kumar et al 2008

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Chemico-Biological Interactions 171 (2008) 332–347

Available online at www.sciencedirect.com

An essential oil and its major constituent isointermedeol induceapoptosis by increased expression of mitochondrial cytochrome c

and apical death receptors in human leukaemia HL-60 cellsAjay Kumar a, Fayaz Malik a, Shashi Bhushan a, Vijay K. Sethi a, Ashok K. Shahi a,

Jagdeep kaur b, Subhash C. Taneja a, Ghulam N. Qazi a, Jaswant Singh a,

a Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, Indiab Department of Biotechnology, Punjab University, Chandigarh 160014, India

Received 20 May 2007; received in revised form 5 October 2007; accepted 18 October 2007Available online 24 October 2007

Abstract

An essential oil from a lemon grass variety of Cymbopogon exuosus (CFO) and its major chemical constituent sesquiterpeneisointermedeol(ISO) were investigated for theirability to induce apoptosis in human leukaemiaHL-60 cellsbecause dysregulation of apoptosis is the hallmark of cancer cells. CFOand ISOinhibited cell proliferation with 48h IC50 of 30and 20 g/ml, respectively.Both induced concentration dependent strong and early apoptosis as measured by various end-points, e.g. annexinV binding, DNAladdering, apoptotic bodies formation and an increase in hypo diploid sub-G0 DNA content during the early 6 h period of study.This could be because of early surge in ROS formation with concurrent loss of mitochondrial membrane potential observed. Both

CFO and ISO activated apical death receptors TNFR1, DR4 and caspase-8 activity. Simultaneously, both increased the expressionof mitochondrial cytochrome c protein with its concomitant release to cytosol leading to caspase-9 activation, suggesting therebythe involvement of both the intrinsic and extrinsic pathways of apoptosis. Further, Bax translocation, and decrease in nuclear NF-

B expression predict multi-target effects of the essential oil and ISO while both appeared to follow similar signaling apoptosispathways. The easy and abundant availability of the oil combined with its suggested mechanism of cytotoxicity make CFO highlyuseful in the development of anti-cancer therapeutics.© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Cymbopogon exuosus ; Essential oil; Isointermedeol; Apoptosis; HL-60 cells

1. Introduction

Cancer is the leading cause of death in the world nextto cardiovascular diseases. Cancer cells cleverly evadeself-demise through apoptosis because of the accumula-

Corresponding author at: Division of Pharmacology, Indian Insti-tute of Integrative Medicine, Canal Road, Jammu 180001, India.Tel.: +91 191 2569000; fax: +91 191 2569111/2569222.

E-mail address: [email protected] (J. Singh).

tion of severalgenetic andepigenetic changes within [1].Agents that can trigger the process of apoptosis in can-cer cells are therefore considered potentially importantfor the development of anti-cancer chemotherapeutics[2]. Of several prescription drugs in use for cancer treat-ment, almost 75%are derived from plant species [3,4]. Itis surprising to note that essential oils, which are foundabundantly in nature, have never been exploited for theiranticancer potential, although they have found extensiveuse in perfumery, aromatherapy, food and avors, etc.since ages. Many essential oils or their constituents are

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A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332–347 333

knownto be thepotent antibacterial as well as anti-fungalagents. Theapplicationof essentialoils in theanti-cancertherapy may appear unconventional however, their easyavailability, pleasant aroma and low or insignicant tox-icity make them more attractive candidates for the longterm treatment of various chronic ailments. In oureffortstowards the development of novel herbal products fortheir anti-cancer potential, we report here for the rsttime, the pro-apoptotic effect of an essential oil and itsusefulness in the development of anticancer therapeuticleads. The essential oil isolated form the lemon grassCymbopogon exuosus is characteristic for its isointer-medeol presence, which constitutes almost 25% of itscontents. This plant is an East Indian perennial herbbelonging to the family poaceae. Essential oil derivedfrom this plant is used in various food and aroma indus-try products. Also present in other diverse essential oils

are some common constituents found in Cymbopogon exuosus oil (CFO) such as geraniol (20%), geranylacetate (12%), limonene (3.5%), -bisabolol (8.4%), allof which individually have been reported for their cancercell cytoxicity [5–7]. Besides, this natural compositionof CFO also contains limonene known for its immunos-timulatory activity [8], and borneol for analgesic andanaesthetic activities [9]. This report is the rst of itskind in providing insight into the basis of cytotoxicity of CFO and its major constituent isointermedeol in cancercell. As such there is no report on the anti-cancer activ-

ity and molecular mechanism involved in the inductionof apoptosis by any other essential oil and therefore thisstudy provides green pasture for development of novelanti-cancer therapeutics.

Apoptosis is a distinct form of cell death [10] thatis regulated by two major pathways. One involves theexecution through cell surface death receptors (TNFR1,DR4 and CD95), recruiting Fas associated death domain(FADD), caspase-8 auto activation [11] with downstream up-regulation of caspase-3, -6 or -7. The sec-ond pathway is mediated through mitochondria wheresmallmoleculeslikecytochrome c arereleasedtocytosolthrough permeability transitionporeor through channelsformed in the mitochondrial membrane by Bax leadingto activation of caspase-9 [12,13] .

