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Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 939810 14 pageshttpdxdoiorg1011552013939810

Research ArticleA Phenylbutenoid Dimercis-3-(3101584041015840-Dimethoxyphenyl)-4-[(E)-31015840101584010158404101584010158401015840-Dimethoxystyryl]Cyclohex-1-ene Exhibits Apoptogenic Properties in T-AcuteLymphoblastic Leukemia Cells via Induction of p53-IndependentMitochondrial Signalling Pathway

Theebaa Anasamy1 Ahmad Bustamam Abdul1 Mohd Aspollah Sukari2

Siddig Ibrahim Abdelwahab34 Syam Mohan3 Behnam Kamalidehghan3

Mohd Zulkhairi Azid2 Nabilah Muhammad Nadzri1 A Reenaa Joys Andas1

Ng Kuan Beng1 A Hamid A Hadi5 and Heshu Sulaiman Rahman16

1 UPM-MAKNA Cancer Research Laboratory Institute of Bioscience University Putra Malaysia 43400 SerdangSelangor Malaysia

2 Department of Chemistry Faculty of Science University Putra Malaysia 43400 Serdang Selangor Malaysia3 Department of Pharmacy Faculty of Medicine University of Malaya 50603 Kuala Lumpur Malaysia4Medical Research Center Faculty of Medicine Jazan University Jazan PO Box 114 Saudi Arabia5 Faculty of Science University of Malaya 50603 Kuala Lumpur Malaysia6Department of Microbiology and Pathology Faculty of Veterinary Medicine University Putra Malaysia43400 UPM Serdang Selangor Malaysia

Correspondence should be addressed to Ahmad Bustamam Abdul ahmadbstmmyahoocom

Received 1 October 2012 Accepted 23 January 2013

Academic Editor Tanawan Kummalue

Copyright copy 2013 Theebaa Anasamy 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

The current studywas designed to evaluate the in vitro cytotoxicity effect of a phenylbutenoid dimer cis-3-(3101584041015840-dimethoxyphenyl)-4-[(E)-3

101584010158401015840

4101584010158401015840

-dimethoxystyryl]cyclohex-1-ene (ZC-B11) isolated from the rhizome of Zingiber cassumunar on various cancer cellline and normal humanbloodmononuclear cells and to further investigate the involvement of apoptosis-related proteins that leadsto the probable pathway in which apoptosis is triggered Cytotoxicity test using MTT assay showed selective inhibition of ZC-B11towards T-acute lymphoblastic leukemia cells CEMss with an IC

50value of 711 plusmn 0240 120583gmL which did not reveal cytotoxic

effects towards normal human blood mononuclear cells (IC50gt 50 120583gmL) Morphology assessments demonstrated distinctive

morphological changes corresponding to a typical apoptosis ZC-B11 also arrested cell cycle progression at S phase and causes DNAfragmentation in CEMss cells Decline of mitochondrial membrane potential was also determined qualitatively In the apoptosis-related protein determination ZC-B11 was found to significantly upregulate Bax caspase 37 caspase 9 cytochrome c and SMACand downregulate Bcl-2 HSP70 and XIAP but did not affect caspase 8 p53 and BIDThese results demonstrated for the first timethe apoptogenic property of ZC-B11 on CEMss cell line leading to the programmed cell death via intrinsic mitochondrial pathwayof apoptosis induction

1 Introduction

Plants in particular have been used by mankind as a sourceof medicine since the times of yore Evidence on plants with

healing properties exists about 5000 years ago which hadbeen documented since Sumerian civilization [1] In about3000 to 1500 years ago curative treatments using plant-based substances had been practiced and increased widely

2 Evidence-Based Complementary and Alternative Medicine

O

OO

O

H3C

H3C

CH3

CH3

Figure 1 The chemical structure of ZC-B11

in ancient Greece followed by useful knowledge of plantshaving therapeutic values in China India and Tibet 1000to 2000 years ago [2] These pieces ancient knowledge werebrought down from one generation to another and withthe advancement of science and technology plant-deriveddrugs have made massive contributions in various clinicalconditions and had provided important leads against variouspharmacological targets including cancer [3]

Zingiber cassumunar (family Zingiberaceae) is com-monly known as ldquoPlairdquo inThailand and ldquoBonglairdquo inMalaysiaIt has long been used in traditional medicine in Thailandbeing the prime ingredient in massage oil to relieve musclepain In Northeast India oral consumption of the rhizomepaste of Z cassumunar was reported to treat dyspepsia andstomach bloating [4 5] whilst in Malaysia it is used forpostpartum medication

cis-3-(3101584041015840-Dimethoxyphenyl)-4-[(E)-31015840101584010158404101584010158401015840-dime-thoxys-tyryl]cyclohex-1-ene (ZC-B11) is a phenylbutenoid dimerisolated from the rhizomes of Z cassumunar (Figure 1) Todate there have been no reported studies on ZC-B11 isolatedfrom the rhizome of Z cassumunar having antileukemicactivity Therefore the current study was conducted toinvestigate the in vitro antileukemic properties of thiscompound to substantiate its anticancer activity

2 Materials and Methods

21 Compound Isolation and Purification The rhizomes of Zcassumunar were collected from Jogjakarta Indonesia in theyear 2007 Voucher specimen was deposited in Herbariumof Faculty of Pharmacy Gajah Mada University JogjakartaBriefly the finely ground rhizomes of Z cassumunar (sim700 g)were soaked in petroleum ether for 72 hours at roomtemperature The extraction was repeated 3 times to removethe nonpolar organic compounds waxes and fats Extractionwas continued with chloroform ethyl acetate and methanolThe solvents were removed under reduced pressure andcrude extracts were obtained Column chromatography oversilica gel using a stepwise gradient elution system wasutilized to fractionate the petroleum ether extract (25 g)The isolation of the crude extract yielded 64 fractions

Fractions 12-13 showed similar pattern on TLC and werelater combined Purification of this fraction was done bycolumn chromatography using mixture of hexane and ethylacetate as eluent Subfraction B11 was collected from hexaneEtOAc (8 2) and was further washed with hexane andmethanol to give white solid cis-3-(3101584041015840-dimethoxyphenyl)-4-[(E)-31015840101584010158404101584010158401015840-dimethoxystyryl]cyclohex-1-eneThemolecu-lar weight of this compound is 380199 gmol with molecularformula C

24H28O4and melting point 91∘C-92∘C [6] The

compound was sent for infrared (IR) and nuclear magneticresonance (NMR) analyses at the laboratory of spectro-scopic analysis Faculty of Science UPM 1H NMR spectrawere recorded on NMR Bruker Avance 400 spectrometerapparatus 13C NMR spectra were reported at Ac 150MHzinstrument and Electron Impact Mass Spectra (EI-MS)were recorded on Finnigan MAT 31 mass spectrometerwith a MATSPECO data system EI-MS analysis indicatedthe presence of molecular ion peak at mz 380 whichcorresponded to the molecular formula of C

24H28O4 The

spectral data were found to be in good agreement withthe published data [6] No previous studies have beenreported on this compound except for its phytochemicalstructure determination and physicochemical characteriza-tion

cis-3-(3101584041015840-Dimethoxyphenyl)-4-[(E)-31015840101584010158404101584010158401015840-dimethoxystyryl]cyclohex-1-ene IR 120592max (cmminus1 UATR)3017 (=CndashH) 2927 (CndashO) 1589 (C=C) 1509 1458 12331138 1019 853 785 680 614 1H NMR (400MHz CDCl

3)

120575 680 (1H d J = 80Hz H-51015840) 676 (3H brs H-61015840 H-6101584010158401015840H-5101584010158401015840) 673 (1H s H-2101584010158401015840) 670 (1H s H-21015840) 626 (1H dJ = 160Hz H-710158401015840) 581 (1H dt J = 101 36Hz H-1) 599(1H d J = 101 Hz H-2) 559 (1H dd J = 160 90Hz H-810158401015840)386 (3H s) 375 (3H s) 386 (3H s) 383 (3H s) 351 (1Hbr s H-3) 272 (1H m H-4) 222 (2H m H-6) 168 (2Ht J = 70Hz H-5) 13C NMR (100MHz CDCl

3) 120575 1489

(C-3101584010158401015840-OCH3) 1482 (C-4101584010158401015840mdashOCH

3) 1480 (C-31015840mdashOCH

3)

