolved in the medium and thecells were incubated for 24 48 and 72 h The cell suspensionwas mixed with an equal volume of AOPI (Sigma-AldrichMO USA) staining solution (1 1) The viable apoptotic andnecrotic cells were observed under Leica fluorescence micro-scope DM 2500 (Leica Microsystem Wetzlar Germany)within 30minwith 100xmagnification Images were capturedby using Alpha Imager (Alpha Innotech CA USA)

25 Flow Cytometric Cell Cycle Analysis The effect of FAAon MCF-7 and MDA-MB-231 cell cycle distribution wasdetermined by flow cytometric analysis Cells were seededat density of 5 times 106 cultured for 24 h prior to treatmentwith IC25 IC50 and IC75 concentrations of FAA exposurefor 24 48 and 72 h The cells were then resuspended in500 120583l PBS prior to being fixed at 70 cold ethanol forat least 2 h at ndash20∘C After centrifuging at 1000 rpm for 10minutes and washing twice with PBS the fixed cells weretreated with 500120583l PIRNase (400 120583l propidium iodide and100 120583l Ribonuclease A) and incubated for 10min at roomtemperature The DNA content per cell was analysed usingBD LSRFortessa Cell Analyzer (Becton Dickinson NJUSA) equipped with 488 nm argon laser light source and630 nm band pass filter for 10000 events per sample Datawas expressed as percentage of cells compared to untreated

Evidence-Based Complementary and Alternative Medicine 3

control population using along with its analytical softwareBD FACSDiva software

26 Annexin-V-FITCPI Apoptotic and necrotic cells weredifferentiated using the Annexin assay V-FITC kit (Sigma-Aldrich MO USA) as per the manufacturerrsquos protocol Cellswere plated at 5 times 106 and incubated for 24 h prior to theindication by IC25 IC50 and IC75 concentrations of FAAexposure for 24 48 and 72 h The cells were centrifuged(1000 rpm for 5 minutes) resuspended in 1x Annexin-Vbinding buffer and stained with 10 120583l of PI and 5120583l ofAnnexin-V-FITC for 10min in darkness prior to analyisby flow cytometry For each experiment 10000 events persample were recorded

27 Assay of Caspase Activity Caspase colorimetric proteaseassay sample kit (Biovision CA USA) was used to determinethe activity of caspases 1 2 3 -5 6 8 and 9 Cells (106ml)were seeded in 6-well plate and incubated for 24 h priorto exposure to FAA in various concentrations The cellswere then transferred into sterile test tube and lysed usingthe cell lysis buffer and incubated on ice for 10min Aftercentrifugation at 5000 rpm for 2min samples (50 120583l) of thelysate were aliquoted into a 96-well microplate to which50 120583l of reaction buffer containing 10Mm DTT was thenadded to the sample Substrates for each of the caspases (5 120583l)were added to the appropriate wells and the plate was thenincubated for 2 h Absorbance at 405 nm was then read ina microplate reader The absorbance of treated samples wascompared with untreated control The selective substratesused were p-nitroaniline

28 qPCR Analysis of Cell Cycle and Apoptosis ResponsiveGenes Genersquos expression was analysed with quantitativereverse transcription-PCR using a real-time SYBR Greengene expression assay kit as described in the protocol TotalRNA was isolated from the MCF-7 and MDA-MB-231 cellstreated with IC25 IC50 and IC75 concentration of FAAfor 24 h using RNeasy Mini kit (Qiagen Inc CA USA)RNA samples were quantitated at OD 260280 by NanoDropSpectrophotometer along with its analytical software V37(Thermo Fisher Scientific DE USA) and RIN numberwas determined by Bioanalyzer (Agilent 2100 Bioanalyzersystem-Agilent Technologies Waldbronn Germany) TheRNA (1 120583g) was introduced to reverse transcription with RT2first strand kit (Qiagen Inc CA USA) and the cDNA wereanalysed using RT2 SYBR Green ROX FAST Mastermix(Qiagen Inc CA USA) qPCR was performed in a reactionvolume of 25 120583L as per the manufacturerrsquos instructionsBriefly 125 120583L of RT2 SYBRGreenMastermix 1120583L of assayprimers (RT2 qPCR Primer Assays Qiagen Inc CA USA)and 1 120583L of template cDNAwere added to each wellThe PCRplate was placed in the real-time PCR instrument CorbettRotor-Gene 6000 (Qiagen Inc CA USA) and run at 95∘Cfor 10 minutes (activating the enzyme) and 40 cycles of 15seconds at 95∘C (denaturation) followed by 30 seconds at60∘C (annealing and synthesis) The expression levels wereexpressed as 119899-fold differences relative to the reference gene

GAPDHThe data were analysed using a comparative thresh-old (Ct) method and the fold inductions of samples werecompared with the untreated samples The gene expressionlevel was then calculated by following formula

2ΔΔCt = 2Ct (treated cells) minusCt (control cells) (2)

where 2 is the amplification efficiency where the templatedoubles in each cycle during exponential amplification

The following primer pairs for target genes and GAPDHwere chosen from the Primer Bank website Bcl-2 51015840-TACCTG AAC CGG CAC CTG-31015840 and 51015840- GCC GTA CAGTTC CAC AAA GG-31015840 Cdk1 51015840- GGGTCAGCTCGC-TACTCAAC-31015840 and 51015840-AAGTTTTTGACGTGGGATGC-31015840 p53 51015840- TGT GGA GTA TTT GGA TGA CA-31015840 and51015840- GAA CAT GAG TTT TTT ATG GC-31015840 GAPDH 51015840-TCCTGCACCA CCAACTGCTTAG-31015840 and 51015840- GGCATG-GACTGTGG TCATGAGT-31015840

29 Evaluation of TAM and FAA Combination Effect Toassess the synergistic effect of FAA extracts in combinationwith TAM the cells were seeded at 1 times 106 cellsml andincubated for 24 h After incubation the cells were treatedwith combination of IC50 values of FAA and TAM (obtainedin our previous study [9]) and incubated for an additional24 48 and 72 h The MTT solution (20 120583l) was added toeach well and incubated for 4 h The MTT-formazan crystalsformedwere dissolved in 100 120583l of DMSO and the absorbancewas measured at 550 nmThe IC50 obtained for single-agentswere compared to the IC50 calculated for their combinationThe combination index (CI) was calculated by CompuSynsoftware (Paramus NJ USA) to analyse synergistic inhibitoryeffect The CI values lt1 =1 and gt1 represent a synergisticadditive and antagonistic effects respectively [12]

210 Liquid Chromatography-Mass Spectrometry (LC-MS)LC-MS was used to identify the major compounds in theFAA extract Chromatographic analysis of FAA was car-ried out by reverse phase elution (Waters Symmetry LC-18 column 250 times 46mm 5120583m) on Agilent 6500 SeriesAccurate-Mass Quadrupole Time-of-Flight (Q-TOF AgilentSanta Clara CA USA) LCMS system with Agilent 1200Series Diode Array Detector (module G1315B detection type1024-element photodiode array light source deuterium andtungsten lamps wavelength range 190ndash950 nm) The mobilephase consisted of (A) formic acid (01 vv) (B) acetonitrile+ 01 formic acid gradient (in solvent B) (i) 20 from0 to 20min (ii) 95 from 20 to 27min and (iii) 35 at27ndash30min of total run time flow rate 02mlmin injectionvolume 3 L ESI parameters both negative and positive ionmode mass range 100ndash1200119898119911 spray voltage 4 kV gastemperature 325∘C gas flow 10 Lmin Nebulizer 40 psi andthe mass was analysed by using Agilent technologies Mass-Hunter software

211 Statistical Analysis Results were analysed by version7 of GraphPad Prism using one-way analysis of variance(ANOVA) and differences were considered statistically sig-nificant at the level of 119901 values le 005 001 and 0001 Gene


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Figure 1 Antiproliferative effect of FAA on breast cancer and 3T3 cells Dose-response curves of FAA treatment on MCF-7 (a) MDA-MB-231(b) and 3T3 (c) [10] Data represent the mean plusmn SD of three independent experiments

expression analysis was done using Studentrsquos 119905-test and thedifferences were considered statistically significant at the levelof 119901 values le 005

3 Results

31 Cell Proliferation Inhibition after FAA Treatment Theproliferation of MCF-7 and MDA-MB-231 cells treated withvarious concentrations of FAA was significantly inhibitedin a dose- and time-dependent manner The estimated IC50value was 655 plusmn 9 120583gml 41 plusmn 0120583gml and 2467 plusmn 35 120583gmlin the case of MCF-7 (Figure 1(a)) and 77 plusmn 529 120583gml 67plusmn 0 120583gml and 51 plusmn 17 120583gml for MDA-MB-231 cell lines(Figure 1(b)) after 24 48 and 72 h respectively Howeverbased on our previous study FAA showed no evident cytotox-icity against the healthy mouse fibroblast 3T3 cells after 72 h(Figure 1(c)) [10]

32 Morphological Feature of Apoptosis The morphologicalappearances of MCF-7 and MDA-MB-231 after 24 48 and

72 h of exposure to FAA were observed under the invertedlight microscope In the case of untreated cells high densityof monolayer cells with intact membrane was observedHowever the cells showed morphological changes suchas autophagy and reduction in the cell volume after thetreatment with IC50 concentration of FAA extracts at 24 hThe features of apoptosis were clearly observed after 48 hof treatment where the treated cells showed the formationof shrinking membrane blebbing and membrane boundedvesicles (apoptotic bodies) The extracts showed more cyto-toxic effects after 72 h of incubation since only debris waspresent in the culture