We report for the rst time that the essential oil aswell as its major chemical constituent isointermedeolinduce apoptosis in human leukaemia HL-60 cells andtherefore are potential candidates for the developmentof novel anti-cancer therapeutics. The anti-proliferativeeffect of CFO and ISO appear to be related to the down-regulation of NF- B expression, caspases activationmediated through both apical receptors and mitochon-

drial signaling pathways. Cytochrome c appeared to be

over expressed in mitochondria affecting simultaneousrelease in the cytosol triggering apoptosis. Our studiesprovide molecular mechanism of action of essential oiland its major constituent ISO in the cytotoxicity of HL-60cells for the rst time, which may befound veryusefulfor further development for anti-cancer activity.

2. Materials and methods

2.1. Isolation of essential oil of Cymbopogon exuosus (Nees ex Steud.) Wats [RRL, (J) CF HP]by hydro-distillation

The Cymbopogonexuosusstraindesignated as RRL(J) CF HP is a perennial densely tufted grass with nosigns of any disease and exhibits high survival underadverse conditions. The grass is grown in our institute

farm. Freshly harvested aerial parts of the Cymbopogon exuosus (500 g) were charged on a Clevenger typehydro-distillation glass apparatus (10 L capacity) con-taining 4 L of water and tted with a condenser. Thecontents were heated to boiling temperature and thehydro-distilled volatile part was collected and separated(upper layer) from the aqueous portion (0.4% yield).Triplicate distillation was performed in succession foreach sample of 500 g of herbage at leang stage. Theessential oil was dried over anhydrous sodium sulphateand stored at 4 ◦ C. The separated oil (density 0.89) was

subjectedto gaschromatography–MS analysison a silicacapillary column (30 mm × 0.25mm), which displayedthe presence of at least 40 peaks; the major constituents(%) identied are geraniol (20.08), geranyl acetate(12.20), -bisabolol (8.42) and isointermedeol (24.97).The other minor constituents include citronellal (0.22),citronellol (0.25), methyl isoeugenol (0.10), linalool(1.70),borneol (1.90), camphor(0.07), camphene(1.33),geranial (0.45), neral (0.43), caryophylleneoxide (0.36),limonene (3.47), methyl ether eugenol (1.25), -phellandrene (0.21), -pinene (0.63), -pinene (0.01),piperitone (0.06), neryl acetate (3.44), sabinene (0.60),allo-ocimene (0.06), myrcene (0.37), caryophyllene(1.78), -humulene (0.26), ( Z )-ocimene (1.78), ( E )-ocimene (0.03), geranyl butyrate (0.31), -farnesen(0.38), elemicin (3.75), piperitol (1.66), carene-2 (1.02),(2- E ), farnesol (0.14), ( Z )-2- p-menthenol (0.06).

2.2. Isolation of isointermedeol from C. exuosusoil

The essential oil (2.0g) obtained from Cymbopogon exuosus was subjected to column chromatography over

silica gel (150g, 60–120 mesh). The elution of the col-

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umn was carried with increasing ratio of ethyl acetatein hexane (1–25%). The lower fractions thus obtainedwere pooled together on the basis of TLC and rechro-matographed over silica gel to separate the fractioncontaining sesquiterpene isointermedeol, isolated as aliquid that solidied after some time as white akes(320mg), mp 41 ◦ C and [α ]D + 1.2 (c, 2.0 MeOH). Thestructure conrmation was completed by comparison of 1H NMR, 13CNMR and physical data [14].

2.3. Chemicals and antibodies

Dihydrorhodamine 123 (DHR123), l -buthionine- S , R-sulfoximine (BSO), ethidium bromide, propid-ium iodide (PI), DNase-free RNase, proteinase K,3-(4,5,-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), staurosporine and camptothecin werepurchased from M/s Sigma chemicals Co., St. Louis.Fetal bovine serum was obtained from M/s GIBCOInvitrogen Corporation, USA. Other reagents used wereof analytical grade and available locally. AnnexinV-FITC apoptosis detection kit, Mitochondrial MembraneSensor Kit and ApoAlert Glutathione detection kit wereobtained from M/s BD Biosciences while ApoAlert cas-pases assay kits were from M/s B.D. Clontech. Mouseanti-human antibodies to Bax (#SC20067), PARP-1(#SC8007), Bcl-2 (SC7382), TNFR1 (#SC8436), actin(#SC-8432), goat anti-human DR4 (#SC-6824), goatanti-rabbit IgG-HRP (#SC2030) and goat anti-mouseIgG-HRP (#SC2031) were from M/s Santa Cruz,USA. Mouse anti-NF- B (#554184, clone G96-337)and cytochrome c (#556433, clone 7H8.2C12) were

from M/s BD, Pharmingin. Anti-COX-IV used wasfrom M/s ApoAlert cell fractionation kit of Clontech,CA (#630105). Electrophoresis reagents and proteinmarkers were from M/s Bio-Rad, USA while Hyper lmand ECL reagents from M/s Amersham Biosciences,UK.

2.4. Cell culture, growth conditions and treatment

Human promyelocytic leukaemia cell line HL-60cells were obtained from NCI, USA. The cells were

grown in RPMI-1640 medium containing 10% FCS,

100 units pencillin/100 g streptomycin perml medium.Cells were grown in CO 2 incubator (Thermocon Elec-tron Corporation, USA) at 37 ◦ C with 95% humidityand 5% CO 2 gas environment. Cells were treated withtest materials dissolved in DMSO while the untreatedcultures received only the vehicle (DMSO, <0.2%, v/v).