1475 (C-41015840mdashOCH3) 1338 (C-11015840) 1324 (C-810158401015840) 1310 (C-1101584010158401015840)

1280 (C-2) 1285 (C-710158401015840) 1290 (C-1) 1219 (C-61015840) 1187(C-6101584010158401015840) 1137 (C-21015840) 1111 (C-5101584010158401015840) 1103 (C-51015840) 1089 (C-2101584010158401015840)458 (C-3) 426 (C-4) 248 (C-6) 243 (C-5) MS mz (intensity) 380 (M+ 15) 300 (2) 229 (2) 190 (100) 175 (17)159 (80) 144 (17)

22 Cell Lines and Reagents All cancer cell lines wereobtained from American Type Culture Collection (ATCC)RPMI 1640 and Fetal Bovine Serum (FBS) were purchasedfrom PAA (Germany) DMSO penicillin and streptomycinsolution were purchased from Sigma (St Louis MO USA)MTT was purchased from Amresco (USA) Quantum PBLmedia was purchased from PAA (Austria) Phosphate buffersaline was obtained from Invitrogen (Carlsbad USA) Allother chemicals and reagents used were of HPLC grade

23 Cell Culture Cancer cells were cultured in RPMI 1640medium supplemented with 10 FBS and 1 100 unitmLpenicillin and 100 120583gmL streptomycin Cultures were main-tained at 37∘C in a humidified 5 CO

2incubator Experi-

ments were performed at concentration of 200000 cellsmL

Evidence-Based Complementary and Alternative Medicine 3

24 Cell Viability Assay on Cancer Cells T-Acute lym-phoblastic leukemia (CEMss) hepatocellular carcinoma(HepG2) human breast adenocarcinoma (MCF-7) humanbreast carcinoma (MDA-MB-231) and cervical carcinoma(HeLa) were used in this study Cell suspension of eachcell line was plated out into 96-well plates and treatedwith different concentrations (1563 3125 625 125 25 and50120583gmL) of ZC-B11 Control wells included vehicle-treatedcells exposed to 01 (wv) DMSO After 68 h incubationMTT (5mgmL) was added to each well and the plate wasincubated for further 4 h Supernatants were removed beforeadding 100120583L DMSO to solubilise the formazan crystalsformed Absorbance was read at wavelength of 595 nmusing a microplate reader (Tecan Sunrise Basic GroedigAustria) Assay was performed in triplicates to calculate IC

50

values (concentration which inhibits 50 of cellular growth)CEMss cells were also treated with 5-fluorouracil used aspositive control

25 Cytotoxicity of ZC-B11 on Human Blood MononuclearCells The ability of ZC-B11 to act selectively on cancer cellsespecially leukemia was evaluated by comparing the cytotox-icity of this compound towards human blood mononuclearcells Briefly blood was collected into the cell preparationtube containing sodium citrate (BD Vacutainer NJ USA)After collection tube was stood upright for 20min at roomtemperature to allow it to equilibrate and later centrifuged at1200timesg for 20min Mononuclear cells and platelets under-neath the plasma layer were collected using a pipette andtransferred into 15mL centrifuge tube Cells were washedtwice with PBS and cultured in complete Quantum PBLmedia with phytohemagglutinin (PAA Pasching Austria)containing 10 FBS supplemented with 100UmL penicillinand 100 120583gmL streptomycin at 37∘C in 5 CO

2atmosphere

Human blood mononuclear cells were treated at variousconcentrations of ZC-B11 in triplicates and cell viability wasmeasured using MTT assay after 72 h of incubation

26 Microscopic Observation of Cellular Morphology UsingPhase-Contrast-Inverted Microscopy This investigationexamines morphologically if cell death induction isimplicated in ZC-B11-treated CEMss cell CEMss cellswere exposed to 711120583gmL (IC

50) of ZC-BII for 24 48 and

72 h Morphological appearances of treated CEMss cells werecompared with untreated control observed under normalphase contrast inverted microscope Cells were identified asundergoing apoptosis cell death if they display condensednuclear fragmented nuclei andor blebbing

27 ConfocalMicroscopy (AcridineOrange AO and PropidiumIodide and PI Double Staining) Morphological assessmentsof treated and untreated CEMss cells were done using adouble-fluorescent dye staining method Briefly CEMss cellswere treated with IC

50concentration of ZC-BII for 24 48

and 72 h After the treatment period cells were washed twiceusing PBS to remove the remaining media Ten 120583L (10 120583L)of fluorescent dyes (AOPI) containing AO (10 120583gmL)and PI (10 120583gmL) was added into the cellular pellet in

equal volumes Freshly stained cell suspension was thendropped onto glass slides and covered by coverslip Slideswere observed under confocal microscope within 30minbefore the fluorescence colour starts to fade The criteria foridentification are as follows (a) green intact nucleus viablecells (b) dense green areas of chromatin condensation in thenucleus early apoptosis (c) dense orange areas of chromatincondensation late apoptosis and (d) orange intact nucleussecondary necrosis [7]

28 Phosphatidylserine Externalisation Study Phosphatidyl-serine (PS) externalisation study was done using AnnexinVFITC assay kit (AbD Serotec USA) CEMss cells weretreated with ZC-BII at IC

50concentration for 24 48

and 72 h while untreated cells were used as negativecontrol After the treatment period the supernatant wasdiscarded and cells were washed twice using PBS Cellswere resuspended in prediluted binding buffer in 1 4ratio (50mL binding buffer + 150mL distilled water) later5 120583L Annexin VFITC was added into 195 120583L of the cellsuspension mixed well and incubated for further 10minin the dark at room temperature Cells were then washedand resuspended with 190 120583L of pre-diluted binding bufferfollowed by the addition of 10 120583L of the PI solution andanalysed with flow cytometer (BD FACS Canto II USA)

29 Cell Cycle Distribution Analysis A time-dependent studyof cell cycle distribution of CEMss cells treated with IC

50

concentrationZC-BIIwas performed in triplicatesUntreatedcells were used as negative control After the incubationperiod (24 48 and 72 h) cells were washed with PBS Torestore cell integrity fixation of cell population for flowcytometry analysis was performed Briefly cell pellets werefixed with 90 cold ethanol by mixing 700120583L of 90 coldethanol and the resulting cell suspension was kept overnightatminus20∘CThe cell suspensionwas then centrifuged at 850 rpmfor 10 minutes and the supernatant containing ethanol wasremoved The cell pellet was washed using 2mL PBS andlater resuspended with 600120583L of PBS + 10mgmL RNase+ 1mgmL Propidium Iodide (PI) PI can bind to RNAmolecule and thus RNAse enzyme was added in orderto allow PI to bind directly to DNA The cells were thenincubated between 30min to 1 h at 37∘C Finally cell cyclekinetics was examined using flow cytometer (BDFACSCantoII USA) Fluorescence intensity of sub-G

0G1cell fraction

represents apoptotic cell population

210 DNA Fragmentation DNA fragmentation was doneusing Suicide-Track DNA Ladder Isolation Kit (CalbiochemGermany) according to the manufacturerrsquos instructionsBriefly CEMss cells were treated with ZC-BII at IC

50

concentration for 48 h After treatment DNA extractionwhich involves the separation of apoptotic DNA from highmolecular weight chromatin and DNA precipitation wereperformed according to the manufacturerrsquos instructionsDNA gel electrophoresis was done by preparing agarose gel(12) All DNA ladder samples (21120583L each) including DNAmarkers (5120583L) were transferred to clean centrifuge tubes and


each sample was added with Novel Juice (GeneDirex USA)(1-part Novel Juice with 5-part DNA sample) Novel Juice isa nonmutagenic fluorescent reagent (alternative to EthidiumBromide) that produces instant visualization of DNA bandsupon UV illumination of agarose gels All samples were thenloaded onto the gel and the gel was run at approximately 50constant volts until the dye front is 1-2 cm from bottom ofthe gel DNA was then visualized by transillumination withUV light (Biospectrum AC Chemi HR 40 UVP Upland CAUSA) and photographed

211 Qualitative Analysis of Mitochondrial Membrane Poten-tial Mitochondrial membrane potential (MMP) was qual-itatively analysed using Rhodamine 123 (Sigma USA) apositive charged molecule that can accumulate in energizedmitochondria resulting in the decline of fluorescence inten-sity Briefly CEMss cells were treated with ZC-B11 compoundat IC50

concentration for 12 24 48 and 72 h Untreatedcells serve as negative control Cells in different treatmentgroups were adjusted to the same density stained with10 120583gmL Rh123 in the dark followed by rinsing in PBS andphotographed under the fluorescent microscope (OlympusBX60F5 Japan)

