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 cells

Ajay 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 flexuosus (CFO) and its major chemical constituent sesquiterpeneisointermedeol (ISO) were investigated for their ability to induce apoptosis in human leukaemia HL-60 cells because dysregulation ofapoptosis is the hallmark of cancer cells. CFO and ISO inhibited cell proliferation with 48 h IC50 of ∼30 and 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. BothCFO 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 thereby

the 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.

ptosis;

Keywords: Cymbopogon flexuosus; Essential oil; Isointermedeol; Apo

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: jsishar1@yahoo.com (J. Singh).

0009-2797/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.cbi.2007.10.003

HL-60 cells

tion of several genetic and epigenetic 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 found

abundantly in nature, have never been exploited for theiranticancer potential, although they have found extensiveuse in perfumery, aromatherapy, food and flavors, etc.since ages. Many essential oils or their constituents are

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nown to be the potent antibacterial as well as anti-fungalgents. The application of essential oils in the anti-cancerherapy may appear unconventional however, their easyvailability, pleasant aroma and low or insignificant tox-city make them more attractive candidates for the longerm treatment of various chronic ailments. In our effortsowards the development of novel herbal products forheir anti-cancer potential, we report here for the firstime, the pro-apoptotic effect of an essential oil and itssefulness in the development of anticancer therapeuticeads. The essential oil isolated form the lemon grassymbopogon flexuosus is characteristic for its isointer-edeol presence, which constitutes almost 25% of its

ontents. This plant is an East Indian perennial herbelonging to the family poaceae. Essential oil derivedrom this plant is used in various food and aroma indus-ry products. Also present in other diverse essential oilsre some common constituents found in Cymbopogonexuosus oil (CFO) such as geraniol (20%), geranylcetate (12%), limonene (3.5%), �-bisabolol (8.4%), allf which individually have been reported for their cancerell cytoxicity [5–7]. Besides, this natural compositionf CFO also contains limonene known for its immunos-imulatory activity [8], and borneol for analgesic andnaesthetic activities [9]. This report is the first of itsind in providing insight into the basis of cytotoxicity ofFO and its major constituent isointermedeol in cancerell. As such there is no report on the anti-cancer activ-ty and molecular mechanism involved in the inductionf apoptosis by any other essential oil and therefore thistudy provides green pasture for development of novelnti-cancer therapeutics.

Apoptosis is a distinct form of cell death [10] thats regulated by two major pathways. One involves thexecution through cell surface death receptors (TNFR1,R4 and CD95), recruiting Fas associated death domain

FADD), caspase-8 auto activation [11] with downtream up-regulation of caspase-3, -6 or -7. The sec-nd pathway is mediated through mitochondria wheremall molecules like cytochrome c are released to cytosolhrough permeability transition pore or through channelsormed in the mitochondrial membrane by Bax leadingo activation of caspase-9 [12,13].

We report for the first time that the essential oil asell as its major chemical constituent isointermedeol

nduce apoptosis in human leukaemia HL-60 cells andherefore are potential candidates for the developmentf novel anti-cancer therapeutics. The anti-proliferative

ffect of CFO and ISO appear to be related to the down-egulation of NF-�B expression, caspases activationediated through both apical receptors and mitochon-

rial signaling pathways. Cytochrome c appeared to be

teractions 171 (2008) 332–347 333

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-60 cells for the first time, which may be found very usefulfor further development for anti-cancer activity.

2. Materials and methods

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

The Cymbopogon flexuosus strain designated 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 institutefarm. Freshly harvested aerial parts of the Cymbopogonflexuosus (500 g) were charged on a Clevenger typehydro-distillation glass apparatus (10 L capacity) con-taining 4 L of water and fitted 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 leafing stage. Theessential oil was dried over anhydrous sodium sulphateand stored at 4 ◦C. The separated oil (density 0.89) wassubjected to gas chromatography–MS analysis on a silicacapillary column (30 mm × 0.25 mm), which displayedthe presence of at least 40 peaks; the major constituents(%) identified 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. flexuosusoil

The essential oil (2.0 g) obtained from Cymbopogonflexuosus was subjected to column chromatography oversilica gel (150 g, 60–120 mesh). The elution of the col-

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334 A. Kumar et al. / Chemico-Biolo

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 solidified after some time as white flakes(320 mg), mp 41 ◦C and [α]D + 1.2 (c, 2.0 MeOH). Thestructure confirmation was completed by comparison of1H 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) werefrom 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 filmand 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 weregrown in RPMI-1640 medium containing 10% FCS,

teractions 171 (2008) 332–347

100 units pencillin/100 �g streptomycin per ml medium.Cells were grown in CO2 incubator (Thermocon Elec-tron Corporation, USA) at 37 ◦C with 95% humidityand 5% CO2 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 48 h. The MTTformazan crystals formed were dissolved in 200 �l ofDMSO; OD measured at 570 nm (reference wave length620 nm). Cell growth as percent viability was calculatedby comparing the absorbance of treated verses untreatedcells.