The morphological changes in the cancer cells nucleiwere observed by fluorescence microscope to understandthe mode of cell death AO and PI were used to differen-tiate between viable apoptotic and necrotic cell based onmembrane integrity The results revealed that FAA triggeredmorphological features that relate to apoptosis in a time-dependent manner The untreated cells showed an intactround nuclear membrane with the nuclei heavily stained by


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Figure 2 Morphological observation of treated MCF7 and MDA-MB-231 cells by phase contrast and fluorescence microscopy (a1)ndash(a4) and(a11015840)ndash(a41015840) indicate the untreated and treated MCF7 cells with FAA for 24 h 48 h and 72 h respectively while (b1)ndash(b4) and (b11015840)ndash(b41015840)display the untreated and treated MDA-MB-231 cells with FAA for 24 h 48 h and 72 h respectively In phase contrast images autophagy(A) shrinkage (S) apoptotic bodies (AB) and debris (D) are shown In fluorescent images the typical characteristics of apoptosis such asnuclear margination (NM) chromatin condensation (CC) nuclear fragmentation (NF) membrane blebbing (MB) apoptotic bodies (AB)and membrane looseness (ML) are presented Similar cellular morphology was observed in three independent experiments (119899 = 3)

AO green fluorescence After the treatment of MCF-7 withFAA at 24 h the cell showed typical morphological featuresof apoptosis including nuclear margination and chromatincondensation After 48 h granulation in the nucleus wasclearly observed The treatment at 72 h with FAA showedmembrane blebbing apoptotic bodies and membrane loose-ness Moreover similarly to MCF-7 MDA-MB-231 cellsexhibited nuclear margination and chromatin condensation

which weremajor consequences of the apoptotic trigger afterthe treatment with FAA at 24 h while the 48 and 72 h oftreatments represented both early (chromatin condensationnuclear fragmentation) and late apoptosis (apoptotic bodiesmembrane looseness) features (Figure 2)

33 Cell Cycle Arrest in MCF-7 and MDA-MB-231 Cells TheDNA content was measured by flow cytometry to evaluate


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Figure 3 Cell cycle analysis of MCF7 and MDA-MB-231 cancer cells treated with FAA at IC25 IC50 and IC75 values The distribution of(a) MCF7 and (b) MDA-MB-231 cells undergoing apoptosis and in various phases of the cell cycle was determined for 24 48 and 72 h incomparison to the respective controls The values are presented as mean plusmn standard error of mean of three determinations indicated by lowastlowastlowast and lowastlowastlowast showing a significant difference (119901 lt 005 119901 lt 001 and 119901 lt 0001 resp)

the cell cycle distribution of MCF-7 and MDA-MB-231cells with or without FAA treatment Treatment of MCF-7 presented a subcellular peak and a dramatic decrease inG0G1 phase in a dose- and time-dependent manner At 24 hdistribution of cells in the S phase significantly incrementsin treated cells with IC25 IC50 and IC75 of FAA whileafter 48 h this value is considerably increased by IC25 andIC50 concentrations of FAA However the 72 h treatmentincreased the S phase percentage only in IC25 (Figure 3(a)see Figure SI in Supplementary Material available online athttpsdoiorg10115520171468957) Moreover 24 h treat-ment by IC25 IC50 and IC75 of FAA represented a significantenhance of MDA-MB-231 cell distribution at G2M phaseThough 48 h treatment increased this value by IC25 and IC50values of FAAThe event occurred after 72 h of treatmentwithIC25 and IC50 of FAA when the cells were accumulated at

the S phase accompanied by a large enhancement of sub-G0and remarkable reduction of G0G1 phase in a time and dosecourse manner (Figures 3(b) SII)

34 Induction of Apoptosis in MCF-7 and MDA-MB-231 Todetermine the effect of FAA in apoptosis induction the FITC-Annexin-V staining was used through flow cytometry Thepercentages of live cell significantly declined in both cell linesafter the treatment with FAA in a dose-dependent mannerAnalysis of MCF-7 cells showed variation in apoptotic andnecrotic cell percentage in time- and dose-dependent man-ner The percentage of early apoptotic cells increased after24 h by IC25 IC50 and IC75 with IC50 and IC75 values after48 h andwith IC50 and IC75 of FAA after 72 hMeanwhile lateapoptosis increased markedly in cells treated with IC50 after24 h with IC75 after 48 h and with IC25 IC50 and IC75 after


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Figure 4Apoptosis study ofMCF-7 andMDA-MB-231 cells exposed to FAA extract at IC25 IC50 and IC75 valuesThe distribution of live cellsand cells undergoing early apoptosis late apoptosis and necrosis was determined in (a)MCF7 and (b)MDA-MB-231 cells following treatmentwith FAA for 24 48 and 72 h in comparison to the respective control using Annexin-V-FITC and propidium iodide flow cytometric analysisThe values are presented as mean plusmn standard error of mean of three determinations

72 h In contrast the percentages of cells entering necrosisdecreased with IC25 and IC75 of FAA after 24 h and with IC50and IC75 after 72 h No reportable change in the percentage ofnecrotic cells was observed after the treatment with FAA for48 h (Figure 4(a))

The proportion of early apoptotic MDA-MB-23 cellsenhanced after 24 h 48 h and 72 h with IC25 IC50 andIC75 concentrations of FAA On the other hand the cellproportion in late apoptosis was significantly raised in cellstreated with IC75 of FAA after 24 h 48 h and 72 h with IC25IC50 and IC75 Percentages of cells entering necrosis weresignificantly enhanced only after 48 h in cells treated withIC50 and IC75 of FAA (Figure 4(b))

35 FAA Treatment Activates the Caspases The activation oftwo inflammatory caspases (caspases 1 and 5) three initiatorcaspases (caspases 2 8 and 9) and two executioner caspase(caspases 3 and 6) was investigated at three concentrationsof FAA extract in 24 h Caspase-1 activity in both treatedcells resulted in a significant reduction compared to theuntreated cellsThe FAA treatment was found to decrease theactivity of caspase-5 in MCF-7 while in MDA-MB-231 cells itincreased at IC25 of FAA The induction of caspase-2 activityincreased significantly in MDA-MB-231 but not in MCF-7 The level of caspase-9 was evaluated significantly at IC75of FAA in MCF-7 In contrast MDA-MB-231 cells exposedto low concentration (IC25) of FAA exhibited a significantlyhigher activation compared to the control FAA could triggercaspase-8 activity in bothMCF-7 andMDA-MB-231 cell linesin IC50 and IC75 concentrations Since both upstream sig-nalling pathways (intrinsic and extrinsic pathway) convergedownstream to activate the final effector caspase mechanism

the activity of caspase-3 and caspase-6 executioner caspaseswas investigated The results demonstrated that FAA sharplyinduced caspase-6 activity in MCF-7 cells as the activitysignificantly increased in all used concentrations On theother hand caspase-6 activity was also enhanced with IC50of FAA in the MDA-MB-231 The FAA extract was found toincrease the activity of caspase-3 significantly only in MCF-7cell line in IC25 value (Figure 5)

36 Expression Levels of Bcl-2 Cdk1 and p53 in MCF-7and MDA-MB-231 Cells The effects of IC25 IC50 and IC75value of FAA extract at 24 h on Bcl-2 Cdk1 and p53 genemodulation were investigated by qRT-PCR The extractedRNA from all the samples exhibited high quality and purityThe expression of Bcl-2 gene in the treated MCF-7 cells wasfound to be dramatically downregulated by 218-fold and 49-fold after treatment with IC50 and IC75 value In MDA-MB-231 cells a significant downregulation ofBcl-2 gene expressionoccurred after the treatment with IC75 of FAA by 215-foldwhen compared to the untreated cellsThe treatment ofMCF-7 cells with IC75 remarkably downregulated the Cdk1 geneexpression with 577-fold Moreover the mRNA level of Cdk1in MDA-MB revealed a 333-fold downregulation at IC75concentration compared to the control However p53 geneexpression was found to be nonsignificant in both cell lines(Figure 6)

37 Combination Effect of FAA and Tamoxifen in MCF-7and MDA-MB-231 Cells Based on the data showing efficacyof FAA in breast carcinoma cells next we assessed thecombinations effect of FAA and tamoxifen (TAM) on MCF-7 and MDA-MB cell growth According to our previous


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Figure 5 Activation of caspases by FAA in MCF-7 and MDA-MB-231 cells The cells were incubated with FAA extract at IC25 IC50 and IC75values for 24 h after which the activity of various caspases was determined as described in Section 2 Data are presented inmeanplusmn SD (119899 = 3)Values for the treated group are significantly different to the untreated control group at the same time point at lowast119901 lt 005 lowastlowast119901 lt 001 andlowastlowastlowast119901 lt 0001


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IC50IC75

Figure 6The expression of Bcl2 Cdk1 and p53 in MCF7 and MDA-MB-231 by real-time quantitative PCR analysis After treatment with theIC25 IC50 and IC75 of FAA for 24 h the relative quantification of the target gene by the delta-delta-Ct method was done using the Qiagensoftware Values for the treated group are significantly different to the untreated control group at lowast119901 le 005

study treatment of the cells with a serial dilution of TAMas a positive control indicated the IC50 value of 98 120583gml72 120583gml and 67 120583gml for MCF-7 and 136 120583gml and11 120583gml and 378 120583gml for MDA-MB-231 after 24 48 and72 h respectively [9] The IC50 values of FAA were thuscombined with the IC50 of TAM which resulted in moregrowth inhibition and less IC50 amount as compared to thetreatment with a single drug at 24 48 and 72 h Combinedtreatment with the IC50 amount of FAA-TAM reduced theIC50 value of MCF-7 significantly to 505120583gml 38 120583gmland 34 120583gml after 24 48 and 72 h respectively The IC50concentration of TAM against MDA-MB-231 significantlyreduced in the presence of FAA to 527120583gml 68 120583gml and073 120583gml at 24 48 and 72 h respectively (Figure 7)