212 Human Apoptosis Proteome Profiler Array To deter-mine the probable pathway of apoptosis induction mediatedby ZC-B11 in CEMss cells detection of several apoptosis-related markers was carried out using the Proteome Pro-filer Array (RayBio Human Apoptosis Antibody Array KitRaybiotech USA) according to the manufacturerrsquos instruc-tions Briefly cells were treated with 711 120583gmL of ZC-B11Untreated cells were used as negative control Three hundredmicro gram proteins from each sample were incubated withthe human apoptosis array overnight The apoptosis arraydatawere quantified by scanning themembrane on aBiospec-trumACChemiHR 40 (UVPUpland CAUSA) and analysisof the array image was performed using image analysissoftware according to the manufacturerrsquos instruction

213 Bioluminescent Assay of Caspases 37 8 and 9 Caspase37 8 and 9 activities of treated and untreated CEMss cellswere measured using a Caspase-Glo assay kit (PromegaCorp Madison WI USA) Briefly CEMss cells were seededin a white-walled 96-well plate and treated with ZC-B11at IC50

concentration for 24 48 and 72 h Untreated cellsserved as negative control The Caspase-Glo 37 8 and9 reagents were mixed well and allowed to equilibrate atroom temperature before starting the assayThe 96-well platecontaining cells was removed from the incubator and allowedto equilibrate to room temperature Then 100120583L of Caspase-Glo 37 8 and 9 reagents were added into each well ofthe 96-well plate containing 100 120583L of blank (vehicle only)negative control cells or treated cells in culture mediumContents inside the wells were gently mixed by using aplate shaker at 300ndash500 rpm for 30 seconds and incubatedat room temperature between 30min to 3 h Luminescenceof each sample was measured in a luminescence microplatereader (Infinite M200 PRO Tecan Austria) Concisely the

proluminescent substrate containing the DEVD LETD andLEHD (sequences are in a single-letter amino acid code)was cleaved by caspases 37 8 and 9 respectively After thecaspase cleavage a substrate for luciferase (aminoluciferin)is released which eventually results in the luciferase reactionand the production of luminescent signal

214 Western Blot This analysis was used to investigatethe expression of apoptosis-related proteins which includedBax Bcl-2 and HSP70 CEMss cells were treated with ZC-B11 for 3 6 12 and 24 h Untreated cells serve as negativecontrol Total proteins of cells were extracted with cell lysisbuffer (50mM Tris-HCL pH 80 120mM NaCl 05 NP-40 1mM PMSF) and 40120583g of protein extract was separatedby 10 SDS PAGE and then transferred to a polyvinyli-denedifluoride (PVDF) membrane (Bio-Rad USA) usingsemidry transfer unit (Hoefer TE 70X USA) blocked with5 nonfat milk in TBS-Tween buffer (012M Tris-base15M NaCl 01 Tween20) for 1 h at room temperatureThe PVDF membrane was then incubated with appropriateprimary antibody overnight at 4∘C and then incubated withhorseradish peroxidase-conjugated secondary antibody for30min at room temperature The bound secondary antibodywas detected using peroxidase-conjugated antirabbit anti-body (1 10000) or antimouse antibody (1 10000) followedby its detection using colorimetric method The followingprimary antibodies 120573-actin (1 10000) Bcl-2 (1 1000) Bax(1 1000) and HSP70 (1 1000) were purchased from SantaCruz Biotechnology Inc CA USA

215 Statistical Analysis Data were expressed as mean plusmn SDStatistical analysis was performed using Studentrsquos 119905-test where119875 lt 005 was considered to be statistically significant

3 Results

31 ZC-B11 Showed Potent Antiproliferative Effect on CEMssCells but Does Not Inhibit Human Blood Mononuclear CellsZC-B11 was found to exert the most potent antiprolifera-tive effect towards CEMss cells with IC

50value of 711 plusmn

024 120583gmL followed by HepG2 MCF-7 MDA-MB-231 andHeLa cells with IC

50values of 1765 plusmn 032 120583gmL 2128 plusmn

025 120583gmL 3238plusmn041 120583gmL andgt50120583gmL respectivelyafter 72 h incubation (Table 1) The antiproliferative activitiesof ZC-B11 on CEMss HepG2 MCF-7 andMDA-MB-231 celllines exhibited IC

50values below 30 120583gmL However the

lowest IC50value of ZC-B11 on CEMss suggested preliminar-

ily that ZC-B11 possesses high anticancer activity which wasto be suggested being useful against T-acute lymphoblasticleukemia Thus further experiments throughout this studywere conducted using this cell line

The crucial objective of expanding molecularly targeteddrugs is to improve the efficacy and selectivity of cancertreatment by exploiting the differences between cancer cellsand normal cells [8] Hence the ability of ZC-B11 to actselectively on cancer cells especially leukemia was evalu-ated by comparing the cytotoxicity of this compound onhuman blood mononuclear cells ZC-B11 did not produce


Table 1 Effect of ZC-B11 on different cell types and the effect of 5-fluorouracil on CEMss cell line expressed as IC50 values inMTT assay after72 hours ZC-B11 potently inhibits the growth of T-acute lymphoblastic leukemic cells

Cell line Tissue of human origin Compound IC50 (120583gmL)ZC-B11 5-Fluorouracil

CEMss T-Acute lymphoblastic leukemia 711 plusmn 0240 154 plusmn 0035

MCF-7 Human breast adenocarcinoma 2128 plusmn 0251 mdashMDA-MB-231 Human breast carcinoma 3238 plusmn 0412 mdashHepG2 Hepatocellular carcinoma 1765 plusmn 0323 mdashHeLa Cervical carcinoma gt50 mdashmdash Human blood mononuclear cells gt50 mdash

Table 2 Flow cytometric analysis of Annexin VFITC assay in CEMss cells treated with IC50 concentration of ZC-B11 for 6 12 24 and 48hours Untreated cells serve as negative control Data were represented as means plusmn SD of at least three independent experiments

Number of cells () plusmn SDUntreated 6 12 24 48

Viable 9730 plusmn 042 9680 plusmn 035 9520 plusmn 057 8670 plusmn 106 8320 plusmn 078

Early apoptosis 220 plusmn 021 300 plusmn 014 380 plusmn 042 1280 plusmn 113lowast

1560 plusmn 120lowast

Late apoptosissecondarynecrosis 050 plusmn 021 030 plusmn 049 060 plusmn 007 110 plusmn 021 120 plusmn 043

lowastindicates a significant difference from the control (119875 lt 005)

any cytotoxic effect on human blood mononuclear cells upto the concentration of 50120583gmL 5-Fluorouracil was used aspositive control and it revealed an inhibitory effect towards T-acute lymphoblasticleukemia cell line (CEMss) with an IC

50

value of 154 plusmn 0035 120583gmL In respect to this the sensitivityof CEMss to 5-fluorouracil correlated to theMTT assay resultobtained for ZC-B11

32 ZC-B11 Causes Morphological Changes Related to Apop-tosis The effect of ZC-B11 on the morphology of CEMsscells was analyzed using normal phase contrast invertedmicroscopy at 24 48 and 72 h incubation Microscopicobservation revealed morphological changes in CEMss cellstreated with 711 120583gmL of ZC-B11 in a time-dependent man-ner ZC-B11-treated CEMss cells showed blebbing of the cellmembrane and shrinkage of the cells These apoptotic effectswere found to be in a time dependentmanner which correlatewell to the phenomenon of cell-death induction consideringthat the number of blebs formation (cytoplasmic protrusion)increases as apoptosis progresses After 24 h treatment somecells remained healthywhile some cells exhibited cytoplasmicprotrusions (Figure 2(b)) The morphological changes weredistinctively clear in treated CEMss cells after 48 and 72 htreatment with features of prominent growth inhibitionincreased blebbing of the cell membrane and shrinkage ofcells (Figures 2(c) and 2(d)) In contrast untreated cellsshowed typical nonadherent cell morphology and remainedhealthy and confluent throughout the treatment period(Figure 2(a)) In confocal microscopy aided with acridineorange and propidium iodide double staining early apoptosisfeatures such as blebbing and chromatin condensation wereseen obviously in treated CEMss cells while untreated cellsshowed even distribution of the acridine orange stain as greenintact nucleus (denotes healthy cells) with well-preservedmorphology (Figure 3(a)) After 24 h exposure to ZC-B11