2.6. Flow cytometric analysis of apoptosis andnecrosis

HL-60 cells (1 × 106/2 ml) were treated with CFOand ISO at 30 �g/ml for indicated time periods, cellswere collected, washed twice with PBS and suspended in0.1 ml binding buffer provided with apoptosis detectionkit (BD Biosciences). Cells were stained with annexinV-FITC antibody and propidium iodide as per instructionsof the manufacturer. Cells were scanned in FL-1 (FITC)versus FL-2 (PI) channels on BD-LSR flow cytometer[15] using quadrant statistics for apoptotic and necroticcell populations.

2.7. Hoechst 33258 staining of cells for nuclearmorphology

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

2.8. Measurement of mitochondrial membranepotential (Ψm)

Mitochondrial membrane potential (�Ψm) 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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ed fluorescence observed in FL-2 channel while cellsith altered mitochondrial membrane potentials, theitoSensor reagent remains as monomers in the cyto-

lasm where it fluoresced green and analyzed on FL-1hannel. The decrease in FL-2 fluorescence also predictsltered mitochondrial membrane potential. In fact JC-1s more specific for mitochondrial membrane potentialnd more consistent in its response to depolarization,hough we also employed another commonly used dyeh-123 where the decrease in FL-1 channel fluorescenceepicts depolarization [15].

.9. Caspase assays

Cells (2 × 106/3 ml/well, 6-well plate) were incu-ated with CFO and ISO for indicated time periods. Athe end of treatment cells were washed in PBS and pel-ets lysed in cell lysis buffer. Activities of caspase-3, -9/6nd -8 in the cell lysates were determined fluorometri-ally, using BD ApoAlert caspase fluorescent assay kits.aspase-3 and -8 employed fluorochome conjugatedeptides DEVD-AFC and IETD-AFC as substrates,espectively, while caspase-9 employed LEHD-AMC.elease of AFC (7-amino-4-trifluoromethyl coumarin)nd AMC (7-aminomethylcoumarin) were assayedccording to the instructions provided in the Manualy the supplier. Specific inhibitors were used as nega-ive control to determine whether fluorescence intensityhanges were specific for the activity of caspases. Theeptide based inhibitors used were DEVD-CHO foraspase-3, IETD-fmk for caspase-8 and LEHD-CHO foraspase-9/6.

.10. DNA cell cycle analysis

Cells were treated for 24 h and collected at 160 × g formin in 5 ml polystyrene tube. Cell pellets were washednce with PBS and fixed in 70% ethanol for overnight at◦C. Cells were again washed, suspended in 250 �l ofBS, incubated with RNaseA at 37 ◦C and stained withI. The cells were analyzed for PI-DNA fluorescence byow cytometerically as described earlier [15].

.11. DNA agarose gel electrophoresis

DNA fragmentation typical of apoptosis was alsossessed by electrophoresis of extracted genomic DNArom HL-60 cells. Briefly, 2 × 106 cells after vari-

us treatments were washed in PBS containing 10 mMDTA. The pellet was lysed in 250 �l of lysis buffer

100 mM NaCl, 5 mM EDTA, 10 mM Tris–HCl, pH 8.0,% Triton X-100, 0.25% SDS) containing 400 �g/ml

teractions 171 (2008) 332–347 335

DNase-free RNase and incubated at 37 ◦C for 90 min fol-lowed by 1 h incubation with proteinase-K (200 �g/ml)at 50 ◦C for 1 h. The DNA was extracted with 200 �l ofphenol: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 was precipitated from aqueous phase with3 volumes of chilled alcohol containing 0.3 M sodiumacetate at 4 ◦C overnight. The precipitate was centrifugedat 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 fluoresces when oxidized by ROS,particularly peroxynitrite, to the positively charged Rho-damine 123 derivative [16], which can be measured byflow cytometery. HL-60 cells (2 × 106/3 ml/well) aftervarious treatments in 6-well plate were incubated withDHR 123 (10 �M) for 30 min. Cells were washed in PBSand analyzed by flow cytometery.