Drug combination effects were analysed by the methodof Chou and Talalay [13] The varying concentrations of thecombination IC125 IC25 and IC50 concentration demon-strated CI lt 1 indicating synergistic effects while IC75showed moderate antagonism with a CI gt 1 The calculated

CI values for MDA-MB-231 cells treated with FAA-TAMshowed a downtrend from IC125 to IC25 IC50 and IC75 at24 and 48 h The result revealed that the interaction of FAA-TAMwas synergism at 24 h even in low concentration whileonly a high concentration (IC75) of the combination at 48 hpromoted synergism but for the doses below that showedantagonism (CI gt 1) Contrariwise the CI values of FAA-TAM showed synergism was obtained only in the lowestconcentration while the higher concentration resulted inantagonism (Figure 8)

38 LC-MS Analysis of FAA Extract Figure 9 shows LC-MSpeaks for FAA and the chemical structure of major com-pounds while Table 1 shows the major compounds presentin the extract with their molecular weight and molecularformula Our findings indicated that the major compoundsin FAA include pirimicarb Asn Gly Met Schizonepeto-side E tamsulosin Lys Gln Ile Istamycin C1 NetilmicinActinodaphnine acetylcaranine sphinganine ripazepam


Table 1 LC-MS characterization of methanolic flower extract of Allium atroviolaceum

Peak number Rt Mass Formula Compound name8 5485 238143 C11H18N4O2 Pirimicarb10 5662 3201157 C11H20N4O5S Asn Gly Met11 5848 3481775 C16H28O8 Schizonepetoside E15 59 4081718 C20H28N2O5S Tamsulosin17 5915 3872487 C17H33N5O5 Lys Gln Ile20 595 4312753 C19H37N5O6 Istamycin C124 5989 4753017 C21H41N5O7 Netilmicin60 8339 7834845 C45H70NO8P PE(226(4Z7Z10Z13Z16Z19Z)184(6Z9Z12Z15Z))61 9846 3111171 C18H17NO4 Actinodaphnine63 12087 313131 C18H19NO4 Acetylcaranine

64 1488 4323255 C27H44O4(23S)-232425- trihydroxyvitamin D3(23S)- 232425-trihydroxycholecalciferol

65 14417 2872835 C17H37NO2 C17 sphinganine

68 18315 4122989 C27H40O3MID42047(22E)-(24S)-112057224-dihydroxy-2627-cyclo-2223- didehydro-20-epivitaminD3(22E)-(24S)-11205722

72 20701 2681322 C15H16N4O Ripazepam75 23633 2782254 C18H30O2 9E12Z15Z-octadecatrienoic acid77 23946 2401161 C16H16O2 Dimethylstilbestrol82 25069 4262032 C18H35O9P Dolichyl phosphate D-mannose

0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Figure 7 Interaction between TAM and FAA in MCF7 and MDA-MB-231 Cells were treated with FA-TAM in a serial dilution for24 48 and 72 h Values are calculated from three independentexperiments lowast119901 lt 005 lowastlowast119901 lt 001 and lowastlowastlowast119901 lt 0001 comparingthe combination of TAM-FAA with TAM alone

9E12Z15Z-octadecatrienoic acid dimethylstilbestrol anddolichyl phosphate D-mannose

4 Discussion

This study represents the cytotoxic activity of FAA on humanbreast carcinoma cancer cell lines Here we have shown

0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ

Figure 8Combination index (CI) of cotreatment with TAMand FAAon MCF7 and MDA-MB-231 Cells were treated with FA-TAM in aserial dilution for 24 48 and 72 h CI value was determined by Chouand Talalay method

the molecular mechanisms of apoptosis induced by FAA inMCF-7 and MDA-MB-231 breast cancer cells Current studyrevealed that FAA inhibits the proliferative activity of MCF-7 and MDA-MB-231 cells It is evident that cells exposed toFAA lose their capability to proliferate in a time- and dose-dependent manner This finding suggests that FAA has the


252

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43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

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385

86420

1208040

121

080604020

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510

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465

Counts versus mass-to-charge (mz) Counts versus mass-to-charge (mz)

Counts versus mass-to-charge (mz)Counts versus mass-to-charge (mz)Counts versus mass-to-charge (mz)

Counts versus mass-to-charge (mz)

Counts versus mass-to-charge (mz) Counts versus mass-to-charge (mz) Counts versus mass-to-charge (mz)





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times106

times106

times105times105times105


times105times105

times105 times105times105


times105times105

times105times105

Figure 9 LC-MS tracings of the major compounds present in methanol extract of FAA

potential to suppress the growth of the breast cancer cellsirreversibly In addition a dramatic reduction in the numberof viable cells suggests that the antiproliferative effect of FAAmay be partly due to its ability to induce cell death Moreover

it has been reported that the FAA extract does not showtoxicity towards the normal cell [10]

The selective preference is fundamentally specific target-ing of tumor cells which serves to enhance the therapeutic


efficacy of cytotoxic drug while at the same time reducingorgan toxicities [14] It is highly desirable to have substanceswhich kill the cancer cells selectively without damaging theadjacent normal cells [15]

Analyses of FAA-treated cells by phase contrast and fluo-rescencemicroscopy are the first evidence demonstrating thatmorphological changes characteristic of apoptotic cell deathwere induced by FAA Apoptotic cells displayed distinctivetypical forms of morphological changes that are widely usedfor the identification of apoptosis [16 17] Visualization ofthe control (untreated) cells by phase contrast microscopeshowed they maintained their original morphology formIn contrast exposure of the cancer cells to FAA for 2448 and 72 h exhibited classical morphologies of apoptosisincluding autophagy and cell rounding due to shrinkageAfter 24 h of treatment where the affected cells emergedautophagy was achieved at maximum In the remaining timeinterval the occurrence of autophagy was not overlappedIt was caused mainly by the fact that the cell tried to gaina delay from cell death by autophagy [18] whereas earlystages of apoptosis which are characterized by the shrinkageof cells and membrane blebbing were clearly observed after48 h The increasing time of exposure to FAA causes thecells to undergo late apoptosisnecrosis The surface changesinto a big single bubble and eventually detached [15] Theapoptosis surface morphology observed was very similar forboth cell types when treated with FAA This implied thatregardless of the induction pathway occurrence of apoptosisis concomitant with the samemorphological alterationsThisis consistent with the well-known highly conserved nature ofapoptosis [19]

The morphological observation by AOPI staining in thecell nuclei of MCF-7 and MDA-MB-231 breast cancer cellsshowed significant morphological alterations when com-pared to untreated control Most AO+ and PIndash apoptotic cellswere noticed with chromatin condensation staining a bright-green colour and nuclearmargination apoptotic granulationand chromatin fragmentation with the appearances of mem-brane blebbing in some samples which can be considered asmoderate apoptosis Increment of cell membrane permeabil-ity was observed after 72 h incubation as evidenced by theapoptotic body separation and presence of reddish-orangecolour due to the binding of AO to denatured DNA whichcan be seen clearly (AO+ PI+) Necrotic (AOndash PI+) cellpopulations appeared very rarely Apoptosis and necrosis arethe two major modes of cell death Apoptosis is the morefavorable mode of cell death because it is a programmedcell death mechanism that does not cause inflammatoryresponses It is triggered and regulated by a variety of cellularsignalling pathways In contrast necrotic cells are charac-terized by vacuolation of the cytoplasm and breakdown ofplasma membrane Due to this the cytoplasmic elementsare dispersed into the extracellular space hence resultingin an acute inflammatory reaction [20] Analysis of the datadisclosed that FAA had the ability to decrease the viabilityof MCF-7 and MDA-MB-231 cancer cells with no obviousnecrotic phenomena implying occurrence of a programmedcell death or induction of cell cycle arrest

The cytotoxicity at the cellular level was assessed bymeasuring the DNA of the cancer cell in terms of distributionin cell cycle phase and ploidy level as it flows through thecytometer [21] Blockade of the cell cycle is considered asan effective strategy for the development of novel cancertherapies [22] Cell cycle analysis of the treated MCF-7revealed that FAA induced an S and G2M phase cell cyclearrest at 24 and 48 h of treatment with an accompanyingdecrease in G1 phase and dose-dependent increase of sub-G0 phase This confirmed that FAA inhibited DNA synthesisand induced a block at the S and G2M phase boundary at 24and 48 h while inducing apoptosis when exposed for longertime whereas FAA arrested cell cycle progression in MDA-MB-231 in S phase only after treatment for 72 h concomitantwith a reduction of the cells presented in G1 phase andinduction of apoptosis by increasing sub-G0phaseThemajorcontrol sites included DNA damage checkpoints (G0G1 Sand G2M) The drug interference with initiation of DNAreplication can permanently arrest the cancer cells at the Sphase Arrest in S phase requires active suppression throughtransactivation of suppressors direct binding of suppressorsto replication machinery and phosphorylation of criticalelements of replication control [23] We noted that FAAdamage DNA and suppress DNA replication and eventuallyarrest the cells in S phase In addition the G2 checkpointallows the cell to repair DNA damage before entering mitosis[24] In MCF-7 cells the result of cell cycle arrest suggeststhat in addition to apoptosis and S phase arrest G2 cell cyclearrest was another factor that contributed to the preferentialcell growth inhibition