blebbing of the cell membrane and dense green nucleuswhich indicate nuclear chromatin condensation were notice-able (Figure 3(b)) The apoptotic characteristics of CEMssbecame more apparent at 48 h of treatment (Figure 3(c))and prominent at 72 h (Figure 3(d)) where most of the cellsexhibited dense green nucleus and blebbing compared tountreated and 24 h treatment Both early apoptosis features(blebbing and chromatin condensation) and late phases ofapoptosis which specify presence of intense reddish-orangecolour due to acridine orange binding to denatured DNAwere observed after 48 and 72 h treatment Apoptotic cellsundergoing secondary necrosis were also detected after 72 hof treatment This provides qualitative evidence to proof thatZC-B11 induces apoptosis in treated CEMss cells AcridineOrange (AO) and Propidium Iodide (PI) are intercalatingnucleic acid-specific fluorochromes which emit green andorange fluorescences respectively when bound to DNAOnly AO can cross the plasma membrane of viable andearly apoptosis cells This criterion of cell morphology iden-tification according to the fluorescence colour density todistinguish apoptosis was previously reported by Ciapetti etal [7]

33 ZC-B11 Induces Phosphatidylserine Externalisation inCEMss Cells In this current investigation PS externalisationof CEMss cells undergoing apoptosis was identified usingAnnexin V-FITC assay according to the manufacturerrsquosinstructions The Annexin V-FITC assay apparently showedinduction of early apoptosis in CEMss cells treated with ZC-B11 in a time-dependent manner (Table 2) The exposuretime chosen for this experiment was 6 12 24 and 48 h forthe purpose of an accurate detection of early apoptotic cellsFor untreated control 973 of cells were viable (AnnexinVnegative PInegative) 22 of cells were in early apop-tosis stage (Annexin Vpositive PInegative) while 05


(a)

BL

CS

(b)

BLCS

CS

(c)

BL

CSCS

CSCS

(d)

Figure 2 Normal phase contrast inverted micrograph of CEMss cells treated withZC-B11 (IC50) for 24 48 and 72 h (a) Control (b) most

of the cells exhibit normal morphology while some cells show cytoplasmic protrusions (24 h) (c) clear apoptogenic morphology such asblebbing and cell shrinkage observed (48 h) and (d) prominent growth inhibition blebbing of the cell membrane and shrinkage of cellsobserved (72 h) BL blebbing of the cell membrane CS cell shrinkage (400x magnification)

were in the late apoptosis (Annexin Vpositive PIpositive)and dead stage (Annexin Vnegative PIpositive) After 6 hof treatment the viable cells decreased to 968 whileearly apoptosis cells were only 3 Viable cells decreasedgradually to 952 867 and 832 after 12 24 and48 h of incubation respectively On the other hand earlyapoptosis cells increased from 3 at 6 h treatment to 38128 and 156 at 12 24 and 48 h respectively (Table 2)The results obtained clearly indicate that ZC-B11 is able toinduce apoptosis and simultaneously exhibit clear apoptosismorphological changes attributed to the induction of apop-tosis reported previously using phase contrast inverted andconfocal microscopy studies

34 ZC-B11 Arrests the Cell Cycle at S-Phase and Induces Apop-tosis As depicted in Figures 4 and 5 there is a significant Sphase arrest in a time-dependent manner as the number ofcells increased significantly from 4243 (untreated control)to 5216 after 24 h of treatment followed by 5413 and6151 for 48 and 72 h of treatment respectively The cells insub-G1G0 phase also increased significantly (119875 lt 005) from001 (untreated control) to 2716 after 48 h of treatmentThese cells are considered as apoptotic cells as the ldquosub-G1G0rdquo peak in DNA histogram denotes hypodiploid DNA

content Subsequently the cells in the G0G1 phase alsodecreased significantly from 4678 (untreated control) to4325 and 3255 after 48 and 72 h of treatment respec-tively promoting cell cycle arrest at S phase

35 ZC-B11 Triggers DNA Fragmentation Which Is the Hall-mark of Apoptosis In the current study the formation ofDNA fragmentation in CEMss cells treated at IC

50con-

centration of ZC-B11 was detected on a 12 agarose gelelectrophoresis after 48 h treatment (Figure 6) FragmentedDNA was clearly observed in treated CEMss cells whilstthe untreated control did not show evidence of laddersThus it is possible that the compound ZC-B11 triggeredapoptosis in CEMss cells as the chromosomal DNA cleavageinto oligonucleosomal size fragments is an integral part ofapoptosis induction

36 ZC-B11 Causes Decline in Mitochondrial MembranePotential In the current study mitochondrial membranepotential (MMP) was assessed by the retention of Rh123 aspecific fluorescent cationic dye that is readily sequesteredby active mitochondria [9 10] Uptake of Rh123 by CEMsscells treated with ZC-B11 was observed qualitatively usingfluorescentmicroscopyThefluorescent intensity of theRh123


VI

VI

20 120583m

(a)

BL

BLCC

20 120583m

(b)

20 120583m

BL AB

SN

(c)

CC

BL

LA

SN

AB

20 120583m

(d)

Figure 3 Confocalmicrograph of acridine orange and propidium iodide double-stainedCEMss cells after 24 48 and 72 h treatmentwith ZC-B11 (IC

50) (a) Control (b) cells exhibit blebbing of the cell membrane and bright green nucleus showing condensation of chromatin (24 h) (c)

blebbing was observed with some orange-coloured cells which denotes late apoptosis (48 h) and (d) more blebbing and late apoptosis orangecolour represents the hallmark of late apoptosis while red color represents secondary necrosis or dead cells (72 h) VI viable cells BL blebbingof the cell membrane CC chromatin condensation AB apoptotic body LA late apoptosis SN secondary necrosis (400x magnification)

dye decreased sequentially in a time dependent manner from12 h to 72 h of treatment whilst untreated cells revealedmaximal dye uptake (Figures 7(a)ndash7(e))The result suggestedthat ZC-B11 disrupts theMMPofCEMss cells after treatment

37 ZC-B11 Upregulates Bax Caspase 3 Cytochrome c andSMAC Downregulates Bcl-2 HSP70 and XIAP but Did NotAffect Caspase 8 p53 and BID To further evaluate themech-anisms of apoptosis induction by ZC-B11 towards CEMsscells screening of several proteins implicated to apoptosisinduction was done using the human apoptosis proteomeprofiler array Bax caspase 3 cytochrome c and SMACshowed significant increase (119875 lt 005) compared to untreatedcontrol cells whilst proteins such as Bcl-2 HSP70 and XIAPdecreased significantly compared to untreated control cells(Figure 8) On the other hand caspase 8 p53 and BID donot show significant difference from untreated control cells

The upregulation of Bax cytochrome c caspase 3 and thedownregulation of Bcl-2 suggest that the compound inducesapoptosis in CEMss via intrinsic pathway This is furtherconfirmedwith unchanged levels of caspase 8 and BID whichplays important role in extrinsic pathway of apoptosis Induc-tion of apoptosis is independent of p53 as the expression ofthis protein remained at basal level after treatment Increasedlevels of cytochrome c and SMAC correlate well with thedecline of mitochondrial membrane potential as mentionedearlier An alteration in the permeability of mitochondrialmembranes promotes translocation of the mitochondrialapoptogenic proteins which included SMAC (an inhibitorof XIAP) and cytochrome c (an activator of caspase 9) intothe cytoplasm Interestingly there was significant decline inthe level of XIAP (apoptosis inhibitor protein) suggestingpossible inhibition of this protein by increased level ofSMAC Hence the evidence gathered by the present studyproposed that SMAC acts as a proapoptotic protein that


0

200

400

600

0 20 40 60 80 100

Num

ber

I II III IV

Channels (PI-A)

(a)

0

200

400

600

800

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(b)

0

100

200

300

400

600

500

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(c)

0

200

400

600

0 20 40 60 80 100

Channels (PI-A)

I II III IVN

umbe

r

(d)

Figure 4 Flow cytometric analysis of cell cycle phase distribution of CEMss cells treated with ZC-B11 (IC50) in a time-dependent manner