2.5. Cell proliferation assay

Cell proliferation was determined using MTT asdescribed earlier [15]. HL-60 cells 2.5 × 104 /200 lmedia in 96-well culture plates were treated with var-ious concentrations of CFO and ISO for 48h. The MTTformazan crystals formed were dissolved in 200 l of DMSO; OD measured at 570 nm (reference wave length620 nm). Cell growth as percent viability was calculatedby comparing the absorbance of treated verses untreated

cells.

2.6. Flow cytometric analysis of apoptosis and necrosis

HL-60 cells (1 × 106 /2 ml) were treated with CFOand ISO at 30 g/ml for indicated time periods, cellswere collected, washedtwice with PBSandsuspended in0.1 ml binding buffer provided with apoptosis detectionkit (BD Biosciences). Cells were stained with annexinV-FITC antibody and propidium iodide as per instructions

of the manufacturer. Cells were scanned in FL-1 (FITC)versus FL-2 (PI) channels on BD-LSR ow cytometer[15] using quadrant statistics for apoptotic and necroticcell populations.

2.7. Hoechst 33258 staining of cells for nuclear morphology

CFO and ISO treated cells (2 × 106 cells/4 ml) werexed for Hoechst 33258 staining [15]. The slides wereobserved for any nuclear morphological changes andapoptoticbodies under inverted uorescence microscope(Olympus IX70).

2.8. Measurement of mitochondrial membrane potential ( Ψ m)

Mitochondrial membrane potential ( Ψ m) was mea-sured by using a Mitochondrial Membrane Sensor Kitcontaining JC-1 dye, as described by the manufac-turer (BD Bioscience, CA). The cells after treatmentwere washed and stained with the dye and analyzedby Flow cytometry. The MitoSensor reagent aggre-

gates in the mitochondria of healthy cells producing

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red uorescence observed in FL-2 channel while cellswith altered mitochondrial membrane potentials, theMitoSensor reagent remains as monomers in the cyto-plasm where it uoresced green and analyzed on FL-1channel. The decrease in FL-2 uorescence also predictsaltered mitochondrial membrane potential. In fact JC-1is more specic for mitochondrial membrane potentialand more consistent in its response to depolarization,though we also employed another commonly used dyeRh-123 where thedecrease in FL-1 channel uorescencedepicts depolarization [15].

2.9. Caspase assays

Cells (2 × 106 /3 ml/well, 6-well plate) were incu-bated with CFO and ISO for indicated time periods. Atthe end of treatment cells were washed in PBS and pel-

lets lysed in cell lysis buffer. Activities of caspase-3, -9/6and -8 in the cell lysates were determined uorometri-cally, using BD ApoAlert caspase uorescent assay kits.Caspase-3 and -8 employed uorochome conjugatedpeptides DEVD-AFC and IETD-AFC as substrates,respectively, while caspase-9 employed LEHD-AMC.Release of AFC (7-amino-4-triuoromethyl coumarin)and AMC (7-aminomethylcoumarin) were assayedaccording to the instructions provided in the Manualby the supplier. Specic inhibitors were used as nega-tive control to determine whether uorescence intensity

changes were specic for the activity of caspases. Thepeptide based inhibitors used were DEVD-CHO forcaspase-3, IETD-fmk for caspase-8andLEHD-CHO forcaspase-9/6.

2.10. DNA cell cycle analysis

Cells were treated for 24 h and collected at160 × g for5 min in 5ml polystyrene tube. Cell pellets were washedonce with PBS and xed in 70% ethanol for overnight at4 ◦ C. Cells were again washed, suspended in 250 l of PBS, incubated with RNaseA at 37 ◦ C and stained withPI. The cells were analyzed for PI-DNA uorescence byow cytometerically as described earlier [15].

2.11. DNA agarose gel electrophoresis

DNA fragmentation typical of apoptosis was alsoassessed by electrophoresis of extracted genomic DNAfrom HL-60 cells. Briey, 2 × 106 cells after vari-ous treatments were washed in PBS containing 10 mMEDTA. The pellet was lysed in 250 l of lysis buffer(100mM NaCl, 5 mM EDTA, 10 mM Tris–HCl, pH 8.0,

5% Triton X-100, 0.25% SDS) containing 400 g/ml

DNase-free RNaseandincubated at 37 ◦ Cfor90minfol-lowed by 1 h incubation with proteinase-K (200 g/ml)at 50 ◦ C for 1 h. The DNA was extracted with 200 l of phenol:chloroform:isoamyl alcohol (25:24:1) for 1 minand centrifuged at 13000 × g for 3 min. The aqueousphase was further extracted with chloroform and cen-trifuged.DNA wasprecipitatedfrom aqueous phase with3 volumes of chilled alcohol containing 0.3 M sodiumacetateat4 ◦ C overnight.Theprecipitate wascentrifugedat 13000 × g for 10 min. The DNA pellet was washed in80% alcohol, dried, dissolved in 50 l TE buffer andelectrophoresed in 1.6% agarose gel at 50 V, stainedwith ethidium bromide and visualized in Bio-Rad geldocumentation system.