Apoptosis confirmation was carried out using the simul-taneous staining of cells with FITC-Annexin-VPI whichallows the discrimination of intact cells (FITCndash PIminus) earlyapoptotic (FITC+ PIminus) late apoptotic (FITC+ PI+) andnecrotic cells (FITCminus PI+) via bivariate analysis [25 26] Inthe earlier events of apoptosismembrane phospholipid phos-phatidylserine (PS) is translocated from the inner to the outerleaflet of the cytoplasmic membrane and thereby exposedthe PS to the external cellular environment Translocationof PS to the outer leaflet is detectable by Annexin-V andcan be quantitated using flow cytometry Plasma membranepermeabilization converts the early apoptotic cells to lateapoptotic cells When the cytoplasmic membrane integrity islost the PI can penetrate to the nucleus and stain the DNA[27 28] Treatment with FAA exhibited that FAA inducedthe exposure of phosphatidylserine on the surface of MCF-7 in dose- and time-dependent manner While in MDA-MB-231 the treatment with low and middle concentrations(IC25 and IC50) of FAA for 24 and 48 h resulted in morepercentages of early apoptotic cells the treatment for a longertime course (72 h) resulted in more cells present in the lateapoptosis stage than early apoptosis These results illustratedinduction of apoptosis through multiple pathway suggestingmany compounds rather than a single component in FAA

Inflammation plays important roles in cancer and cancercells produce many inflammatory mediators [29] AlthoughCaspases 1 and 5 play important roles in both inflammationand apoptosis [30] the role of caspases 1 and 5 in apoptosiswas considered minor compared to the other caspases in


both MCF-7 and MDA-MB-231 cells The caspase activity intreated MCF-7 revealed that FAA increased the activation ofcaspases 8 and 6 in all three used concentrations while inonly one concentration it increased caspase 9 (only in IC75)and caspase 3 (only in IC25) activities The results illustratedthat the apoptotic effects of FAA on MCF-7 cells were viathe activation of caspase-8 which led to the activation ofthe final effector caspase-6 that finally caused apoptosisTheonly soluble cytosolic caspase which could cleave caspase-8is caspase-6 [31] In addition caspase-6 is required for thedownstream activation of caspase-8 [32] Caspase-6 acts asan initiator caspase by processing the initiator caspases 8and 10 during apoptosis [33] On the other hand release ofcytochrome c from mitochondria activates the downstreamcaspases 2 and 9 that are early biomarkers of apoptosis andeventually lead to caspase-3 activation responsible for DNAfragmentation and morphological changes [34] Howevercaspases 8 and 3 can cleave procaspase-2 without priordimerization [35] On the other hand caspase-1 and caspase-5 are called inflammatory caspases but they are also involvedin the hydrolysis of procaspase-3 to the activated caspase-3 [29] It is known that caspase-3 is a key mediator ofnuclease activation that is triggered after the disruptionof the mitochondrial membrane Thus caspase-3 activationdepends on release of cytochrome c [36] Therefore this wasaligned with our results where in the absence of caspase-1 caspase-5 caspase-2 and caspase-9 activities the executorcaspase-3 has been activated by IC25 probably from the otherpathway

In MDA-MB-231 cells between the initiator caspases(caspases 2 8 and 9) caspase-2 in IC50 and caspase-8in IC50 and IC75 caspase-9 in only the IC25 of FAA hadsignificantly higher activity compared to the control Amongthe executioner caspases (6 and 3) only caspase-6 activitywas raised in the IC50 of FAA It can be concluded that indifferent concentrations various caspases are activated Inthe IC50 concentration of FAA initiator caspases 2 and 8resulted in the activation of caspases 6 and 3 respectively InIC25 and IC75 concentrations of FAA none of the executionercaspases 3 or 6 was activated while caspase-9 and caspase-8 were activated in at least one of these concentrationsFirstly the results gave the possibility that apoptosis inMDA-MB-231 cells in these concentrations was not goingthrough the caspase activation pathway More and morestudies have illustrated that apoptosis is not synonymouswith the activation of caspases [37] Secondly maybe theexecutioner caspase-7 was activated in these concentrationsBoth caspases 3 and 7 could be activated by caspases 8 and9 during apoptosis Caspases 3 and 7 have overlapping butalso distinct roles in apoptosis that is cell type and stimulusdependent [38]

Bcl-2 family of genes plays a critical role in the regulationof the apoptotic process initiated at themitochondria [39] andgoverned the mitochondrial outer membrane permeabiliza-tion [40] Given that FAA has been identified as inducers ofapoptosis we sought to detect the Bcl-2mRNA level inMCF-7 and MDA-MB-231 after treatment with FAA The expres-sion of Bcl-2 exhibited an initial strong decrease illustratingthe potential of FAA to affect the transcriptional regulation of

Bcl-2 and the degradation of its mRNA Assessment of Bcl-2levels in both cells upon treatment by FAA also demonstratedsome significant modulations depending on the initial doseof exposureThe increment of FAA concentrations from IC25to IC75 in MCF-7 cells showed a downward trend of Bcl-2 expression On the other hand MDA-MB-231 displayeda dramatic downregulation of Bcl-2 in IC75 of FAA Themechanism of apoptosis inhibition by Bcl-2 is through thesuppression of caspase proteins [41] Caspase-9 induces lossof mitochondrionmembrane concomitant with the Bcl-2 andBcl-xL cleavage and therefore inactivates the antiapoptoticfunctions of these molecules and will potentially disruptthe balance between pro- and antiapoptotic molecules andinduce cell death [42] The Bcl-2 expression observed inMCF-7 cells was aligned with the activity level of caspase-9since downregulation of Bcl-2was concomitant with caspase-9 activity In contrast the result of MDA-MB-231 which wasin an argument with the observation of caspase-9 activitydemonstrated that FAA lead to Bcl-2 downregulation with-out impairing themitochondrial functionswhich confirm theextramitochondrial roles of Bcl-2

Cell cycle regulatory proteins had been considered aspredictive markers in cancers Cyclin-dependent proteinkinases (Cdks) are key signalling molecules in the regulationof cell cycle [43] for entry into mitosis and a core componentin spindle morphogenesis [44] Cdk1 is the only necessaryCdk in the process of cell proliferation through mitosisAs Cdk1 inhibitors effectively arrested tumor cell growthfinding the new Cdk1 inhibitors is a new target in theresearch and development of anticancer drugs [45 46] Ithas been evident that Cdk1 which is overexpressed and haveenhanced kinase activity inmany tumor types is the potentialtargets for cancer therapy [47] FAA extract succeeded inreducing the expression of Cdk1 in both cells in a dose-dependent manner as IC75 showed the significantly lowerexpression in MCF-7 and MDA-MB-231 The inhibition ofG2M progression induced by FAA in MCF-7 suggests thatthe Cdk1-cyclin B complex regulates the G2 to M transitionand resulted in the antiproliferative effect of FAA In contrastalthough downregulation of Cdk1 occurred in MDA-MB-231 only sub-G0G1 phase arrest was observed (in the sameconcentration at 24 h) that demonstrated apoptosis in this cellline Since Cdk1 transcription is controlled by various factorswe hypothesize that treatment with FAA in MDA-MB-231 may affect Cdk1 transcription via inhibition of proteinkinases involved in the upstream regulation of this gene [48]Moreover it has been shown that Cdk1 may be required forapoptosis independent of the cell cycle regulation [49]

The p53 tumor suppressor gene includes various func-tional categories and plays a role in repair of DNA damagecell cycle progression and apoptosis [50] The activation ofspecific p53 response could be determined by the specifictranscriptional targets and roles in subcellular pathways [51]In the current study p53 expression in MCF-7 and MDA-MB-231 was not significantly upregulated The current studydid not exclude the possibility that some of the cells thatwere negative for nuclear p53 expression may have a p53gene mutation [52] About 50 of all cancer cases possessa p53 mutation with loss of its tumor suppressor activities


therefore these cases are resistant to apoptosis and requirednew drugs that act in a p53-independent pathways [53] Thenovel plant extract FAAdemonstrated the ability to kill breastcancer cells by a p53-independent mechanism while it wasassociated with extrinsic or intrinsic signalling pathway anddownregulation of Bcl-2 depending on the concentrations

Since cancer is the result of numerous mutations repo-sition combination of two or more drugs with differentmechanisms of action may lead to enhancing cell death[54] FAA could be useful in synergizing the efficacy ofTAM in bothMCF-7 andMDA-MB231 cellsThe significanceof this finding lies in the fact that besides its therapeuticpotency TAM cause high toxicity to the normal tissues andharmful effects on health and development of primary andsecondary drug resistance limiting its clinical success in thecancer chemotherapy In this regard current report showsthat combination of chemotherapeutic agents with naturallyoccurring dietary supplements is a preferable modality forcancer treatment than single-agent that might also reducesystemic toxicity of the chemotherapeutic agents [55]

LC-MS was utilized to separate and evaluate chemicalconstituents in FAA which indicated the presence of dif-ferent phytochemicals in the respective extract with vari-ous biological actions such as antioxidant and anticanceractivates For instance sulfonamide derivatives tamsulosinreduces prostate smoothmuscle tone and thereby inhibits thedynamic component of the obstruction utilized in prostatecancer [56] Actinodaphnine an aporphine alkaloid has beenshown to possess antitopoisomerases antibacterial antifun-gal and anticancer properties It also possesses antiplateletaggregation and vasorelaxant activity [57] Acetylcarnitineis an acetylated form of carnitine which has antioxidantactivities through direct scavenging free radicals and chelat-ing catalytic metals-promoters of reactive oxidative species(ROS) [58] TrihydroxyvitaminD3 is a product of vitaminD3metabolism several vitamin D analogs are entering clinicaltrials and are showing great promise as potential therapeu-tic agents for the treatment of cancer and other diseases[59] Sphingoid bases may function as chemotherapeutic aswell as chemopreventive agents by preferentially inhibitingcancer cells and eliminating stem cells from which mostbreast cancer cells arise [60] 91215-Octadecatrienoic acid isanother major compound in the studied plant extracts andhas been reported to have antimicrobial antioxidant andanti-inflammatory activities [61] The results show that theoccurrence of the variable compoundsmay be responsible forthe different extent of anticancer activity shown by the testextract