(a) Control (b) 24 h (c) 48 h and (d) 72 h Region I is ldquosub-G0G1rdquo peak denoting apoptotic cells with hypodiploid DNA content Region IIis ldquoG0G1rdquo phase Region III is S phase and Region IV is ldquoG2Mrdquo phase

binds and neutralizes the activity of XIAP Alternatively thelevel of HSP70 showed significant decrease compared tothe untreated control HSP70 is an inhibitor of apoptosissince cellular-stress response can mediate cellular protectionthrough the expression of HSP70 which in turn can interferewith the induction of apoptotic cell death hence resultingin tumour cells often expressing elevated levels of HSP70[11 12] A previous study has found that HSP70 inhibitsapoptosis downstream of cytochrome c release and upstreamof caspase3 activation [13] Hence in the current studytreatment of ZC-B11 towards CEMss cells decreases HSP70protein activity thus preventing its inhibition on cytochromec release and caspase 3 activation The current findings from

the apoptosis proteome profiler array suggest that ZC-B11induces apoptosis in CEMss cells which is independent ofp53 protein expression and may not involve the extrinsicpathway Further to this ZC-B11 may possibly activate thecaspase cascades in CEMss cells accompanied by the releaseof SMACand the subsequent suppression ofXIAP andHSP70proteins

38 ZC-B11 Increases the Activity of Caspases Involved inIntrinsic Pathway CEMss cells treated with IC

50concen-

tration of ZC-B11 significantly (119875 lt 005) increased theactivities of caspase 37 (Figures 9(a) and 9(c)) in a time


Negative control24 h

48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

Cell cycle phase distribution

Figure 5 Graphical presentation of cell cycle phase distributionanalysis Induction of S phase arrest in the cell cycle progression ofCEMss cells treated with ZC-B11 (IC

50) Results were represented

as means plusmn SD of three independent experiments ldquolowastrdquo indicates asignificant difference from the control (119875 lt 005)

A B C D

Figure 6 Electrophoresis separation of fragmented DNA ofuntreated and treated CEMss cells for 48 hours with ZC-B11 (IC

50)

Lane A negative control (untreated CEMss cells) Lane B 48 hourstreatment Lane C DNA marker Lane D positive control

dependent manner whilst the activity of caspase 8 (Fig-ure 9(b)) remained unchanged throughout the treatmentperiod Caspases 37 and 9 are caspases that are involvedin the intrinsic pathway and this strongly suggests that ZC-B11 induces apoptosis in CEMss cells via intrinsic pathwayThe formation of apoptosome a catalytic multiprotein com-plex consisting of Apaf-1 cytochrome c and procaspase 9within the intrinsic apoptotic pathway activates caspase 9 in

response to the apoptotic signals which contributes later tothe activation of caspase 3 [14 15] In relation to this thecurrent study suggests the possibility of ZC-B11 inducing theformation of apoptosome complex in treated CEMss cellssince this protein complex is implicated in activating bothcaspases 3 and 9 of the intrinsic pathway

39 Western Blotting Confirms the upregulation of Bax anddownregulation of Bcl-2 HSP70 Induced by ZC-B11 ZC-B11 increased the expression of Bax while the expressionof Bcl-2 and HSP70 decreased after treatment in a time-dependent manner compared to untreated control cells 120573-Actinwas used as the internal control to confirmequal sampleloading and protein concentration in all samples The resultsobtained from the Western blot analysis confirmed that ZC-B11 induced up regulation of Bax and down regulation ofBcl-2 and HSP70 proteins in a time dependent manner(Figure 10) and these were concurrent with previous humanapoptosis proteome profiler array results As Bax and Bcl-2are the main orchestrators of apoptosis regulation the abilityto upregulate and downregulate these proteins optimally toinduce apoptosis is a crucial attribute for an anticancer agentand ZC-B11 has demonstrated this capability when used totreat CEMss cells

4 Discussion and Conclusion

Over the last decade countless studies have revealed that theresponse to current cancer therapies crucially depends onfunctional cell death pathways in cancer cells Recognition ofkey regulators of apoptosis in childhood cancers has providedthe basis for the advancement of experimental strategiesaiming at restoring intact cell death programs in cancer cells[16]The present study elucidates themechanism of apoptosisprovoked by ZC-B11 in CEMss cells

The antiproliferative assay used in this study is the MTTassay an in vitro tetrazolium-based colorimetric assay Themethodwas first described byMosmann in 1983 for detectingmammalian cell survival and proliferation [17] MTT (3-[45-dimethylthiazol-2-yl]-25-diphenyltetrazolium bromide) is awater soluble tetrazolium salt which is reduced to an insol-uble formazan product by cleavage of the tetrazolium ringby succinate dehydrogenase enzymewithin themitochondriaof metabolically active cells [18] The amount of formazangenerated is directly proportional to the viable cell numberwhen using homogenous cell populationsThis technique hassince been accepted largely for its accuracy and speediness inquantifying cell survival and proliferation [19 20] It is wellacknowledged that different cell lines demonstrate differentsensitivities to cytotoxic compounds [21 22] The use ofmore than one cell line is therefore necessary in screeningthe antiproliferative activity of ZC-B11 The five differentcancer cell lines used are from four different origins (bloodbreast liver and cervix) that possess different morphologyand tumorigenic properties [22] The data obtained in thisexperiment demonstrated the antiproliferative effects of ZC-B11 on CEMss cell line selectively without affecting thenormal blood mononuclear cells


20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)

Figure 7 Fluorescent micrograph of CEMss cells treated with ZC-B11 (IC50) for 12 24 48 and 72 h stained with Rh123 dye (a) Control (b)

12 h (c) 24 h (d) 48 h and (e) 72 h (400x magnification)

The antileukemic activities of ZC-B11 were furtherestablished using various microscopic analyses and AOPIstaining which showed distinctive morphological changescorresponding to typical apoptosis features such as chro-matin condensation DNA fragmentation cell membraneblebbing and separated apoptotic bodies The inclusion ofPS externalization study and DNA fragmentation analysisfurther confirmed the induction of apoptosis as the eventof causing cell death towards CEMss cells Annexin V

is a 35-kDa Ca2+-binding protein initially described byReutelingsperger et al (1985) as a vasculature-derived pro-tein with strong anticoagulant properties [23] Annexin Vbinds preferentially to PS which is normally absent in theouter leaflet of the plasma membrane and is only exposedon the cell surface upon induction of apoptosis Once onthe cell surface it can be specifically detected by stainingwith fluorescein isothiocyanate-(FITC-) labeled annexin V(annexin V-FITC) a protein with high affinity for PS This


Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID

minus2000 minus1500 minus1000 minus500 0 500 1000 1500 2000

Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70

Figure 8 Human apoptosis proteome profiler array in CEMss cells treated with ZC-B11 (IC50) for 48 hours Graph shows the difference

between treated and untreated control cells Results were represented as means plusmn SD for three independent experiments ldquolowastrdquo indicates asignificant difference from the control (119875 lt 005)

occurs specifically in early phases of apoptotic cell deathduring which the cell membrane itself remains intact [24 25]Fragmentation of DNA also appears to be part of an earlyevent in apoptosis prior to the complete digestion of DNAintomultiples of nucleosomal size fragments Demonstrationof internucleosomal DNA fragmentation has been the majoradvance in the detection of apoptosis induction [26] DNAfragments are detectable as a ladder pattern in gel elec-trophoresis of isolated DNA Necrosis on the other hand ischaracterized by accidental DNA fragmentation which formsa ldquosmearrdquo on agarose gels [27]

ZC-B11 also causes DNA damage in CEMss cells byarresting the cell cycle at S phase This is in accordance toseveral studies elsewhere that reported compounds isolatedfrom natural products which arrested cell cycle at S phasesimilarly induce apoptosis Triptolide a diterpenoid obtainedfrom Tripterygum wilfordii Hook if was reported to induceS phase cell cycle arrest in human melanoma cells andapoptosis that probably is induced through the intrinsicpathway [28] Resveratrol a polyphenolic phytoalexin foundin the skin of red grapes various other fruits and rootextracts of the weed Polygonum cuspidatum also inhibitsproliferation causes S-phase arrest and induces apoptosisin acute myeloid leukemia and several other cancer cells[29 30]

Mitochondria are the main producers of ATP in eukary-otic aerobic cells In addition mitochondria also implicatedcell physiology and pathology including involvement in ionshomeostasis regulation of the cell redox state and transportof metabolites including import of proteins synthesized inthe cytosol lipid and amino acid metabolism and cell death