2.12. Measurement of intracellular peroxides (ROS)in HL-60 cells

The level of intracellular peroxides was determinedusing dihydrorhodamine 123 (DHR123). DHR localizesto mitochondria and uoresces when oxidized by ROS,particularly peroxynitrite, to the positively charged Rho-damine 123 derivative [16], which can be measured byow cytometery. HL-60 cells (2 × 106 /3 ml/well) aftervarious treatments in 6-well plate were incubated withDHR 123 (10 M)for 30min. Cells were washed in PBSand analyzed by ow cytometery.

2.13. Measurement of GSH contents in cells

Intracellular levels of GSH were estimated usingthe BD ApoAlert TM Glutathione detection kit employ-ing monochlorobimane (MCB) reagent. Briey, cellsafter various treatments were lysed according to man-ufacturer’s protocol. Cell lysates were incubated with2 mM MCB for 3 h at 37 ◦ C. Reduced glutathione levelswere assayed uorometrically at 395 nm excitation and480nm emissions.

2.14. Preparation of cell lysates for western blotsanalysis

Treatedanduntreated HL-60 cells were centrifuged at400 × g at 4 ◦ C, washed in PBSand cell pellets processedfor preparation of cytosolic, mitochondrial and wholecell lysates as described [17].

2.15. Preparation of cytosolic and nuclear extracts for NF- κ B immunoblot analysis

HL-60 cells (5 × 106) were washed with ice-cold

phosphate-buffered saline after various treatments and

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centrifuged. Cell pellets were homogenized in 200 l of the buffer and processed for the preparation of cytosolicand nuclear lysates [18].

2.16. Western blot analysis of PARP1, DR4,TNFR1, NF- κ B, cytochrome c, COX-IV, Bax, Bcl-2and actin proteins

The above lysates were resolved on SDS-PAGE anal-ysis and electro-transferred to polyvinylidene diuoride(PVDF) membranes (Bio-Rad) over night at 30 V, 4 ◦ C.The membraneswere blocked in blocking buffer (10 mMTris–HCl, 150mM NaCl, 0.1% Tween-20) containing5% milk for 1 h and blotted with respective anti-humanprimary antibodies for 2 h. Blots were washed in TBSand incubated with horseradish peroxidase-conjugatedsecondary antibody. Protein bands were detected using

enhancedchemiluminescence’s reagent,ECLkit (Amer-sham Biosciences). The density of the bands wasarbitrarily quantied using Quantity One software of Bio-Rad gel documentation system.

The protein contents were determined using Brad-ford reagent (Bio-Rad protein assay kit) and aliquotsnormalized to equal quantities before loading.

2.17. Statistical analysis

Data are presented as mean ± S.D. of the number

of experiments indicated. The comparisons were madebetween control and treated cultures using unpaired stu-dent ‘t ’ test and the difference was considered to bestatistically signicant if the P -value was <0.05.

3. Results

3.1. Cymbopogon exuosus oil (CFO) and ISOinhibit cell proliferation

Both CFO and ISO were able to inhibit HL-60 cellproliferation after 48 h with IC 50 values of approxi-mately 30 and 20 g/ml, respectively. DMSO used asdelivery vehicle (<0.2%, v/v), did not affect the cellgrowth when treated for the same time period ( Fig. 1A).Further, theeffectofCFOand ISOon time dependentcellviability through 48 h was also examined at 30 g/ml;both inhibited cell proliferation by about 27% at 6 h(Fig. 1B).

3.2. CFO and ISO induce apoptosis

In order to understand the mechanism of cytotox-

icity of the essential oil and its major constituent

isointermedeol, we thought that the herbal composi-tion might be inducing apoptosis in cancer cells. Forthis purpose various biological end-points of apop-tosis were analyzed during an early 6 h period of exposure.

3.2.1. Induction of DNA fragmentation typical of apoptosis

Both CFO and ISO induced concentration dependentDNA laddering of 180–200bp in HL-60 cells treated for6 h. The minimal concentration inducing DNA fragmen-tation was evident at 3 g/ml (Fig. 1C).

3.2.2. CFO and ISO alter nuclear morphologyobserved by Hoechst staining

Hoechst 33258 stain selectively binds DNA andallows monitoring of nuclear morphological changesunder uorescence microscopy. Both CFO and ISO at30 g/ml induced nuclear condensation and blebbing inHL-60 cells after 6 h of treatment ( Fig. 1C). The pres-ence of apoptotic bodies observed in cells treated withboth CFO and ISO conrmed that these herbal productstrigger cell demise by apoptosis.

3.2.3. CFO induces externalization of phosphatydylserine in HL-60 cells

AnnexinV binding of phosphatydyl serine of exposedcells is an early indicator of cells undergoing apoptosis.Therefore, HL-60 cells were incubated with differentconcentrations of CFO for 6 h, and the percentage of cells undergoing apoptosis/necrosis was determined bystaining with annexinV-FITC and PI by ow cytome-tery [15]. Both CFO and ISO produced comparable timedependentincreasein apoptoticcell populationandat theend of 6 h treatment more than 30% cells were apoptotic(Fig. 2). It was interesting to note that neither CFO norISO produced any necrosis and that cell death occurredmainly by apoptosis.