5 Conclusion

In conclusion FAA inhibited cell proliferation MCF-7 andMDA-MB-231 cells through stimulation of cell-specific S andG2M arrest in MCF-7 and S cell cycle phase arrest in MDA-MB-231 accompanied by induction of apoptosis through bothextrinsic and intrinsic apoptotic pathways probably in theresult of biological properties of phytochemicals present inFAA extract In addition the FAA-induced apoptosis in bothcells is likely to be caspase-dependent Bcl-2-dependent and

p53-transcription-independent FAA also exhibited synergis-tic effect when combined with TAM suggesting that it cancomplement current chemotherapeutic treatment

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

The authors acknowledge the Malaysian Genome Institutefor the technical assistance in the use of the BioanalyserSpecial thanks are due to Dr Dehpour from Azad Universityof Ghaemshahr Iran for identification of the plant speciesand voucher number

References

[1] E Azizi M H Abdolmohammadi S Fouladdel A Shafiee GAmin and S M Ghaffari ldquoEvaluation of p53 and BCL-2 genesand proteins expression in human breast cancer T47D cellstreated with extracts of Astrodaucus persicus (Boiss) drudein comparison to tamoxifenrdquo DARU Journal of PharmaceuticalSciences vol 17 no 3 pp 181ndash186 2009

[2] B Pratumvinit T Srisapoomi P Worawattananon N Opartki-attikul W Jiratchariyakul and T Kummalue ldquoIn vitro antineo-plastic effect of Ficus hispida L plant against breast cancer celllinesrdquo Journal ofMedicinal Plants Research vol 3 no 4 pp 255ndash261 2009

[3] A I Elkady O A Abuzinadah N A Baeshen and T RRahmy ldquoDifferential control of growth apoptotic activity andgene expression in human breast cancer cells by extractsderived from medicinal herbs Zingiber officinalerdquo Journal ofBiomedicine and Biotechnology vol 2012 Article ID 614356 14pages 2012

[4] A Saetung A Itharat C Dechsukum C WattanapiromsakulN Keawpradub and P Ratanasuwan ldquoCytotoxic activity ofThaimedicinal plants for cancer treatmentrdquo Songklanakarin J SciTechnol vol 27 no 2 pp 469ndash478

[5] A Afonin S Greene N Dzyubenko and A Frolov Interactiveagricultural ecological atlas of Russia and neighboring coun-tries Economic plants and their diseases pests andweeds 2008

[6] D Mitroi D Anton C Nicu and M Manda ldquoVariability ofdecorative morphological characteristics in the species papaverrhoeas of spontaneous vegetationrdquo South-Western Journal ofHorticulture Biology and Environment vol 1 no 1 pp 21ndash27

[7] A A Dehpour B Babakhani S Khazaei and M AsadildquoChemical composition of essential oil and antibacterial activityof extracts from flower of Allium atroviolaceumrdquo Journal ofMedicinal Plants Research vol 5 no 16 pp 3667ndash3672 2011

[8] A Sengupta S Ghosh and S Bhattacharjee ldquoAllium vegetablesin cancer prevention An overviewrdquo Asian Pacific Journal ofCancer Prevention vol 5 no 3 pp 237ndash245 2004

[9] S Khazaei NM Esa V Ramachandran et al ldquoIn vitro antipro-liferative and apoptosis inducing effect of Allium atroviolaceumbulb extract on breast cervical and liver cancer cellsrdquo Frontiersin Pharmacology vol 8 article no 5 2017

[10] S Khazaei R Abdul Hamid N Mohd Esa et al ldquoPromotionof HepG2 cell apoptosis by flower of Allium atroviolaceum andthemechanismof actionrdquoBMCComplementary andAlternativeMedicine vol 17 no 1 article no 104 2017


[11] S Khazaei V Ramachandran R Abdul hamid et al ldquoFlowerextract of Allium atroviolaceum triggered apoptosis activatedcaspase-3 and down-regulated antiapoptotic Bcl-2 gene inHeLacancer cell linerdquo Biomedicine amp Pharmacotherapy vol 89 pp1216ndash1226 2017

[12] C-C Chang Y-C Liang A Klutz et al ldquoReversal of multidrugresistance by two nordihydroguaiaretic acid derivatives M4Nand maltose-M3N and their use in combination with doxoru-bicin or paclitaxelrdquo Cancer Chemotherapy and Pharmacologyvol 58 no 5 pp 640ndash653 2006

[13] J Zhao K Kelnar and A G Bader ldquoIn-depth analysis showssynergy between erlotinib and miR-34ardquo PLoS ONE vol 9 no2 Article ID e89105 2014

[14] H M M Arafa ldquoPossible contribution of 120573-glucosidase andcaspases in the cytotoxicity of glufosfamide in colon cancercellsrdquo European Journal of Pharmacology vol 616 no 1-3 pp58ndash63 2009

[15] S N Syed Abdul Rahman N Abdul Wahab and S N AbdMalek ldquoIn vitromorphological assessment of apoptosis inducedby antiproliferative constituents from the rhizomes of cur-cuma zedoariardquo Evidence-Based Complementary and Alterna-tive Medicine vol 2013 Article ID 257108 14 pages 2013

[16] H J Brady Apoptosis Methods and Protocols vol 282 HumanaPress New York NY USA 2004

[17] P Moongkarndi N Kosem S Kaslungka O Luanratana NPongpan and N Neungton ldquoAntiproliferation antioxidationand induction of apoptosis by Garcinia mangostana (mangos-teen) on SKBR3 human breast cancer cell linerdquo Journal ofEthnopharmacology vol 90 no 1 pp 161ndash166 2004

[18] L Mazalova J Balvan H Polanska and Z Sladek Occurrenceof cell death in cancer cell line PC-3 after treatment of plumba-gin

[19] S Rello J C Stockert V Moreno et al ldquoMorphological criteriato distinguish cell death induced by apoptotic and necrotictreatmentsrdquo Apoptosis vol 10 no 1 pp 201ndash208 2005

[20] A L Edinger and C BThompson ldquoDeath by design apoptosisnecrosis and autophagyrdquoCurrentOpinion inCell Biology vol 16no 6 pp 663ndash669 2004

[21] R Mathur S K Gupta N Singh S Mathur V Kochupillai andT Velpandian ldquoEvaluation of the effect of Withania som-nifera root extracts on cell cycle and angiogenesisrdquo Journal ofEthnopharmacology vol 105 no 3 pp 336ndash341 2006

[22] S Gu and J He ldquoDaphnoretin induces cell cycle arrest andapoptosis in human osteosarcoma (HOS) cellsrdquoMolecules vol17 no 1 pp 598ndash612 2012

[23] F Borel F B Lacroix and R L Margolis ldquoProlonged arrest ofmammalian cells at the G1S boundary results in permanent Sphase stasisrdquo Journal of Cell Science vol 115 no 14 pp 2829ndash2838 2002

[24] R S DiPaola et al ldquoTo arrest or not to G2-M cell-cycle arrestcommentary re AK Tyagi et al silibinin strongly synergizeshuman prostate carcinomaDU145 cells to doxorubicin-inducedgrowth inhibition G2-M arrest and apoptosis Clin CancerRes 8 3512ndash3519 2002rdquo Clinical cancer research vol 8 no 11pp 3512ndash3519 2002

[25] M H Abdolmohammadi S Fouladdel A Shafiee G AminS M Ghaffari and E Azizi ldquoAntiproliferative and apoptoticeffect of Astrodaucus orientalis (L) drude on T47D humanbreast cancer cell line Potential mechanisms of actionrdquo AfricanJournal of Biotechnology vol 8 no 17 pp 4265ndash4276 2009

[26] W-S Juan H-W Lin Y-H Chen et al ldquoOptimal Percollconcentration facilitates flow cytometric analysis for annexin

Vpropidium iodine-stained ischemic brain tissuesrdquo CytometryPart A vol 81 no 5 pp 400ndash408 2012

[27] T S Hawley andRGHawley FlowCytometry Protocols vol 91Humana Press New York NY USA 2 edition 2004

[28] I K H Poon M D Hulett and C R Parish ldquoMolecular mech-anisms of late apoptoticnecrotic cell clearancerdquo Cell Death ampDifferentiation vol 17 no 3 pp 381ndash397 2010

[29] E Babas M T Ekonomopoulou I Karapidaki A DoxakisG Betsas and Z Iakovidou-Kritsi ldquoIndication of participationof caspase-2 and caspase-5 in mechanisms of human cervicalmalignancyrdquo International Journal of Gynecological Cancer official journal of the International Gynecological Cancer Societyvol 20 no 8 pp 1381ndash1385 2010

[30] Y H Soung E G Jeong C H Ahn et al ldquoMutational analysisof caspase 1 4 and 5 genes in common human cancersrdquoHumanPathology vol 39 no 6 pp 895ndash900 2008

[31] V Cowling and J Downward ldquoCaspase-6 is the direct activatorof caspase-8 in the cytochrome c-induced apoptosis pathwayAbsolute requirement for removal of caspase-6 prodomainrdquoCell Death amp Differentiation vol 9 no 10 pp 1046ndash1056 2002

[32] M Kruidering and G I Evan ldquoCaspase-8 in apoptosis thebeginning of rsquothe endrsquordquo IUBMB Life vol 50 no 2 pp 85ndash902000

[33] J H Cho P Y Lee W-C Son et al ldquoIdentification of thenovel substrates for caspase-6 in apoptosis using proteomicapproachesrdquo BMB Reports vol 46 no 12 pp 588ndash593 2013