These important functions are extremely dependent on theelectrochemical transmembrane potential of the mitochon-dria [31 32] The disruption of mitochondrial membraneintegrity is one of the early events leading towards irreversibleapoptosis [33] Mitochondrial inner membrane is negativelycharged for being rich of negatively charged glycoprotein Alarge migration of protons out of the inner membrane causesthe transmembrane potential to decrease substantially [34]Decline ofMMP causes leakage of Rh123 frommitochondriaresulting in the decline of fluorescence intensity [35] Therate of fluorescence decay is proportional to the decline ofMMP [34] The decline of MMP in ZC-B11-treated CEMsscells was also found to be concomitant with the upregulationof cytochrome c and SMAC which are the key apoptotic pro-teins from mitochondrial intermembrane space Thereforeone possible mechanism by which ZC-B11 induces apoptosisis through changes in the MMP which would lead to therelease of cytochrome c and SMAC from the mitochondrialeading to sequential activation of caspase 9 and caspase 3Furthermore regulation of several apoptosis-related proteinswas also observed in the human proteome profiler arraywhich suggest collectively that ZC-B11 mediates cell death inCEMss via intrinsic pathway of apoptosis These results werelater confirmed by caspase bioluminescent assay and westernblot analysis HSP70 which shows decline in ZC-B11-treatedCEMss cells is a protein that is abnormally expressed in differ-ent malignancies and has emerged as a promising new targetfor anticancer therapy [36] The expression of p53 was foundto be unaffected in ZC-B11-treated CEMss cells which suggesta probable p53-independent pathway of apoptosis Since over50 of human tumours contain a functionally defective p53


450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)

Figure 9 The bioluminescent assay of caspases 37 (a) 8 (b) and 9 (c) in CEMss cells treated with ZC-B11 (IC50) after 24 48 and 72 hours

of treatment Untreated cells serve as negative control (a) (c) Caspase 37 and 9 activities increased significantly (lowast119875 lt 005) compared tountreated control (b) Caspase 8 activity remained at the basal level throughout the treatment period Results were represented as means plusmnSD for three independent experiments

that reduces sensitivity to commonly used chemotherapeuticagents such as etoposide and cisplatin [37] the aptitude ofZC-B11 to induce apoptosis independently of p53 may offeran advantage in anticancer therapy Ultimately this studyhas shown that ZC-B11 possesses antiproliferative propertieson acute lymphoblastic leukemia and is potential to be

developed as an antileukemic and chemotherapy agent as itinduces cell death via apoptosis signalling pathway

However in vivo preclinical studies and possibly furtherproduct development are needed to ascertain its ability andusefulness as a natural drug for the treatment of leukemiaDirect engagements of apoptotic pathways or combinations


0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin

Hour(s) aftertreatment

Figure 10 Western blot analysis of Bax Bcl-2 and HSP70 levels in CEMss cells treated with ZC-B11 (IC50) for 3 6 12 and 24 hours and

compared with negative control (0 hours) The expression of Bax increased while Bcl-2 and HSP70 decreased after treatment in a time-dependent manner 120573-actin was used as the internal control to confirm equal sample loading

of agents that lower the threshold for apoptosis inductionby conventional anticancer agents are alternatives to exploitapoptosis induction pathways for paediatric oncology such asALL [16]These findings will therefore help in understandingthe mechanism by which apoptosis pathway is regulated inZC-B11-treatedALL and consequently provides valuable linksfor developing strategies to improve the efficacy of anticancertherapy

Abbreviations

ALL Acute lymphoblastic leukemiaApaf-1 Apoptotic protease-activating factor-1Bax Bcl-2-associated X proteinBcl-2 B-cell lymphoma 2BID BH3 interacting domain death agonistDMSO Dimethyl sulfoxideEI-MS Electron impact-mass spectraEtOAc Ethyl acetateFITC Fluorescein isothiocyanateHPLC High-performance liquid chromatographyHSP70 Heat shock protein 70IC50 Half-maximal inhibitory concentration

IR InfraredMMP Mitochondrial membrane potentialMTT 3-(45-Dimethylthiazol-2-yl)-25-

diphenyltetrazoliumbromide

NMR Nuclear magnetic resonanceSMAC Second mitochondria-derived activator of

caspaseXIAP X-Linked inhibitor of apoptosis protein

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

The authors thank Universiti Putra Malaysia for renderingfinancial support (RUGS) The authors would also like toexpress their utmost gratitude and appreciation to Universityof Malaya and Ministry of Higher Education (HIR GrantF00009-21001) for providing grant to conduct this study

References

[1] P K Mukherjee S Rai V Kumar K Mukherjee P J Hylandsand R C Hider ldquoPlants of Indian origin in drug discoveryrdquoExpert Opinion on Drug Discovery vol 2 no 5 pp 633ndash6572007

[2] R A Halberstein ldquoMedicinal plants historical and cross-cultural usage patternsrdquo Annals of Epidemiology vol 15 no 9pp 686ndash699 2005

[3] M J Balunas and A D Kinghorn ldquoDrug discovery frommedicinal plantsrdquo Life Sciences vol 78 no 5 pp 431ndash441 2005

[4] Tushar S Basak G C Sarma and L Rangan ldquoEthnomedicaluses of Zingiberaceous plants of Northeast Indiardquo Journal ofEthnopharmacology vol 132 no 1 pp 286ndash296 2010

[5] B Saikia S K Borthakur and N Saikia ldquoMedico-ethnobotanyof Bodo tribals in Gohpur of Sonitpur district Assamrdquo IndianJournal of Traditional Knowledge vol 9 no 1 pp 52ndash54 2010

[6] M Kuroyanagi S Fukushima and K Yoshihira ldquoFurthercharacterization of the constituents of a Thai medicinal plantZingiber cassumunar Roxbrdquo Chemical and Pharmaceutical Bul-letin vol 28 no 10 pp 2948ndash2959 1980

[7] G Ciapetti D Granchi L Savarino et al ldquoIn vitro testing ofthe potential for orthopedic bone cements to cause apoptosisof osteoblast-like cellsrdquo Biomaterials vol 23 no 2 pp 617ndash6272002

[8] FM Balis ldquoEvolution of anticancer drug discovery and the roleof cell-based screeningrdquo Journal of the National Cancer Institutevol 94 no 2 pp 78ndash79 2002

[9] S Davis M J Weiss and J R Wong ldquoMitochondrial andplasma membrane potentials cause unusual accumulation and


retention of rhodamine 123 by human breast adenocarcinoma-derived MCF-7 cellsrdquo Journal of Biological Chemistry vol 260no 25 pp 13844ndash13850 1985

[10] H Hong and G Q Liu ldquoProtection against hydrogen peroxide-induced cytotoxicity in PC12 cells by scutellarinrdquo Life Sciencesvol 74 no 24 pp 2959ndash2973 2004

[11] H M Beere B B Wolf K Cain et al ldquoHeat-shock protein 70inhibits apoptosis by preventing recruitment of procaspase-9 tothe Apaf-1 apoptosomerdquo Nature Cell Biology vol 2 no 8 pp469ndash475 2000

[12] Y Q Wei X Zhao Y Kariya K Teshigawara and A UchidaldquoInhibition of proliferation and induction of apoptosis byabrogation of heat-shock protein (HSP) 70 expression in tumorcellsrdquo Cancer Immunology Immunotherapy vol 40 no 2 pp73ndash78 1995

[13] C H Li J S Lee Y G Ko J I Kim and J S Seo ldquoHeatshock protein 70 inhibits apoptosis downstream of cytochromec release and upstream of caspase-3 activationrdquo Journal ofBiological Chemistry vol 275 no 33 pp 25665ndash25671 2000

[14] H Zou Y Li X Liu and X Wang ldquoAn APAf-1 sdot cytochrome Cmultimeric complex is a functional apoptosome that activatesprocaspase-9rdquo Journal of Biological Chemistry vol 274 no 17pp 11549ndash11556 1999

[15] L Czerski and G Nunez ldquoApoptosome formation and caspaseactivation is it different in the heartrdquo Journal of Molecular andCellular Cardiology vol 37 no 3 pp 643ndash652 2004

[16] S Fulda ldquoTargeting apoptosis pathways in childhoodmalignan-ciesrdquo Cancer Letters 2010

[17] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[18] G Fotakis and J A Timbrell ldquoIn vitro cytotoxicity assayscomparison of LDH neutral red MTT and protein assay inhepatoma cell lines following exposure to cadmium chloriderdquoToxicology Letters vol 160 no 2 pp 171ndash177 2006

