Food and Chemical Toxicology 62 (2013) 246–254

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Food and Chemical Toxicology

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Disruption of the PI3K/AKT/mTOR signaling cascade and induction ofapoptosis in HL-60 cells by an essential oil from Monarda citriodora

0278-6915/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fct.2013.08.037

Abbreviations: MC, essential oil from Monarda citriodora; Dwmt, mitochondrialmembrane potential; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazoliumbromide; PI, propidium iodide; PTP, permeability transition pore; Rh-123, Rhoda-mine-123; PBS, phosphate buffer saline; PARP, poly-ADP-ribose polymerase; VEGF,vascular endothelial growth factor; FBS, fetal bovine serum; mTOR, mammaliantarget of rapamycin.⇑ Corresponding authors. Address: Division of Cancer Pharmacology, Indian

Institute of Integrative Medicine, CSIR, Canal Road, Jammu 180001, India. Tel.: +91191 25690001; fax: +91 191 2569333 (S. Bhushan).

E-mail address: sbhushan@iiim.ac.in (S. Bhushan).

Anup Singh Pathania a,b, Santosh Kumar Guru b, M.K. Verma c, Chetna Sharma d, Sheikh Tasduq Abdullah a,e,Fayaz Malik a,b, Suresh Chandra f, Meenu Katoch d,⇑, Shashi Bhushan a,b,⇑a Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi, Indiab Cancer Pharmacology Division, Indian Institute of Integrative Medicine, CSIR, Jammu 180001, Indiac Instrumentation Division, Indian Institute of Integrative Medicine, CSIR, Jammu 180001, Indiad Microbial Biotechnology Division, Indian Institute of Integrative Medicine, CSIR, Jammu 180001, Indiae PK-PD-Toxicology Division, Indian Institute of Integrative Medicine, CSIR, Jammu 180001, Indiaf Genetic Resources & Agrotech Division, Indian Institute of Integrative Medicine, CSIR, Jammu 180001, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 June 2013Accepted 17 August 2013Available online 30 August 2013

Keywords:Essential oilMonarda citriodoraApoptosisPI3K/AKT/mTOR

We have isolated an essential oil from Monarda citriodora (MC) and characterized its 22 chemical constit-uents with thymol (82%), carvacrol (4.82%), b-myrcene (3.45%), terpinen-4-ol (2.78%) and p-cymene(1.53%) representing the major constituents. We have reported for the first time the chemotherapeuticpotential of MC in human promyelocytic leukemia HL-60 cells by means of apoptosis and disruption ofthe PI3K/AKT/mTOR signaling cascade. MC and its major constituent, thymol, inhibit the cell proliferationin different types of cancer cell lines like HL-60, MCF-7, PC-3, A-549 and MDAMB-231. MC was found tobe more cytotoxic than thymol in HL-60 cells with an IC50 value of 22 lg/ml versus 45 lg/ml for thymol.Both MC and thymol induce apoptosis in HL-60 cells, which is evident by Hoechst staining, cell cycleanalysis and immuno-expression of Bcl-xL, caspase-3,-8,-9 and PARP-1 cleavage. Both induce apoptosisby extrinsic and intrinsic apoptotic pathways that were confirmed by enhanced expression of deathreceptors (TNF-R1, Fas), caspase-9, loss of mitochondrial membrane potential and regression of Bcl-2/Bax ratio. Interestingly, both MC and thymol inhibit the downstream and upstream signaling of PI3K/AKT/mTOR pathway. The degree of apoptosis induction and disruption of the PI3K signaling cascadeby MC was significantly higher when compared to thymol.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Cancer represents a disorder that involves deregulation of apop-tosis, proliferation, invasion, angiogenesis and metastasis. Exten-sive research during the past 30 years has revealed that themajority of human cancers are induced by carcinogenic factorspresent in our environment including the food we eat (Aggarwaland Shishodia, 2006; Lopez et al., 2004). The incidence and mortal-ity is increasing on daily bases with alarming numbers. Therefore,