3.2.4. CFO and ISO increase sub-G0 populationwithout blocking any cell cycle phase

Based on our above results, we used 30 g/ml as thesuitable dose of CFO and ISO for further interrogat-ing in vitro mechanistic studies. Treatment of HL-60cells with CFO and ISO observed a time dependentincrease in hypodiploid sub-G0 DNA fraction indicat-ing apoptotic fraction due to DNA fragmentation. Therelative increase in this fraction was more with ISOover CFO ( Fig. 3). No initial blockage of G1, S andG2/M cell cycle phases was elicited either by CFO or

ISO.

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Fig. 1. CFO and ISO inhibit cell proliferation and induce apoptosis in HL-60 cells. (A) Cell proliferation: HL-60 cells (2.5 × 104 /well) grown in96-well culture plate were incubated with indicated concentrations of CFO and ISO for 48 h. Cell proliferation was assessed by MTT reductionassay as described in Section 2. Data are mean value ± S.D. of three similar experiments. (B) Time dependent effect of CFO and ISO on HL-60 cellproliferation: cells were incubated with CFO and ISO at 30 g/ml for different time periods. The treatment was staggered to incubate cells withMTT at the same time. All other conditions were same as described in (A). (C) CFO and ISO induce DNA fragmentation. Fragmentation of genomicDNA was studied in HL-60 cells exposed to the indicated concentrations of CFO and ISO for 6 h. Genomic DNA was isolated and electrophoresedas described in Section 2. DNA from camptothecin (4 M) treated cells was used as positive control. The data are representative one of three similarexperiments. (D) Effect of CFO and ISO on the nuclear morphology of HL-60 cells. HL-60 cells were treated with 30 g/ml of CFO and ISO each

for 6 h and subsequently stained with Hoechst 33258 as described in Section 2. Cells were observed under uorescence microscopy (30 × ). BothCFO and ISO induced the formation of apoptotic bodies as indicated by arrows. Data are one of two similar experiments.

3.3. CFO and ISO bring about early surge of ROS formation in HL-60 cells

A vast majority of cellular ROS produced in cellsoriginates from mitochondria during conditions that dis-rupt mitochondrial electron transport [19]. Cells treatedwithCFOandISOat30 g/ml for different time periods.Cells were stained with DHR-123 [16] and FAC scanned

in FL-1 channel of the ow cytometer for rhodamine-

123 positive cell population. Both CFO and ISO werefound to enhance oxidative stress during the rst 30min,followed by gradual decline over next 6 h of treatment.The level of ROS generation was found to be marginallyhigher in CFO treated cells compared to ISO treatedcells (Fig. 4A). The initial upsurge in ROS followedby gradual decrease was also conrmed by another dyeDCFH-DA. The relative magnitude of the initial ROS

measured by this dye however was almost 4-fold lower

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Fig. 2. Flow cytometric analysis of CFO and ISO induced apoptosis and necrosis in HL-60 cells using annexinV-FITC and PI double staining. (A)HL-60 cells (1 × 106 /ml) were incubated with 30 g/ml of CFO and ISO for indicated time periods and stained with annexinV-FITC/PI as describedin Section 2. Quadrant analysis of uorescence intensity of gated cells in FL-1 vs. FL-2 channels was from 10,000 events. Cells in the lower rightquadrant represented apoptosis while in the upper right quadrant indicated post-apoptotic necrosis. FACScan is representative one of three similarexperiments. (B) Quantitative presentation of apoptosis induced by CFO and ISO in HL-60 cells taken from the lower right quadrant of the given dotplot analysis. Data are mean ± S.D. of three similar experiments. Other conditions were same as described in (A). Statistical signicance: * P -value<0.001 compared to untreated control.

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Fig. 3. CFO and ISO increased hypodiploid Sub-G0 cell population in HL-60 cells measured by ow cytometry. HL-60 cells (1 × 106 /ml) in culturewere treated with CFO and ISO each (30 g/ml) for indicated time periods. Cells were stained with PI to determine DNA uorescence by owcytometery as described in Section 2. Sub-G0 population indicative of DNA damage was analyzed from the hypo diploid fraction (<2 n DNA) of DNA cell cycle analysis. Data are representative one of three similar experiments.

(not shown) than observed with DHR-123. It is knownthat DHR-123 is more specic for mitochondrial ROSwhile DCFH-DA measures ROS largely localized in thecytoplasm where DCF is mainly trapped. This furthersuggested that mitochondria may be the primary targetof CFO toxicity, and prompted us to nd out whetherearly surge in ROS may be involved in the apoptoticsignal triggered by CFO and ISO.

3.4. CFO induced apoptosis is not protected byanti-oxidants

To know if the apoptosis induced by CFO and ISO isbecause of early ROS generation, we treated HL-60 cellswith various antioxidants for 1 h prior to treatment withCFO or ISO at 30 g/ml for 6 h. The studies revealedthat various antioxidants used did not have any inu-ence on the extent of sub-G0 hypodiploid DNA fractionin CFO and ISO treated HL-60 cells ( Fig. 4B and C). Onthe contrary prior treatment with ascorbate increased thesub-G0 population in cells. These studies suggested thatany ROS formed are not protected by the anti-oxidants

during this early period of exposure and may thus not

be involved directly in the apoptotic death by CFO. Cor-respondingly, there was hardly any potential decline inGSH level (Fig. 5), while at this time most of the cellssuffered remarkable apoptotic features. GSH is impor-tant for maintaining redox state in the cell and this wassignicantly decreased in cells treated with BSO used aspositive control.