[19] J M Edmondson L S Armstrong and A O MartinezldquoA rapid and simple mtt-based spectrophotometric assay fordetermining drug sensitivity in monolayer culturesrdquo Methodsin Cell Science vol 11 no 1 pp 15ndash17 1988

[20] S P C Cole ldquoRapid chemosensitivity testing of human lungtumor cells using the MTT assayrdquo Cancer Chemotherapy andPharmacology vol 17 no 3 pp 259ndash263 1986

[21] A Saleem M Husheem P Harkonen and K Pihlaja ldquoInhibi-tion of cancer cell growth by crude extract and the phenolics ofTerminalia chebula retz fruitrdquo Journal of Ethnopharmacologyvol 81 no 3 pp 327ndash336 2002

[22] S Kaur H Michael S Arora P L Harkonen and S KumarldquoThe in vitro cytotoxic and apoptotic activity of Triphala anIndian herbal drugrdquo Journal of Ethnopharmacology vol 97 no1 pp 15ndash20 2005

[23] C P M Reutelingsperger G Hornstra and H C HemkerldquoIsolation and partial purification of a novel anticoagulantfrom arteries of human umbilical cordrdquo European Journal ofBiochemistry vol 151 no 3 pp 625ndash629 1985

[24] G Zhang V Gurtu S R Kain and G Yan ldquoEarly detectionof apoptosis using a fluorescent conjugate of annexin VrdquoBioTechniques vol 23 no 3 pp 525ndash531 1997

[25] G Koopman C P M Reutelingsperger G A M Kuijten RM J Keehnen S T Pals and M H J van Oers ldquoAnnexin Vfor flow cytometric detection of phosphatidylserine expression

on B cells undergoing apoptosisrdquo Blood vol 84 no 5 pp 1415ndash1420 1994

[26] J H Zhang and M Xu ldquoDNA fragmentation in apoptosisrdquo CellResearch vol 10 no 3 pp 205ndash211 2000

[27] J A Collins C A Schandl K K Young J Vesely and MC Willingham ldquoMajor DNA fragmentation is a late event inapoptosisrdquo Journal of Histochemistry and Cytochemistry vol 45no 7 pp 923ndash934 1997

[28] Y Tao M L Zhang P C Ma and J F Sun ldquoTriptolide inhibitsproliferation and induces apoptosis of human melanoma A375cellsrdquo Asian Pacific journal of cancer prevention vol 13 no 4pp 1611ndash1615 2012

[29] Z Estrov S Shishodia S Faderl et al ldquoResveratrol blocksinterleukin-1120573-induced activation of the nuclear transcriptionfactor NF-120581B inhibits proliferation causes S-phase arrest andinduces apoptosis of acute myeloid leukemia cellsrdquo Blood vol102 no 3 pp 987ndash995 2003

[30] A K Joe H Liu M Suzui M E Vural D Xiao and IB Weinstein ldquoResveratrol induces growth inhibition S-phasearrest apoptosis and changes in biomarker expression inseveral human cancer cell linesrdquo Clinical Cancer Research vol8 no 3 pp 893ndash903 2002

[31] G Solaini G Sgarbi G Lenaz and A Baracca ldquoEvaluatingmitochondrial membrane potential in cellsrdquo Bioscience Reportsvol 27 no 1ndash3 pp 11ndash21 2007

[32] D B Zorov N K Isaev E Y Plotnikov et al ldquoThe mitochon-drion as Janus Bifronsrdquo Biochemistry vol 72 no 10 pp 1317ndash1384 2007

[33] J L Scarlett P W Sheard G Hughes E C LedgerwoodH H Ku and M P Murphy ldquoChanges in mitochondrialmembrane potential during staurosporine-induced apoptosisin Jurkat cellsrdquo FEBS Letters vol 475 no 3 pp 267ndash272 2000

[34] A Baracca G Sgarbi G Solaini and G Lenaz ldquoRhodamine123 as a probe ofmitochondrialmembrane potential evaluationof proton flux through F0 during ATP synthesisrdquo Biochimica etBiophysica Acta vol 1606 no 1ndash3 pp 137ndash146 2003

[35] Y Gong and X D Han ldquoNonylphenol-induced oxidativestress and cytotoxicity in testicular Sertoli cellsrdquo ReproductiveToxicology vol 22 no 4 pp 623ndash630 2006

[36] M Kaiser A Kuhnl S Fischer et al ldquoAntileukemic activity oftheHSP70 inhibitor pifithrin-l in acute leukemiardquoBlood CancerJournal vol 1 no 7 article e28 2011

[37] R D Abeysinghe B T Greene R Haynes et al ldquop53-independent apoptosis mediated by tachpyridine an anti-cancer iron chelatorrdquo Carcinogenesis vol 22 no 10 pp 1607ndash1614 2001

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


O

OO

O

H3C

H3C

CH3

CH3

Figure 1 The chemical structure of ZC-B11

in ancient Greece followed by useful knowledge of plantshaving therapeutic values in China India and Tibet 1000to 2000 years ago [2] These pieces ancient knowledge werebrought down from one generation to another and withthe advancement of science and technology plant-deriveddrugs have made massive contributions in various clinicalconditions and had provided important leads against variouspharmacological targets including cancer [3]

Zingiber cassumunar (family Zingiberaceae) is com-monly known as ldquoPlairdquo inThailand and ldquoBonglairdquo inMalaysiaIt has long been used in traditional medicine in Thailandbeing the prime ingredient in massage oil to relieve musclepain In Northeast India oral consumption of the rhizomepaste of Z cassumunar was reported to treat dyspepsia andstomach bloating [4 5] whilst in Malaysia it is used forpostpartum medication

cis-3-(3101584041015840-Dimethoxyphenyl)-4-[(E)-31015840101584010158404101584010158401015840-dime-thoxys-tyryl]cyclohex-1-ene (ZC-B11) is a phenylbutenoid dimerisolated from the rhizomes of Z cassumunar (Figure 1) Todate there have been no reported studies on ZC-B11 isolatedfrom the rhizome of Z cassumunar having antileukemicactivity Therefore the current study was conducted toinvestigate the in vitro antileukemic properties of thiscompound to substantiate its anticancer activity

2 Materials and Methods

21 Compound Isolation and Purification The rhizomes of Zcassumunar were collected from Jogjakarta Indonesia in theyear 2007 Voucher specimen was deposited in Herbariumof Faculty of Pharmacy Gajah Mada University JogjakartaBriefly the finely ground rhizomes of Z cassumunar (sim700 g)were soaked in petroleum ether for 72 hours at roomtemperature The extraction was repeated 3 times to removethe nonpolar organic compounds waxes and fats Extractionwas continued with chloroform ethyl acetate and methanolThe solvents were removed under reduced pressure andcrude extracts were obtained Column chromatography oversilica gel using a stepwise gradient elution system wasutilized to fractionate the petroleum ether extract (25 g)The isolation of the crude extract yielded 64 fractions

Fractions 12-13 showed similar pattern on TLC and werelater combined Purification of this fraction was done bycolumn chromatography using mixture of hexane and ethylacetate as eluent Subfraction B11 was collected from hexaneEtOAc (8 2) and was further washed with hexane andmethanol to give white solid cis-3-(3101584041015840-dimethoxyphenyl)-4-[(E)-31015840101584010158404101584010158401015840-dimethoxystyryl]cyclohex-1-eneThemolecu-lar weight of this compound is 380199 gmol with molecularformula C

24H28O4and melting point 91∘C-92∘C [6] The

compound was sent for infrared (IR) and nuclear magneticresonance (NMR) analyses at the laboratory of spectro-scopic analysis Faculty of Science UPM 1H NMR spectrawere recorded on NMR Bruker Avance 400 spectrometerapparatus 13C NMR spectra were reported at Ac 150MHzinstrument and Electron Impact Mass Spectra (EI-MS)were recorded on Finnigan MAT 31 mass spectrometerwith a MATSPECO data system EI-MS analysis indicatedthe presence of molecular ion peak at mz 380 whichcorresponded to the molecular formula of C