[34] M F Abu Bakar M Mohamad A Rahmat S A Burr and JR Fry ldquoCytotoxicity cell cycle arrest and apoptosis in breastcancer cell lines exposed to an extract of the seed kernel ofMangifera pajang (bambangan)rdquoFood andChemical Toxicologyvol 48 no 6 pp 1688ndash1697 2010

[35] M E DelgadoM Olsson F A Lincoln B Zhivotovsky andMRehm ldquoDetermining the contributions of caspase-2 caspase-8and effector caspases to intracellular VDVADase activities dur-ing apoptosis initiation and executionrdquoBiochimica et BiophysicaActa (BBA) -Molecular Cell Research vol 1833 no 10 pp 2279ndash2292 2013

[36] Y Liang C Yan and N F Schor ldquoApoptosis in the absence ofcaspase 3rdquo Oncogene vol 20 no 45 pp 6570ndash6578 2001

[37] L C-M Chiu T-S Ho E Y-L Wong and V E C Ooi ldquoEthylacetate extract of Patrinia scabiosaefolia downregulates anti-apoptotic Bcl-2Bcl-X119871 expression and induces apoptosis inhuman breast carcinomaMCF-7 cells independent of caspase-9activationrdquo Journal of Ethnopharmacology vol 105 no 1-2 pp263ndash268 2006

[38] M Lamkanfi and T-D Kanneganti ldquoCaspase-7 a protease in-volved in apoptosis and inflammationrdquo The International Jour-nal of Biochemistry amp Cell Biology vol 42 no 1 pp 21ndash24 2010

[39] E I Papadopoulos G M Yousef and A Scorilas ldquoCytotoxicactivity of sunitinib and everolimus in Caki-1 renal cancer cellsis accompanied by modulations in the expression of apoptosis-relatedmicroRNAclusters andBCL2 family genesrdquoBiomedicineamp Pharmacotherapy vol 70 no C pp 33ndash40 2015

[40] O Cakir M Pekmez E Cepni B Candar and K FidanldquoEvaluation of biological activities of physalis peruviana ethanolextracts and expression of Bcl-2 genes in HeLa cellsrdquo FoodScience and Technology vol 34 no 2 pp 422ndash430 2014

[41] L Shi H Teng M Zhu et al ldquoPaeoniflorin inhibits nucleuspulposus cell apoptosis by regulating the expression of Bcl-2family proteins and caspase-9 in a rabbitmodel of intervertebraldisc degenerationrdquo Experimental andTherapeuticMedicine vol10 no 1 pp 257ndash262 2015


[42] M Chen A D Guerrero L Huang et al ldquoCaspase-9-inducedmitochondrial disruption through cleavage of anti-apoptoticBCL-2 family membersrdquo The Journal of Biological Chemistryvol 282 no 46 pp 33888ndash33895 2007

[43] R M Golsteyn ldquoCdk1 and Cdk2 complexes (cyclin dependentkinases) in apoptosis A role beyond the cell cyclerdquo CancerLetters vol 217 no 2 pp 129ndash138 2005

[44] S ChenXChen Y-L Xiu K-X Sun andY Zhao ldquoMicroRNA-490-3P targets CDK1 and inhibits ovarian epithelial carcinomatumorigenesis and progressionrdquo Cancer Letters vol 362 no 1pp 122ndash130 2015

[45] M A Al-SharsquoEr and M O Taha ldquoDiscovery of novel CDK1inhibitors by combining pharmacophore modeling QSARanalysis and in silico screening followed by in vitro bioassayrdquoEuropean Journal of Medicinal Chemistry vol 45 no 9 pp4316ndash4330 2010

[46] H-M Guo Y-M Sun S-X Zhang et al ldquoMetabolism andpharmacokinetics of 8-hydroxypiperidinylmethyl-baicalein(BA-j) as a novel selective CDK1 inhibitor in monkeyrdquoFitoterapia vol 107 pp 36ndash43 2015

[47] Y Matthess M Raab R Knecht S Becker and K StrebhardtldquoSequential Cdk1 and Plk1 phosphorylation of caspase-8 trig-gers apoptotic cell death during mitosisrdquo Molecular Oncologyvol 8 no 3 pp 596ndash608 2014

[48] W-I Jang Z-L Lin S H Lee S Namgoong and N-HKim ldquoA specific inhibitor of CDK1 RO-3306 reversibly arrestsmeiosis during in vitro maturation of porcine oocytesrdquo AnimalReproduction Science vol 144 no 3-4 pp 102ndash108 2014

[49] F Hongo N Takaha M Oishi et al ldquoCDK1 and CDK2activity is a strong predictor of renal cell carcinoma recurrencerdquoUrologic Oncology Seminars and Original Investigations vol 32no 8 pp 1240ndash1246 2014

[50] C Balachandran B Sangeetha V Duraipandiyan et al ldquoAflavonoid isolated from Streptomyces sp (ERINLG-4) inducesapoptosis in human lung cancer A549 cells through p53 andcytochrome c release caspase dependant pathwayrdquo Chemico-Biological Interactions vol 224 pp 24ndash35 2014

[51] A A Alshatwi P Subash-Babu and P Antonisamy ldquoViolaceininduces apoptosis in human breast cancer cells through upregulation of BAX p53 and down regulation of MDM2rdquoExperimental and Toxicologic Pathology vol 68 no 1 pp 89ndash97 2016

[52] M Jiang and J Milner ldquoBcl-2 constitutively suppresses p53-dependent apoptosis in colorectal cancer cellsrdquo Genes amp Devel-opment vol 17 no 7 pp 832ndash837 2003

[53] C F Lima M Costa M F Proenca and C Pereira-WilsonldquoNovel structurally similar chromene derivatives with opposingeffects on p53 and apoptosis mechanisms in colorectal HCT116cancer cellsrdquo European Journal of Pharmaceutical Sciences vol72 article no 3202 pp 34ndash45 2015

[54] F C Wong C C Woo A Hsu and B K H Tan ldquoThe Anti-Cancer Activities of Vernonia amygdalina Extract in HumanBreast Cancer Cell Lines Are Mediated through Caspase-Dependent and p53-Independent Pathwaysrdquo PLoS ONE vol 8no 10 Article ID e78021 2013

[55] G Sharma A K Tyagi R P Singh D C F Chan and RAgarwal ldquoSynergistic anti-cancer effects of grape seed extractand conventional cytotoxic agent doxorubicin against humanbreast carcinoma cellsrdquo Breast Cancer Research and Treatmentvol 85 no 1 pp 1ndash12 2004

[56] A Tahmatzopoulos R G Rowland and N Kyprianou ldquoTherole of120572-blockers in themanagement of prostate cancerrdquoExpertOpinion on Pharmacotherapy vol 5 no 6 pp 1279ndash1285 2004

[57] T-J Hsieh T-Z Liu F-J Lu P-Y Hsieh and C-H ChenldquoActinodaphnine induces apoptosis through increased nitricoxide reactive oxygen species and down-regulation of NF-120581B signaling in human hepatoma Mahlavu cellsrdquo Food andChemical Toxicology vol 44 no 3 pp 344ndash354 2006

[58] P F Surai ldquoAntioxidant action of carnitine molecular mecha-nisms and practical applicationsrdquo EC Veterinary Science vol 2pp 66ndash84 2015

[59] W Li J Chen Z Janjetovic et al ldquoChemical synthesis of 20S-hydroxyvitamin D3 which shows antiproliferative activityrdquoSteroids vol 75 no 12 pp 926ndash935 2010

[60] H A Eun C-C Chang and J J Schroeder ldquoEvaluation of sph-inganine and sphingosine as human breast cancer chemothera-peutic and chemopreventive agentsrdquo Experimental Biology andMedicine vol 231 no 10 pp 1664ndash1672 2006

[61] S Mickymaray M S Al Aboody P K Rath P Annamalai andT Nooruddin ldquoScreening and antibacterial efficacy of selectedIndian medicinal plantsrdquo Asian Pacific Journal of TropicalBiomedicine vol 6 no 3 pp 185ndash191 2016

Submit your manuscripts athttpswwwhindawicom

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


a source of vitamins although useful properties of this specieshave not been studied adequately [6] Gas chromatography-mass spectrophotometry study on A atroviolaceum revealedthe presence of various bioactive components in the plantextracts which might be the reason of antioxidant andantibacterial potency and perhaps other biological activ-ities [7] Notably Allium vegetables are rich in flavonolsand organosulfur compounds which have exhibited tumorinhibitory properties in laboratory studies Some compo-nents ofAllium vegetables are reported to block several stagesof carcinogenesis although the underlying mechanisms ofaction are generally unclear [8] The previous studies on Aatroviolaceum bulb extract against breast cervical and livercancers [9] and flower extracts against HepG2 [10] and Hela[11] cells demonstrated potential anticancer activity

In current study methanol extract from flower of Aatroviolaceum (FAA) has been used in scientific research toreveal the therapeutic properties of this plant against twohuman breast cancer cell lines MCF-7 and MDA-MB-231We hypothesized that FAA extract induces anticancer effectsthrough the induction of cell cycle arrest and apoptosis inhuman breast cancer cells Based on this hypothesis the studyaimed to investigate the possible mechanisms of action inMCF-7 and MDA-MB-231 cells

2 Materials and Methods

21 Preparation of Plant Extract The process of extractpreparation was described previously [10] Fresh flowers ofA atroviolaceum were collected in July of 2013 from KojurIran (latitude = 363825 longitude = 517106 Lat = 36 degrees230 minutes north Long = 51 degrees 426 minutes east)The plant was compared with the voucher specimen number720ndash722 deposited at the Faculty of BiologyHerbarium AzadUniversity of Ghaemshahr Iran Harvested fresh flowerswere rinsed and air dried at room temperature The driedmaterial was homogenized to obtain a coarse powder andextracted by 70 methanol (CheMarCo Inc SC USA) for6 h using Soxhlet apparatus (Electrothermal Eng RochfordUK) The extract was concentrated in a rotary evaporator(Buchi Labortechnik AG Flawil Switzerland) The resultantcondensate was subjected to freeze-drying (VirTis Bench-Top K freeze dryer NY USA)The stock solution of extractwas prepared by dimethyl sulfoxide (DMSO) (Sigma-AldrichMO USA) prior to usage