3.5. CFO and ISO bring about early activation of initiator and executioner caspases leading to PARP1cleavage

Both CFO and ISO produced an early time depen-dent increase in caspase-3 activity ( Fig. 6A), the effectsbeing comparable and registered almost 4-fold increaseafter 6 h treatment of cells. In the caspase-dependentpathway of apoptosis, caspase-3 serves as an execu-tioner molecule in cleaving proteins including the poly(ADP-ribose) polymerase1 (PARP1) [20]. The increas-ing caspase-3 activity brought about further proteolyticcleavage of downstream substrate PARP1, an end-pointof apoptosis ( Fig. 6D). Caspase-3 activity is known to

be up-regulated through a variety of signaling cascades

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Fig. 4. (A) Effect of CFO and ISO on the generation of ROS in HL-60 cells. HL-60 cells (1 × 106 /ml) were treated with 30 g/ml of CFO andISO each for indicated time periods. Cells were stained with DHR123 (5 M) and 10000 events acquired, and gated population analyzed usingBD-LSR ow cytometer. Other conditions were same as described in Section 2. Representative data of three independent experiments are shown. (Band C) Effect of antioxidants on Sub-G0 DNA fraction of CFO and ISO treated HL-60 cells. HL-60 cells (1 × 106 /ml) were treated with 30 g/mlof CFO (B) and ISO (C) in 12-well culture plates for 6 h. One hour prior to the treatment, cells were incubated with various antioxidants (Tiron,1 mM; Trolox, 200 M; NAC, 5 mM; Ascorbate, 5 mM). After treatment cells were stained with PI for FACS analysis. Other conditions are sameas described in Section 2. Data represented are mean ± S.D. of three similar experiments taken from sub-G0 population of the histogram FLA-2 vs.cell counts. * P < 0.05; **P < 0.01 for treated vs. control.

emanating from caspase-8 and -9 activation. We there-fore, examined for any corresponding increase in theactivity of either caspase-8 or -9 following treatmentof HL-60 cells with CFO and ISO over a period of 6 h

(Fig. 6A–C). HL-60 exposed to CFO and ISO displayed

higher than 2-fold caspase-8 activity at 6 h. Similarly theactivity of caspase-9 observed a time dependent increaseof about 2.5-fold ( Fig. 6C). In general, ISO treated cellsexpressed higher caspase-8 and -9 activities over CFO

treated cells.

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Fig. 5. Effect of CFO and ISO on GSH content in HL-60 cells. HL-60 cells (3 × 106 /well, 6-well plate) were treated with CFO and ISO(30 g/ml) for indicated time periods. Cells were extracted for deter-mination of reduced glutathione contents as described in Section 2.Data are mean ± S.D. of three similar experiments.

3.6. CFO and ISO appeared to have similar effectsin regulating the expression of apical deathreceptors DR4 and TNFR1 in HL-60 cells

Because of increased activity of caspase-8, it wasinteresting to analyze the expression of TNFR1 and DR4proteins in CFO and ISO treated cells. Death receptorsDR4 (TRAIL-R1) and TNFR1 can deliver a powerfuland rapid pro-apoptotic signal through death domain(DD) mediating recruitment of FADD and formationof so called death inducing signaling complex (DISC)[21] . FADD in turn canactivate procaspase-8anddeliverthe pro-apoptotic signal down stream by either directlyactivating executioner caspases or indirectly by helpingrelease of cytochrome c. Both CFO and ISO produced asignicant increase in theexpression of both TNFR1 andDR4(TRAIL-R1) in a time dependentmanner( Fig.7A).Expression of both the receptors was found to be higherin ISO treated cells when compared to CFO. We alsoanalyzed the level of transcription factor NF- B in thenucleus of both CFO and ISO treated cells. The nuclearlevel of NF- B observed a time-related decline with

maximal effect being at 6 h of study while ISO exertedalmost similar effect. On the contrary, the transcriptionfactor was highlyover expressed in thecytosol ( Fig.7A).

3.7. Translocation of Bax from cytosol tomitochondria without affecting the expression of Bcl-2 by CFO and ISO in HL-60 cells

Bax is a pro-apoptotic protein that exists as a sol-uble monomer in cytosol or is loosely associated withmitochondria. However, uponapoptotic stimulation,Bax

translocates to mitochondria where it forms oligomers

that are inserted into the outer mitochondrial membrane[22], and forms pores leading to release of cytochromec from mitochondria to cytosol. Bcl-2 on the other handis an anti-apoptotic protein, which prevents the releaseof cytochrome c from the mitochondria. In fact this isthe ratio rather than amount of pro-apoptotic and anti-apoptotic proteins that determines whether apoptoticsignaling can proceed [23,24] . We therefore analyzedthe expression of Bax in the cytosolic and mitochon-drial fractions by immunoblot analysis. Both CFO andISO decreased the cytosolic expression of Bax with cor-responding rise in the mitochondria ( Fig. 7B). It wasinteresting to note that Bcl-2 expression wasnot changedeither by CFO or ISO ( Fig. 7B).