24H28O4 The

spectral data were found to be in good agreement withthe published data [6] No previous studies have beenreported on this compound except for its phytochemicalstructure determination and physicochemical characteriza-tion

cis-3-(3101584041015840-Dimethoxyphenyl)-4-[(E)-31015840101584010158404101584010158401015840-dimethoxystyryl]cyclohex-1-ene IR 120592max (cmminus1 UATR)3017 (=CndashH) 2927 (CndashO) 1589 (C=C) 1509 1458 12331138 1019 853 785 680 614 1H NMR (400MHz CDCl

3)

120575 680 (1H d J = 80Hz H-51015840) 676 (3H brs H-61015840 H-6101584010158401015840H-5101584010158401015840) 673 (1H s H-2101584010158401015840) 670 (1H s H-21015840) 626 (1H dJ = 160Hz H-710158401015840) 581 (1H dt J = 101 36Hz H-1) 599(1H d J = 101 Hz H-2) 559 (1H dd J = 160 90Hz H-810158401015840)386 (3H s) 375 (3H s) 386 (3H s) 383 (3H s) 351 (1Hbr s H-3) 272 (1H m H-4) 222 (2H m H-6) 168 (2Ht J = 70Hz H-5) 13C NMR (100MHz CDCl

3) 120575 1489

(C-3101584010158401015840-OCH3) 1482 (C-4101584010158401015840mdashOCH

3) 1480 (C-31015840mdashOCH

3)

1475 (C-41015840mdashOCH3) 1338 (C-11015840) 1324 (C-810158401015840) 1310 (C-1101584010158401015840)

1280 (C-2) 1285 (C-710158401015840) 1290 (C-1) 1219 (C-61015840) 1187(C-6101584010158401015840) 1137 (C-21015840) 1111 (C-5101584010158401015840) 1103 (C-51015840) 1089 (C-2101584010158401015840)458 (C-3) 426 (C-4) 248 (C-6) 243 (C-5) MS mz (intensity) 380 (M+ 15) 300 (2) 229 (2) 190 (100) 175 (17)159 (80) 144 (17)

22 Cell Lines and Reagents All cancer cell lines wereobtained from American Type Culture Collection (ATCC)RPMI 1640 and Fetal Bovine Serum (FBS) were purchasedfrom PAA (Germany) DMSO penicillin and streptomycinsolution were purchased from Sigma (St Louis MO USA)MTT was purchased from Amresco (USA) Quantum PBLmedia was purchased from PAA (Austria) Phosphate buffersaline was obtained from Invitrogen (Carlsbad USA) Allother chemicals and reagents used were of HPLC grade

23 Cell Culture Cancer cells were cultured in RPMI 1640medium supplemented with 10 FBS and 1 100 unitmLpenicillin and 100 120583gmL streptomycin Cultures were main-tained at 37∘C in a humidified 5 CO

2incubator Experi-

ments were performed at concentration of 200000 cellsmL



50



2atmosphere













50


0G1cell fraction



50












3 Results























50






(a)

BL

CS

(b)

BLCS

CS

(c)

BL

CSCS

CSCS

(d)







50con-




VI

VI

20 120583m

(a)

BL

BLCC

20 120583m

(b)

20 120583m

BL AB

SN

(c)

CC

BL

LA

SN

AB

20 120583m

(d)








0

200

400

600

0 20 40 60 80 100

Num

ber

I II III IV

Channels (PI-A)

(a)

0

200

400

600

800

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(b)

0

100

200

300

400

600

500

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(c)

0

200

400

600

0 20 40 60 80 100

Channels (PI-A)

I II III IVN

umbe

r

(d)






50concen-




48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast





A B C D


50)









20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















50



2atmosphere













50


0G1cell fraction



50












3 Results























50






(a)

BL

CS

(b)

BLCS

CS

(c)

BL

CSCS

CSCS

(d)







50con-




VI

VI

20 120583m

(a)

BL

BLCC

20 120583m

(b)

20 120583m

BL AB

SN

(c)

CC

BL

LA

SN

AB

20 120583m

(d)








0

200

400

600

0 20 40 60 80 100

Num

ber

I II III IV

Channels (PI-A)

(a)

0

200

400

600

800

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(b)

0

100

200

300

400

600

500

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(c)

0

200

400

600

0 20 40 60 80 100

Channels (PI-A)

I II III IVN

umbe

r

(d)






50concen-




48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast





A B C D


50)









20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results























50






(a)

BL

CS

(b)

BLCS

CS

(c)

BL

CSCS

CSCS

(d)







50con-




VI

VI

20 120583m

(a)

BL

BLCC

20 120583m

(b)

20 120583m

BL AB

SN

(c)

CC

BL

LA

SN

AB

20 120583m

(d)








0

200

400

600

0 20 40 60 80 100

Num

ber

I II III IV

Channels (PI-A)

(a)

0

200

400

600

800

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(b)

0

100

200

300

400

600

500

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(c)

0

200

400

600

0 20 40 60 80 100

Channels (PI-A)

I II III IVN

umbe

r

(d)






50concen-




48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast





A B C D


50)









20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























50






(a)

BL

CS

(b)

BLCS

CS

(c)

BL

CSCS

CSCS

(d)







50con-




VI

VI

20 120583m

(a)

BL

BLCC

20 120583m

(b)

20 120583m

BL AB

SN

(c)

CC

BL

LA

SN

AB

20 120583m

(d)








0

200

400

600

0 20 40 60 80 100

Num

ber

I II III IV

Channels (PI-A)

(a)

0

200

400

600

800

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(b)

0

100

200

300

400

600

500

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(c)

0

200

400

600

0 20 40 60 80 100

Channels (PI-A)

I II III IVN

umbe

r

(d)






50concen-




48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast





A B C D


50)









20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

BL

CS

(b)

BLCS

CS

(c)

BL

CSCS

CSCS

(d)







50con-




VI

VI

20 120583m

(a)

BL

BLCC

20 120583m

(b)

20 120583m

BL AB

SN

(c)

CC

BL

LA

SN

AB

20 120583m

(d)








0

200

400

600

0 20 40 60 80 100

Num

ber

I II III IV

Channels (PI-A)

(a)

0

200

400

600

800

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(b)

0

100

200

300

400

600

500

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(c)

0

200

400

600

0 20 40 60 80 100

Channels (PI-A)

I II III IVN

umbe

r

(d)






50concen-




48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast





A B C D


50)









20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VI

VI

20 120583m

(a)

BL

BLCC

20 120583m

(b)

20 120583m

BL AB

SN

(c)

CC

BL

LA

SN

AB

20 120583m

(d)








0

200

400

600

0 20 40 60 80 100

Num

ber

I II III IV

Channels (PI-A)

(a)

0

200

400

600

800

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(b)

0

100

200

300

400

600

500

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(c)

0

200

400

600

0 20 40 60 80 100

Channels (PI-A)

I II III IVN

umbe

r

(d)






50concen-




48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast





A B C D


50)









20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

200

400

600

0 20 40 60 80 100

Num

ber

I II III IV

Channels (PI-A)

(a)

0

200

400

600

800

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(b)

0

100

200

300

400

600

500

0 20 40 60 80 100

I II III IV

Num

ber

Channels (PI-A)

(c)

0

200

400

600

0 20 40 60 80 100

Channels (PI-A)

I II III IVN

umbe

r

(d)






50concen-




48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast





A B C D


50)









20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















48 h72 h

70

50

40

30

20

10

0

60

minus10

Num

ber o

f cel

ls (

)

Sub G0G1 S G2MG0G1

lowastlowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast





A B C D


50)









20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















20 120583m

(a)

20 120583m

(b)

20 120583m

(c)

20 120583m

(d)

20 120583m

(e)






Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Bax lowast

lowast

lowast

lowast

lowast

lowast

lowast

SMAC

XIAP

BID


Signal intensity

Bcl-2

Caspase 8

Caspase 3

Cytochrome c

p53

HSP70








450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















450000

400000

350000

300000

250000

200000

150000

100000

50000

0

Lum

ines

cenc

e (RL

U)

lowast

lowastlowast

Negativecontrol

24 48 72

Time (hours)

(a)

45000

40000

35000

30000

25000

20000

15000

10000

5000

0

Lum

ines

cenc

e (RL

U)

Negativecontrol

24 48 72

Time (hours)

(b)

60000

50000

40000

30000

20000

10000

0

Lum

ines

cenc

e (RL

U)

lowastlowast

lowast

Negativecontrol

24 48 72

Time (hours)

(c)







0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 3 6 12 24

Bax

Bcl-2

HSP70

120573-actin





Abbreviations








Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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