2.13. Measurement of GSH contents in cells

Intracellular levels of GSH were estimated usingthe BD ApoAlertTM Glutathione detection kit employ-ing monochlorobimane (MCB) reagent. Briefly, 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 fluorometrically at 395 nm excitation and480 nm emissions.

2.14. Preparation of cell lysates for western blotsanalysis

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

2.15. Preparation of cytosolic and nuclear extracts

for NF-κB immunoblot analysis

HL-60 cells (5 × 106) were washed with ice-coldphosphate-buffered saline after various treatments and

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ing apoptotic fraction due to DNA fragmentation. The

336 A. Kumar et al. / Chemico-Biolo

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

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

The above lysates were resolved on SDS-PAGE anal-ysis and electro-transferred to polyvinylidene difluoride(PVDF) membranes (Bio-Rad) over night at 30 V, 4 ◦C.The membranes were blocked in blocking buffer (10 mMTris–HCl, 150 mM 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 usingenhanced chemiluminescence’s reagent, ECL kit (Amer-sham Biosciences). The density of the bands wasarbitrarily quantified using Quantity One software ofBio-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 numberof experiments indicated. The comparisons were madebetween control and treated cultures using unpaired stu-dent ‘t’ test and the difference was considered to bestatistically significant if the P-value was <0.05.

3. Results

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

Both CFO and ISO were able to inhibit HL-60 cellproliferation after 48 h with IC50 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, the effect of CFO and ISO on time dependent cellviability 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

teractions 171 (2008) 332–347

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 ofexposure.

3.2.1. Induction of DNA fragmentation typical ofapoptosis

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 fluorescence 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 confirmed 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 ofcells undergoing apoptosis/necrosis was determined bystaining with annexinV-FITC and PI by flow cytome-tery [15]. Both CFO and ISO produced comparable timedependent increase in apoptotic cell population and at 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-

relative 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 orISO.

A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332–347 337

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 similare f HL-60f SectionC by arrow

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xperiments. (D) Effect of CFO and ISO on the nuclear morphology oor 6 h and subsequently stained with Hoechst 33258 as described inFO and ISO induced the formation of apoptotic bodies as indicated

.3. CFO and ISO bring about early surge of ROSormation in HL-60 cells

A vast majority of cellular ROS produced in cellsriginates from mitochondria during conditions that dis-

upt mitochondrial electron transport [19]. Cells treatedith CFO and ISO at 30 �g/ml for different time periods.ells were stained with DHR-123 [16] and FAC scanned

n FL-1 channel of the flow cytometer for rhodamine-

cells. HL-60 cells were treated with 30 �g/ml of CFO and ISO each2. Cells were observed under fluorescence microscopy (30×). Boths. Data are one of two similar experiments.

123 positive cell population. Both CFO and ISO werefound to enhance oxidative stress during the first 30 min,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 treated

cells (Fig. 4A). The initial upsurge in ROS followedby gradual decrease was also confirmed by another dyeDCFH-DA. The relative magnitude of the initial ROSmeasured by this dye however was almost 4-fold lower

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

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 fluorescence 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 significance: *P-value<0.001 compared to untreated control.

A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332–347 339

Fig. 3. CFO and ISO increased hypodiploid Sub-G0 cell population in HL-60 cells measured by flow cytometry. HL-60 cells (1 × 106/ml) in culturew eriods.c f DNAD xperim

(twcsoes

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ere treated with CFO and ISO each (30 �g/ml) for indicated time pytometery as described in Section 2. Sub-G0 population indicative oNA cell cycle analysis. Data are representative one of three similar e

not shown) than observed with DHR-123. It is knownhat DHR-123 is more specific for mitochondrial ROShile DCFH-DA measures ROS largely localized in the

ytoplasm where DCF is mainly trapped. This furtheruggested that mitochondria may be the primary targetf CFO toxicity, and prompted us to find out whetherarly surge in ROS may be involved in the apoptoticignal triggered by CFO and ISO.