3.8. Enhanced mitochondrial expression of cytochrome c prevents early loss of mitochondrial

membrane potential

Our data on annexinV analysis, sub-G0 DNA frac-tion and activation of caspases suggested that inductionof apoptosis by CFO and ISO in HL-60 cells is an earlyevent beginning within rst hour of treatment; we askedif the perturbation of mitochondrial membrane poten-tial is correlated with these events. We analyzed thechange in mitochondrial membrane potential ψ m byow cytometry using the uorescent lipophilic cationicprobe JC-1 (5,5,6,6-tetrachloro-1,1,3,3 tetraethyl ben-

zimidazolcarbocyanine iodide), and found that CFOtreated cells observed a signicant loss of mitochondrialmembrane potential, maximum being at 3 h followed bya gradual decrease through 6 h periodofstudywhile ISO,which followed similar trend, relatively was less effec-tive (Fig. 8A). The loss of ψ m was also conrmed byanotherdyerhodamine-123and theresults obtainedwerecomparable to JC-1 ( Fig. 8B). On the contrary activa-tion of caspase-9 requires cytochrome c that is releasedthrough permeabilized outer mitochondrial membraneor due to loss mitochondrial membrane potential ψ m,when cytochrome c is an important component of elec-tron transport chain [25]. In this concern we made twoimportantobservations,1) that both CFOandISOenablecells to over express cytochrome c in the mitochondriaand 2) that outer mitochondrial membrane simultane-ously enabledtherelease ofcytochrome c intothecytosolwithout undergoing any signicant early loss of ψ m(Fig. 9). Similarly enhanced mitochondrial expressionof cytochrome c was observed in Molt-4 and HeLa celllines (Data not shown).

In order to understand whether other proteins relatedto mitochondrial electron transportchain such as Cox-IV

are also affected by the treatment with CFO and ISO, we

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Fig. 6. (A–C) CFO and ISO induce caspases activation in HL-60 cells. HL-60 cells (2 × 106 /3 ml) were exposed to CFO and ISO at 30 g/ml forindicated time periods for assay of caspase-3 (A), caspase-8 (B) and caspase-9 (C) activities. The activities were determined uorimetrically in thecell lysates of HL-60 cells using BD ApoAlert caspase uorescent assay kits. Specic peptide based inhibitors provided along with the assay kitswere used for negative control (data not shown) to determine whether uorescence intensity changes were specic for the activity of caspases asdescribed in Section 2. Data are mean ± S.D. from three similar experiments. * P < 0.05; **P < 0.01 for treated vs. control. (D) CFO and ISO inducePARP1 cleavage. HL-60 cells were treated with CFO and ISO (30 g/ml) for indicated time periods. Equal amounts of total cell lysate proteinswere resolved on 10% SDS-PAGE, then transferred to PVDF membrane and probed with anti-PARP antibody. Anti-body to actin served as sampleloading control for protein level. Other conditions were same as described in Section 2. Western blot is representative from one of three similarexperiments.

determined the expression of COX-IV in HL-60 cells.COX-IV is a nuclear-encoded subunit among 13 dif-ferent subunits of the cytochrome c oxidase complexIV. This complex is one of the major sites for oxidativephosphorylation [26]. We observed a down-regulation of COX-IV with time in both CFO and ISO treated HL-60cells (Fig. 9A). Suppression of COX-IV has been relatedto the inhibition of electron transport chain [27] and itsdecline in mitochondria may sensitize cells to undergo

apoptosis [27].

4. Discussion

Much of the contemporary research in the develop-ment of anticancer therapeutics from plants has beenfocused on investigating the molecular mechanism bywhich an agent induces cytotoxicity and apoptosis incancer cells. This study provides valuable insight intothe mechanism of action of the Cymbopogon exuosusoil (CFO) and its major chemical constituent isointer-

medeol (ISO) that are able to trigger apoptosis in cancer

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Fig. 8. CFO and ISO induced loss of mitochondrial membrane potential ( Ψ m ) in HL-60 cells. (A) Measurement of ψ m using JC-1 dye: FACScananalysis of ψ m loss in HL-60 cells treated with 30 g/ml of CFO and ISO for indicated time periods. Cells were stained with JC-1 dye and un-gatedpopulation analyzed by Flow cytometry as described in Section 2. A decrease in FL-2 uorescence and a concurrent increase in FL-1 uorescenceare indicative of mitochondrial membrane depolarization as described in Section 2. Data are representative of one of three similar experiments. (B)Measurement of ψ m using Rhodamine-123 dye: HL-60 cells as in (A) were incubated for indicated time periods with 30 g/ml CFO. Cells werestained with RH-123, 30 min before the termination, and the un-gated population analyzed for decrease in FL-1 uorescence. Cell population in thelower left quadrant indicated mitochondrial depolarized cells. Other conditions were same as described in (A).