22 Cell Lines The human breast cancer cell lines MCF-7 (human hormone-dependent breast cancer cell lineATCC HTB-22) and MDA-MB-231 (human non-hormone-dependent breast cancer cell line ATCC HTB-26) andthe normal 3T3 cell line (mouse embryo fibroblast ATCCCRL-1658) were purchased from American Type CultureCollection (VA USA) Cell lines were grown in tissue cultureflasks at 37∘C 5 CO2 and 90 humidity (IR censoredCO2 incubator) in RPMI-1640 medium (Sigma-AldrichSteinheim Germany) containing 10 fetal bovine serum(Sigma-Aldrich Steinheim Germany) and 100 IUml peni-cillin streptomycin (Sigma-Aldrich Steinheim Germany)The cells were grown confluence which could be observed

under an inverted microscope and subcultured at three tofour days interval

23 MTT Cell Proliferation Test The antiproliferative effectsof FAA on MCF-7 and MDA-MB-231 cells were examinedusing the MTT colorimetric assay Cells were seeded in 96-well plates (TPP Switzerland) at a density of 106 cellsml for24 h before exposure to the indicated concentrations of FAAfor 24 48 and 72 hMedia without FAA extract were used as anegative control After removal of the medium 20 120583l of MTTreagent (Sigma-Aldrich MO USA) was added to each welland followed by 4 h incubationThe purple formazan crystalswere dissolved in 100 120583l of DMSO The optical density (OD)of each well was measured at 570 nm on FLUOstar Omegamicroplate reader (BMG Labtech Ortenberg Germany) Anexperiment was done in triplicate and cell viability has beencalculated by following equation

Cytotoxicity = OD of treated cellsOD of negative control

times 100 (1)

24 Microscopic Examination To determine the effects ofFAA on the cell morphology the cells were seeded in 6-well plates (106ml) After 24 h 5ml of media containingIC50 concentrations of the FAA was added while the controlcontained untreated media After 24 48 and 72 h the cellswere then washed with PBS and observed by OlympusCulture Microscope model CK40 (Olympus CorporationTokyo Japan)

Moreover acridine orange (AO) and propidium iodide(PI) double staining was applied to observe the apoptoticfeature of the cells by fluorescent microscope MCF-7 andMDA-MB-231 cells were seeded at density of 5 times 106 cellswelland incubated for 24 h then the medium was replaced withIC50 concentration of FAA dissolved in the medium and thecells were incubated for 24 48 and 72 h The cell suspensionwas mixed with an equal volume of AOPI (Sigma-AldrichMO USA) staining solution (1 1) The viable apoptotic andnecrotic cells were observed under Leica fluorescence micro-scope DM 2500 (Leica Microsystem Wetzlar Germany)within 30minwith 100xmagnification Images were capturedby using Alpha Imager (Alpha Innotech CA USA)

25 Flow Cytometric Cell Cycle Analysis The effect of FAAon MCF-7 and MDA-MB-231 cell cycle distribution wasdetermined by flow cytometric analysis Cells were seededat density of 5 times 106 cultured for 24 h prior to treatmentwith IC25 IC50 and IC75 concentrations of FAA exposurefor 24 48 and 72 h The cells were then resuspended in500 120583l PBS prior to being fixed at 70 cold ethanol forat least 2 h at ndash20∘C After centrifuging at 1000 rpm for 10minutes and washing twice with PBS the fixed cells weretreated with 500120583l PIRNase (400 120583l propidium iodide and100 120583l Ribonuclease A) and incubated for 10min at roomtemperature The DNA content per cell was analysed usingBD LSRFortessa Cell Analyzer (Becton Dickinson NJUSA) equipped with 488 nm argon laser light source and630 nm band pass filter for 10000 events per sample Datawas expressed as percentage of cells compared to untreated














0 20 40 60 80 1000

20

40

60

80

100

Concentration (gml)

o

f liv

e cel

ls

24 Ｂ

48 Ｂ

72 Ｂ

(a)

0 20 40 60 80 1000

20

40

60

80

100

Concentration (gml)

o

f liv

e cel

ls

24 Ｂ

48 Ｂ

72 Ｂ

(b)

0 20 40 60 80 1000

20

40

60

80

100

Concentration (gml)

o

f liv

e cel

ls

(c)



3 Results






NM

CC

NF

MB

ML

AB

CC

NM

ML CC

NF

ML AB

A

A

S

AB S

AB

D D

(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)






0

20

40

60

80

100

o

f cou

nted

cells

24 Ｂ 48 Ｂ 72 Ｂ

lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

SubG0G0G1

SG2M

(a)

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowast

lowastlowastlowast


lowastlowastlowast

24 Ｂ 48 Ｂ 72 Ｂ

SubG0G0G1

SG2M

(b)






0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

24 Ｂ 48 Ｂ 72 Ｂ

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

NecrosisLate

EarlyIntact cell

(a)

0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

24 Ｂ 48 Ｂ 72 Ｂ

NecrosisLate

EarlyIntact cell

(b)









MCF7 MDA-MB-2310

20406080

100120140

Caspase-6

Concentration

Casp

ase-

6 ac

tivity

()

lowastlowast


lowastlowast


ControlIC25

IC50IC75

0

20

40

60

80

100

120

Caspase-1

Casp

ase-

1 ac

tivity



lowastlowastlowast


0

20

40

60

80

100

120

Caspase-5

Casp

ase-

5 ac

tivity

()

lowastlowast



020406080

100120140

Caspase-8

Casp

ase-

8 ac

tivity

()


lowastlowastlowast

lowast



0

20

40

60

80

100

120

Caspase-2

Casp

ase-

2 ac

tivity

() lowast


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-9

Casp

ase-

9 ac

tivity

()


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-3

Casp

ase-

3 ac

tivity

()

ControlIC25

IC50IC75

lowast

lowastlowastlowast







minus10

minus8

minus6

minus4

minus2

0

2

4Cdk1

Concentration

Fold

chan

ge

lowast

lowast

MCF7 MDA-MB-231

ControlIC25

IC50IC75


012 Bcl-2

Concentration

Fold

chan

ge

lowast

lowast

lowast

MDA-MB-231

ControlIC25

IC50IC75

minus4

minus3

minus2

minus1

0

1

2

3

4 p53

Concentration

Fold

chan

ge

MCF7 MDA-MB-231

ControlIC25

IC50IC75













0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion


0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ




252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























0 20 40 60 80 1000

20

40

60

80

100

Concentration (gml)

o

f liv

e cel

ls

24 Ｂ

48 Ｂ

72 Ｂ

(a)

0 20 40 60 80 1000

20

40

60

80

100

Concentration (gml)

o

f liv

e cel

ls

24 Ｂ

48 Ｂ

72 Ｂ

(b)

0 20 40 60 80 1000

20

40

60

80

100

Concentration (gml)

o

f liv

e cel

ls

(c)



3 Results






NM

CC

NF

MB

ML

AB

CC

NM

ML CC

NF

ML AB

A

A

S

AB S

AB

D D

(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)






0

20

40

60

80

100

o

f cou

nted

cells

24 Ｂ 48 Ｂ 72 Ｂ

lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

SubG0G0G1

SG2M

(a)

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowast

lowastlowastlowast


lowastlowastlowast

24 Ｂ 48 Ｂ 72 Ｂ

SubG0G0G1

SG2M

(b)






0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

24 Ｂ 48 Ｂ 72 Ｂ

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

NecrosisLate

EarlyIntact cell

(a)

0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

24 Ｂ 48 Ｂ 72 Ｂ

NecrosisLate

EarlyIntact cell

(b)









MCF7 MDA-MB-2310

20406080

100120140

Caspase-6

Concentration

Casp

ase-

6 ac

tivity

()

lowastlowast


lowastlowast


ControlIC25

IC50IC75

0

20

40

60

80

100

120

Caspase-1

Casp

ase-

1 ac

tivity



lowastlowastlowast


0

20

40

60

80

100

120

Caspase-5

Casp

ase-

5 ac

tivity

()

lowastlowast



020406080

100120140

Caspase-8

Casp

ase-

8 ac

tivity

()


lowastlowastlowast

lowast



0

20

40

60

80

100

120

Caspase-2

Casp

ase-

2 ac

tivity

() lowast


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-9

Casp

ase-

9 ac

tivity

()


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-3

Casp

ase-

3 ac

tivity

()

ControlIC25

IC50IC75

lowast

lowastlowastlowast







minus10

minus8

minus6

minus4

minus2

0

2

4Cdk1

Concentration

Fold

chan

ge

lowast

lowast

MCF7 MDA-MB-231

ControlIC25

IC50IC75


012 Bcl-2

Concentration

Fold

chan

ge

lowast

lowast

lowast

MDA-MB-231

ControlIC25

IC50IC75

minus4

minus3

minus2

minus1

0

1

2

3

4 p53

Concentration

Fold

chan

ge

MCF7 MDA-MB-231

ControlIC25

IC50IC75













0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion


0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ




252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NM

CC

NF

MB

ML

AB

CC

NM

ML CC

NF

ML AB

A

A

S

AB S

AB

D D

(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)

(a1)

(a2)

(a3)

(a4)

(b1)

(b2)

(b3)

(b4)






0

20

40

60

80

100

o

f cou

nted

cells

24 Ｂ 48 Ｂ 72 Ｂ

lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

SubG0G0G1

SG2M

(a)