.4. CFO induced apoptosis is not protected bynti-oxidants

To know if the apoptosis induced by CFO and ISO isecause of early ROS generation, we treated HL-60 cellsith various antioxidants for 1 h prior to treatment withFO or ISO at 30 �g/ml for 6 h. The studies revealed

hat various antioxidants used did not have any influ-nce on the extent of sub-G0 hypodiploid DNA fractionn CFO and ISO treated HL-60 cells (Fig. 4B and C). On

he contrary prior treatment with ascorbate increased theub-G0 population in cells. These studies suggested thatny ROS formed are not protected by the anti-oxidantsuring this early period of exposure and may thus not

Cells were stained with PI to determine DNA fluorescence by flowdamage was analyzed from the hypo diploid fraction (<2n DNA) ofents.

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 wassignificantly decreased in cells treated with BSO used aspositive control.

3.5. CFO and ISO bring about early activation ofinitiator 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 tobe up-regulated through a variety of signaling cascades

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

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 flow 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,

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1 mM; Trolox, 200 �M; NAC, 5 mM; Ascorbate, 5 mM). After treatmas described in Section 2. Data represented are mean ± S.D. of three scell 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

s were stained with PI for FACS analysis. Other conditions are samexperiments taken from sub-G0 population of the histogram FLA-2 vs.

higher than 2-fold caspase-8 activity at 6 h. Similarly the

activity 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 CFOtreated cells.

A. Kumar et al. / Chemico-Biological In

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(mD

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30 �g/ml) for indicated time periods. Cells were extracted for deter-ination of reduced glutathione contents as described in Section 2.ata are mean ± S.D. of three similar experiments.

.6. CFO and ISO appeared to have similar effectsn regulating the expression of apical deatheceptors DR4 and TNFR1 in HL-60 cells

Because of increased activity of caspase-8, it wasnteresting to analyze the expression of TNFR1 and DR4roteins in CFO and ISO treated cells. Death receptorsR4 (TRAIL-R1) and TNFR1 can deliver a powerful

nd rapid pro-apoptotic signal through death domainDD) mediating recruitment of FADD and formationf so called death inducing signaling complex (DISC)21]. FADD in turn can activate procaspase-8 and deliverhe pro-apoptotic signal down stream by either directlyctivating executioner caspases or indirectly by helpingelease of cytochrome c. Both CFO and ISO produced aignificant increase in the expression of both TNFR1 andR4 (TRAIL-R1) in a time dependent manner (Fig. 7A).xpression of both the receptors was found to be higher

n ISO treated cells when compared to CFO. We alsonalyzed the level of transcription factor NF-�B in theucleus of both CFO and ISO treated cells. The nuclearevel of NF-�B observed a time-related decline with

aximal effect being at 6 h of study while ISO exertedlmost similar effect. On the contrary, the transcriptionactor was highly over expressed in the cytosol (Fig. 7A).

.7. Translocation of Bax from cytosol toitochondria without affecting the expression ofcl-2 by CFO and ISO in HL-60 cells

Bax is a pro-apoptotic protein that exists as a sol-ble monomer in cytosol or is loosely associated withitochondria. However, upon apoptotic stimulation, Bax

ranslocates to mitochondria where it forms oligomers

teractions 171 (2008) 332–347 341

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 was not changedeither by CFO or ISO (Fig. 7B).

3.8. Enhanced mitochondrial expression ofcytochrome c prevents early loss of mitochondrialmembrane 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 first hour of treatment; we askedif the perturbation of mitochondrial membrane poten-tial is correlated with these events. We analyzed thechange in mitochondrial membrane potential �ψm byflow cytometry using the fluorescent lipophilic cationicprobe JC-1 (5,5,6,6-tetrachloro-1,1,3,3 tetraethyl ben-zimidazolcarbocyanine iodide), and found that CFOtreated cells observed a significant loss of mitochondrialmembrane potential, maximum being at 3 h followed bya gradual decrease through 6 h period of study while ISO,which followed similar trend, relatively was less effec-tive (Fig. 8A). The loss of �ψm was also confirmed byanother dye rhodamine-123 and the results obtained werecomparable 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 �ψm,when cytochrome c is an important component of elec-tron transport chain [25]. In this concern we made twoimportant observations, 1) that both CFO and ISO enablecells to over express cytochrome c in the mitochondriaand 2) that outer mitochondrial membrane simultane-ously enabled the release of cytochrome c into the cytosolwithout undergoing any significant early loss of �ψm(Fig. 9). Similarly enhanced mitochondrial expressionof cytochrome c was observed in Molt-4 and HeLa cell

lines (Data not shown).