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Fig. 9. Expression of Cyt-c and COX-IV in CFO and ISO treated HL-60 cells. HL-60 cells were treated with CFO and ISO (30 g/ml) forindicatedtime periods. Immunoblotanalysisof Cyt-cand COX-IV wasperformed in designated sub-cellular lysate. Equal amount of proteinswere loaded and resolved on 15% SDS-PAGE. Other conditions aredescribed in Section 2.

death of cancer cells by CFO and ISO. Increased mito-chondrialcytochrome c expression bycertain anti-cancerdrugs [30] and as observed in this study may be a mech-anism to compensate its immediate loss to the cytosol,although release of cytochrome c is a ‘point of no return’forcancercell to trigger self-demise.This salient event ishighly prominent during the initial period of exposure of cells to CFO and ISO perhaps as means of self-defense toanti-cancer drugs as the cells were not exposed beyond6 h in our studies.

It may also be mentioned that cytochrome c consti-

tutes an important link in the electron transport chainwhich shuttles electrons between complex III and com-plex IV of the respiratory chain, this movement of electrons is accompanied by pumping out protons fromthe inner membrane, resulting in a potential differenceacross the mitochondrial membranes [31]. The respira-tory protein subunit IV of COX is a part of cytochromec oxidase complex IV of electron transport chain anddown-regulation of COX-IV has been associated withslow down or complete inhibition of electron transportchain [27], leading to loss of Ψ m . Both CFO and ISOwere also able to bring about a signicant ψ m loss withgradual decrease of COX-IV expression. The late effectson COX-IV seemingly appeared less important consid-ering that onset of apoptosis is a much advanced and anearlyevent that distinctlyis associatedwith translocationof cytochrome c. Thus both CFO and ISO produce strongstress in the mitochondrial functionalityby affecting var-ious components of electron transport chain particularlycytochrome c. Unlike its function as a respiratory elec-tron carrier, cytochrome c is also reported as a potentantioxidant [32]. With rise in mitochondrial ROS levels,cytochrome c detaches from inner mitochondrial mem-

brane and is capable of reducing O 2•−

to molecular

oxygen. ROS stress generated in mitochondria duringrst hour of treatment with CFO followed by decline at3 or 6 h of treatment may be attributed to increased mito-chondrialexpression of cytochrome c, which byvirtue of its strong antioxidant activity possibly caused quenchingof ROS to rescue cells from DNA damage. Activation of the intrinsic and extrinsic signaling pathways may thusbe important for apoptotic death, as various antioxidantsused in the study could not prevent DNA fragmentationinduced by CFO or ISO.

Many apoptotic molecules relocate subcellularly incells undergoing apoptosis [33] while pro-apoptotic pro-tein Bax plays an important role. Bax is implicated in theformation of pores in the outer mitochondrial membraneafter translocation from cytosol allowing subsequentrelease of cytochrome c and apoptosis activating fac-tors [34] leading to caspase-9 activation. We observed

decline in the cytosolic level of Bax with correspondingincrease in mitochondria in both CFO and ISO treatedcells. The level of Bcl-2 however, remained unchangedin the treated cells. But this is the ratio of Bax/Bcl-2rather than the level that appears to decide the directionof apoptotic signal. Our results indicate that the ratio of Bax/Bcl-2 in CFO and ISO treated cells to be in favorof apoptosis. Up-regulation of caspase-8 and -9 lead toactivation of downstream executioner caspase-3 and itscleavage of PARP1 is consequential events culminat-ing into apoptosis; both CFO and ISO were distinctly

effective in these actions.NF- B family of transcription factor plays a centralrole in the regulation of apoptosis, oncogenesis, inam-matory and immune responses and is activated by a widerange of stimuli [35,36] . Inhibition of NF- B is sug-gested to be a useful strategy for cancer therapy [35,36] .Both CFO and ISO decreased the expression of nuclearNF- B dimer p65/rel. The decrease could be related tothe inhibition of activation and therefore poor transloca-tion to nucleus; it would be our interest to understandfurther the effect of CFO and other constituents on NF-

B regulated genes expression in cancer cells. NF- Bin the cytosol normally remains in complex form withits inhibitory regulatory proteins, which upon activa-tion by various stimuli is released to nucleus. The overexpression observedin cytosol in thesestudies suggestedthat the protein is in complexation with the inhibitoryregulatory proteins restraining its availability for tran-scription activation of several genes. This may be thereason thatdecreasedexpressionof this transcriptionfac-tor is observed in the nucleus. It may be mentioned thatboth CFOandISO followed similar pathways of produc-ing cytotoxicity in cancer cells. Our studies demonstrate

that the essential oil and its major constituents may nd

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useful application in thedevelopmentofanti-cancer ther-apeutics because of its stability, abundant availabilitycombined with safety, and oral LD50 >2000 mg/kg b.wt.mice (not shown).

In conclusion, apoptosis induced by Cymbopogon exuosus oil (CFO) and isointermedeol (ISO) utilize awide range of molecular targets that include both api-cal receptors and mitochondrial dependent pathwaysbesides molecules like NF- B involved in cell prolifer-ation and differentiation. Being an herbal composition,which already is being used in aromatherapy perfumeryand neutraceuticals, CFO and isointermedeol providegreat potentials to be developed into anticancer thera-peutics.

Acknowledgements

Thanks are due to the Council ofScientic and Indus-trial Research, India, for nancial support for seniorresearch fellowships to Ajay Kumar and Fayaz Malik.We greatly appreciate the help of Dr. Sarang Bani of ourinstitute in the use of Flow Cytometery and analysis of results.

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