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowast

lowastlowastlowast


lowastlowastlowast

24 Ｂ 48 Ｂ 72 Ｂ

SubG0G0G1

SG2M

(b)






0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

24 Ｂ 48 Ｂ 72 Ｂ

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

NecrosisLate

EarlyIntact cell

(a)

0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

24 Ｂ 48 Ｂ 72 Ｂ

NecrosisLate

EarlyIntact cell

(b)









MCF7 MDA-MB-2310

20406080

100120140

Caspase-6

Concentration

Casp

ase-

6 ac

tivity

()

lowastlowast


lowastlowast


ControlIC25

IC50IC75

0

20

40

60

80

100

120

Caspase-1

Casp

ase-

1 ac

tivity



lowastlowastlowast


0

20

40

60

80

100

120

Caspase-5

Casp

ase-

5 ac

tivity

()

lowastlowast



020406080

100120140

Caspase-8

Casp

ase-

8 ac

tivity

()


lowastlowastlowast

lowast



0

20

40

60

80

100

120

Caspase-2

Casp

ase-

2 ac

tivity

() lowast


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-9

Casp

ase-

9 ac

tivity

()


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-3

Casp

ase-

3 ac

tivity

()

ControlIC25

IC50IC75

lowast

lowastlowastlowast







minus10

minus8

minus6

minus4

minus2

0

2

4Cdk1

Concentration

Fold

chan

ge

lowast

lowast

MCF7 MDA-MB-231

ControlIC25

IC50IC75


012 Bcl-2

Concentration

Fold

chan

ge

lowast

lowast

lowast

MDA-MB-231

ControlIC25

IC50IC75

minus4

minus3

minus2

minus1

0

1

2

3

4 p53

Concentration

Fold

chan

ge

MCF7 MDA-MB-231

ControlIC25

IC50IC75













0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion


0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ




252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

o

f cou

nted

cells

24 Ｂ 48 Ｂ 72 Ｂ

lowastlowast

lowast


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast




lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

SubG0G0G1

SG2M

(a)

0

20

40

60

80

100

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

lowastlowast

lowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast


lowastlowast

lowastlowastlowast


lowastlowastlowast

24 Ｂ 48 Ｂ 72 Ｂ

SubG0G0G1

SG2M

(b)






0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

24 Ｂ 48 Ｂ 72 Ｂ

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

NecrosisLate

EarlyIntact cell

(a)

0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

24 Ｂ 48 Ｂ 72 Ｂ

NecrosisLate

EarlyIntact cell

(b)









MCF7 MDA-MB-2310

20406080

100120140

Caspase-6

Concentration

Casp

ase-

6 ac

tivity

()

lowastlowast


lowastlowast


ControlIC25

IC50IC75

0

20

40

60

80

100

120

Caspase-1

Casp

ase-

1 ac

tivity



lowastlowastlowast


0

20

40

60

80

100

120

Caspase-5

Casp

ase-

5 ac

tivity

()

lowastlowast



020406080

100120140

Caspase-8

Casp

ase-

8 ac

tivity

()


lowastlowastlowast

lowast



0

20

40

60

80

100

120

Caspase-2

Casp

ase-

2 ac

tivity

() lowast


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-9

Casp

ase-

9 ac

tivity

()


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-3

Casp

ase-

3 ac

tivity

()

ControlIC25

IC50IC75

lowast

lowastlowastlowast







minus10

minus8

minus6

minus4

minus2

0

2

4Cdk1

Concentration

Fold

chan

ge

lowast

lowast

MCF7 MDA-MB-231

ControlIC25

IC50IC75


012 Bcl-2

Concentration

Fold

chan

ge

lowast

lowast

lowast

MDA-MB-231

ControlIC25

IC50IC75

minus4

minus3

minus2

minus1

0

1

2

3

4 p53

Concentration

Fold

chan

ge

MCF7 MDA-MB-231

ControlIC25

IC50IC75













0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion


0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ




252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

24 Ｂ 48 Ｂ 72 Ｂ

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

NecrosisLate

EarlyIntact cell

(a)

0

20

40

60

80

100

Concentration

Cel

l pop

ulat

ion

()

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

Con

trol

IC25

IC50

IC75

24 Ｂ 48 Ｂ 72 Ｂ

NecrosisLate

EarlyIntact cell

(b)









MCF7 MDA-MB-2310

20406080

100120140

Caspase-6

Concentration

Casp

ase-

6 ac

tivity

()

lowastlowast


lowastlowast


ControlIC25

IC50IC75

0

20

40

60

80

100

120

Caspase-1

Casp

ase-

1 ac

tivity



lowastlowastlowast


0

20

40

60

80

100

120

Caspase-5

Casp

ase-

5 ac

tivity

()

lowastlowast



020406080

100120140

Caspase-8

Casp

ase-

8 ac

tivity

()


lowastlowastlowast

lowast



0

20

40

60

80

100

120

Caspase-2

Casp

ase-

2 ac

tivity

() lowast


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-9

Casp

ase-

9 ac

tivity

()


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-3

Casp

ase-

3 ac

tivity

()

ControlIC25

IC50IC75

lowast

lowastlowastlowast







minus10

minus8

minus6

minus4

minus2

0

2

4Cdk1

Concentration

Fold

chan

ge

lowast

lowast

MCF7 MDA-MB-231

ControlIC25

IC50IC75


012 Bcl-2

Concentration

Fold

chan

ge

lowast

lowast

lowast

MDA-MB-231

ControlIC25

IC50IC75

minus4

minus3

minus2

minus1

0

1

2

3

4 p53

Concentration

Fold

chan

ge

MCF7 MDA-MB-231

ControlIC25

IC50IC75













0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion


0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ




252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MCF7 MDA-MB-2310

20406080

100120140

Caspase-6

Concentration

Casp

ase-

6 ac

tivity

()

lowastlowast


lowastlowast


ControlIC25

IC50IC75

0

20

40

60

80

100

120

Caspase-1

Casp

ase-

1 ac

tivity



lowastlowastlowast


0

20

40

60

80

100

120

Caspase-5

Casp

ase-

5 ac

tivity

()

lowastlowast



020406080

100120140

Caspase-8

Casp

ase-

8 ac

tivity

()


lowastlowastlowast

lowast



0

20

40

60

80

100

120

Caspase-2

Casp

ase-

2 ac

tivity

() lowast


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-9

Casp

ase-

9 ac

tivity

()


lowastlowastlowast


0

20

40

60

80

100

120

Caspase-3

Casp

ase-

3 ac

tivity

()

ControlIC25

IC50IC75

lowast

lowastlowastlowast







minus10

minus8

minus6

minus4

minus2

0

2

4Cdk1

Concentration

Fold

chan

ge

lowast

lowast

MCF7 MDA-MB-231

ControlIC25

IC50IC75


012 Bcl-2

Concentration

Fold

chan

ge

lowast

lowast

lowast

MDA-MB-231

ControlIC25

IC50IC75

minus4

minus3

minus2

minus1

0

1

2

3

4 p53

Concentration

Fold

chan

ge

MCF7 MDA-MB-231

ControlIC25

IC50IC75













0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion


0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ




252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minus10

minus8

minus6

minus4

minus2

0

2

4Cdk1

Concentration

Fold

chan

ge

lowast

lowast

MCF7 MDA-MB-231

ControlIC25

IC50IC75


012 Bcl-2

Concentration

Fold

chan

ge

lowast

lowast

lowast

MDA-MB-231

ControlIC25

IC50IC75

minus4

minus3

minus2

minus1

0

1

2

3

4 p53

Concentration

Fold

chan

ge

MCF7 MDA-MB-231

ControlIC25

IC50IC75













0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion


0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ




252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























0

5

10

15

Time (h)

IC50

MCF7 MDA-MB-231

TAMTAM + FAA

24 48 72 24 48 72

lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast



4 Discussion


0

1

2

3

Concentration

Com

bina

tion

inde

x (C

I)

MCF7 MDA-MB-231

IC50

IC75

IC12

5

IC25

IC50

IC75

IC12

5

IC25

24 Ｂ

48 Ｂ

72 Ｂ




252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















252

151

050

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

295

290

43210

86420

43210

45

3210

45

5

3210

43210

43210

4

6

2

0

140

160

180

200

220

240

260

280

300

320

340

252

151

050

252

151

050

252

151

050

252

151

050

252

151

050

253

2151

050

252

151

050

2151

050

320

325

330

335

340

345

350

355

360

365

370

375

380

385

86420

1208040

121

080604020

180

200

220

240

260

280

300

320

340

360

380

400

420

440

355

360

365

370

375

380

385

390

395

400

405

410

415

420

400

405

410

415

420

425

430

435

440

445

450

455

460

465

445

450

455

460

465

470

475

480

485

490

495

500

505

510

400

405

410

415

420

425

430

435

440

445

450

455

460

465









400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

400

405

410

415

420

425

430

435

440

445

450

455

460

465

470

445

450

455

460

465

470

475

480

485

490

495

500

505

510

200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600 200

300

400

500

600

700 800

900

1000

1100

1200

1300

1400

1500

1600280

285

290

295

300

305

310

315

320

325

330

335

340

345

255

260

265

270

275

280

285

290

295

300

305

310

315

320

380

385

390

395

400

405

410

415

420

425

430

435

440

445

450

240

245

250

255

260

265

270

275

280

285

290

295

30 0 30 5

250

255

260

265

270

275

280

285

290

295

300

305

310

315

210

215

220

225

230

235

240

245

250

255

260

265

270

275

395

400

405

410

415

420

425

430

435

440

445

450

455

460

times106

times106



times105times105



times105times105

times105times105























5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































5 Conclusion





Acknowledgments


References






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















ResearchArticle - Hindawi Publishing...

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