In order to understand whether other proteins relatedto mitochondrial electron transport chain such as Cox-IVare also affected by the treatment with CFO and ISO, we

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

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 fluorimetrically in thecell lysates of HL-60 cells using BD ApoAlert caspase fluorescent assay kits. Specific peptide based inhibitors provided along with the assay kitswere used for negative control (data not shown) to determine whether fluorescence intensity changes were specific 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 proteins

ne andcribed i

were resolved on 10% SDS-PAGE, then transferred to PVDF membraloading control for protein level. Other conditions were same as desexperiments.

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 ofCOX-IV with time in both CFO and ISO treated HL-60

cells (Fig. 9A). Suppression of COX-IV has been relatedto the inhibition of electron transport chain [27] and itsdecline in mitochondria may sensitize cells to undergoapoptosis [27].

probed with anti-PARP antibody. Anti-body to actin served as samplen Section 2. Western blot is representative from one of three similar

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 in

cancer cells. This study provides valuable insight intothe mechanism of action of the Cymbopogon flexuosusoil (CFO) and its major chemical constituent isointer-medeol (ISO) that are able to trigger apoptosis in cancer

A. Kumar et al. / Chemico-Biological In

Fig. 7. (A) Immunoblot analysis of TNF-R1, DR-4 and NF-�B. HL-60cells treated with CFO and ISO (30 �g/ml) for indicated time periods.Total cell lysates were prepared for TNFR-1 and DR-4 while nuclearextract for NF-�B analysis. Protein samples 50 �g were loaded onSDS-PAGE gel for western blot analysis as described in Section 2.Relative density of each band for NF-�B indicates arbitrary units ofexpression analyzed by Quantity One software of Bio-Rad gel doc-umentation system. P-values: *P < 0.05; **P < 0.01; CFO and ISOtreated vs. untreated control cells. (B) CFO and ISO induced transloca-tion of Bax with no effect on Bcl-2. HL-60 cells were treated with CFOand ISO (30 �g/ml) for indicated time periods. Immunoblot analysisof Bax, Bcl-2 was performed in designated sub-cellular lysate. Equalamount of proteins were loaded and resolved on 15% SDS-PAGE.Other conditions are described in Section 2. Density of each band forBax was calculated as described in (A). Data are mean ± S.D. of threesimilar experiments. *P < 0.05; **P < 0.01 for treated vs. control.

teractions 171 (2008) 332–347 343

cells. We report for the first time that the essential oilfrom this elite variety, present in abundance in nature,has other useful important pharmacological actions too,other than its requirement in food flavor, perfumery andaromatherapy. Our studies demonstrated that CFO is apotential pro-apoptotic agent and hence can be devel-oped into an important anti-cancer lead of therapeuticpotential. This is evidenced from measurement of severalbiological end-points of the apoptosis such as annexinVbinding, appearance of apoptotic bodies, DNA fragmen-tation and increase in sub-G0 DNA faction in HL-60 cellstreated with CFO and ISO within a brief exposure of 1 h.We also used ISO along with CFO as it dominates thecontents of this essential oil and that the pro-apoptoticproperties of the oil may be contributed largely becauseof ISO, though there are several other constituents in thisoil responsible for cell cytotoxicity.

The essential oil produced oxidative stress for a veryshort while, which did not appear to partake in cyto-toxicity because the effect was short lived. Moreoverthe increase in sub-G0 DNA fraction was not rescuedby several anti-oxidants used in the study suggestingother signaling pathways activated by CFO or ISOmight be contributing to the onset of apoptosis. On thecontrary, use of anti-oxidants and in particular of ascor-bate augmented the sub-G0 DNA fraction probably theantioxidants diverted necrotic cells to apoptotic popu-lation. Such an enhanced action of ascorbate on thesusceptibility of HL-60 cells to chemotherapeutic agentshas also been noticed recently by others [28].

Requirement of signaling pathways activated byCFO or ISO appeared to involve both extrinsic andintrinsic cascades as evidenced by strong activation ofboth caspase-8 and -9, respectively. The activation ofcaspase-8 has been associated with upstream increasedexpression of apical death receptors TNFR1 and DR4(TRAIL-R1), which observed time dependent increasein both CFO and ISO treated HL-60 cells, indicating thatactivation of caspase-8 is related to the activated expres-sion of TNFR1 and DR4 (TRAIL-R1). The activationof these receptors might engage Fas associated deathdomain (FADD) and death inducing signaling complex(DISC) allowing the release of active form of caspase-8[29].

The release of cytochrome c from mitochondria tocytosol is essential for the formation of apoptosome andconsequent activation of caspase-9 [12]. Interestinglyboth CFO and ISO augmented the mitochondrial expres-

sion of cytochrome c in HL-60 cells. However, release ofcytochrome c into the cytosol is not accompanied by anyconcomitant decrease in the mitochondria, suggestingthat over expression of cytochrome c precedes apoptotic

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

Fig. 8. CFO and ISO induced loss of mitochondrial membrane potential (Ψm) in HL-60 cells. (A) Measurement of�ψm using JC-1 dye: FACScananalysis of�ψm 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 fluorescence and a concurrent increase in FL-1 fluorescenceare indicative of mitochondrial membrane depolarization as described in Section 2. Data are representative of one of three similar experiments. (B)Measurement of �ψm 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 fluorescence. Cell population in thelower left quadrant indicated mitochondrial depolarized cells. Other conditions were same as described in (A).

A. Kumar et al. / Chemico-Biological In

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) foripwd

dcdaafhca6

twpetatcdscwgoeeosictacb

ndicated time periods. Immunoblot analysis of Cyt-c and COX-IV waserformed in designated sub-cellular lysate. Equal amount of proteinsere loaded and resolved on 15% SDS-PAGE. Other conditions areescribed in Section 2.

eath of cancer cells by CFO and ISO. Increased mito-hondrial cytochrome c expression by certain anti-cancerrugs [30] and as observed in this study may be a mech-nism to compensate its immediate loss to the cytosol,lthough release of cytochrome c is a ‘point of no return’or cancer cell to trigger self-demise. This salient event isighly prominent during the initial period of exposure ofells to CFO and ISO perhaps as means of self-defense tonti-cancer drugs as the cells were not exposed beyondh in our studies.

It may also be mentioned that cytochrome c consti-utes an important link in the electron transport chainhich shuttles electrons between complex III and com-lex IV of the respiratory chain, this movement oflectrons is accompanied by pumping out protons fromhe inner membrane, resulting in a potential differencecross the mitochondrial membranes [31]. The respira-ory protein subunit IV of COX is a part of cytochrome

oxidase complex IV of electron transport chain andown-regulation of COX-IV has been associated withlow down or complete inhibition of electron transporthain [27], leading to loss of �Ψm. Both CFO and ISOere also able to bring about a significant�ψm loss withradual decrease of COX-IV expression. The late effectsn COX-IV seemingly appeared less important consid-ring that onset of apoptosis is a much advanced and anarly event that distinctly is associated with translocationf cytochrome c. Thus both CFO and ISO produce strongtress in the mitochondrial functionality by affecting var-ous components of electron transport chain particularlyytochrome c. Unlike its function as a respiratory elec-

ron carrier, cytochrome c is also reported as a potentntioxidant [32]. With rise in mitochondrial ROS levels,ytochrome c detaches from inner mitochondrial mem-rane and is capable of reducing O2

•− to molecular

teractions 171 (2008) 332–347 345

oxygen. ROS stress generated in mitochondria duringfirst hour of treatment with CFO followed by decline at3 or 6 h of treatment may be attributed to increased mito-chondrial expression of cytochrome c, which by virtue ofits strong antioxidant activity possibly caused quenchingof ROS to rescue cells from DNA damage. Activation ofthe 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 observeddecline 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 ofBax/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 distinctlyeffective in these actions.

NF-�B family of transcription factor plays a centralrole in the regulation of apoptosis, oncogenesis, inflam-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 observed in cytosol in these studies suggestedthat the protein is in complexation with the inhibitoryregulatory proteins restraining its availability for tran-scription activation of several genes. This may be thereason that decreased expression of this transcription fac-

tor is observed in the nucleus. It may be mentioned thatboth CFO and ISO followed similar pathways of produc-ing cytotoxicity in cancer cells. Our studies demonstratethat the essential oil and its major constituents may find

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useful application in the development of anti-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 Cymbopogonflexuosus 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 of Scientific and Indus-trial Research, India, for financial 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 ofresults.

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