discovery of novel chemotherapeutic agents is highly needed andplants offer a potential source for novel drug discovery. Plants havebeen utilized for their aromatic and medicinal properties for centu-ries with important implications due to their safety and availabilityprofile. Over three-quarters of the world population rely mainly onplants for health care and only 30% of the entire plant species arecurrently used for medicinal purposes. Aromatic plants have beenused for a long time due to their pharmacological importance,which is entirely attributed to their essential oil fractions (Rajeshet al., 2003). Essential oils are constituted by lipophilic compounds,mainly monoterpenes and sesquiterpines hydrocarbons and oxy-genated products of hydrocarbons like alcohols, aldehydes, esters,ethers, ketones, phenols and oxides (Merle et al., 2004). The as-sorted therapeutic potentials of essential oils have attracted theattention of many researchers to explore their anticancer activity.In our experiments we have isolated an essential oil, MC, fromthe aerial parts of Monarda citriodora and characterized its 22chemical constituents in which thymol (82%) was the major com-ponent. We are here reporting the cytotoxic and apoptotic

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potential of essential oil (MC) and thymol in human leukemia HL-60 cells via disruption of the PI3K/AKT/mTOR signaling cascade.

Monarda is a genus consisting of roughly sixteen species oferect, herbaceous, annual or perennial plants of Lamiaceae family.M. citriodora is a flowering plant of the mint family, native to theUnited States and Mexico; common names include lemon bee-balm, lemon mint and purple horsemint. It is typically found inrocky or sandy prairies, pastures and roadsides. The plant heightranges in between 50 and 90 cm, leaves are 3–8 cm long, soft tex-tured, lance shaped and flowers are pink in color. When crushed ithas a very strong and pleasant fragrance like Trachyspermum ammi(ajwain, in Hindi). It is used in salads and as a flavoring agent in tea,wine, liqueurs, cakes, cheesecakes, sauces, pies, certain seafood andmeat dishes such as crab and chicken. This genus has a long historyof use as a medicinal plant with expectorant, anti-bacterial andanti-fungal properties along with suitability as a perfume and fla-voring agent in the food industry. It is the natural source of theantiseptic thymol, a primary active ingredient in modern commer-cial mouthwash formulations. Various investigators had previouslyreported the chemical composition of diverse species and varietiesof Monarda genus e.g. geraniol is a major component of the Mon-arda fistulosa L. var. menthaefolia oil (Marshall and Scora, 1972;Mazza et al., 1987), whereas hybrids M. fistulosa and Monarda didy-ma were rich in geraniol (>92%), linalool (67%), thymol (31%) andcarvacrol (73.5%) (Mazza and Marshall, 1992). The essential oil ofMonarda fragrans contained twenty-five components which in-cluded the a-pinene (22%), b-pinene (21.5%), dabinene(15.4%),myristicin (9.4%) and terpinen-4-ol (5.7%). The essential oil of M.citriodora flowers (1.5% w/w) contains 30 constituents (97.23%) inwhich thymol (44.6%), 1,8-cineole (23.6%), a-phellandrene (4.9%)and b-cymene (4.1%) were the major compounds (Lu et al.,2011). In another study, eighteen compounds were identified inthe essential oil of aerial herbage of M. citriodora in which thymol(70.6%), p-cymene (10.6%), carvacrol (6.1%), 7-octen-4-ol and terpi-nen-4-ol (1.2%) were identified as dominant components (Dormanand Deans, 2004). Essential oil isolated from different variety of M.citriodora has different chemical compositions but thymol remainsone of its major components. Therefore, we have explored the che-motherapeutic potential of MC along with thymol and investigatedwhether the activity of MC was due to thymol or its synergy withthe other components.

2. Materials and methods

2.1. Chemicals and antibodies

3-(4,5,-Dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), propi-dium iodide (PI), DNase-free RNase, bovine serum albumin, Rhodamine-123(Rh123), Hoechst 33258, proteinase-K, penicillin, kanamycin were purchased fromM/s Sigma Chemicals Co., India. Fetal bovine serum was obtained from M/s GIBCOInvitrogen Corporation, USA. Apo-Alert caspases assay kits were purchased from M/s B.D. Clontech. Anti-human antibodies to Bcl-2, Bax, Bcl-xL, PARP-1, caspase-3, cas-pase-8, caspase-9, actin, goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRPwere purchased from M/s Santa Cruz, USA. Antibodies to p110a, p-PTEN (S380/T382), Akt, p-Akt (S473), mTOR, p-mTOR, p-P70S6kinase (T389), p-eIF4E (S209),VEGFR2 were purchased from M/s Cell signalling technology, USA. Electrophoresisreagents and protein markers were purchased from M/s Bio-Rad, USA while ECL re-agents and Hyperfilm were purchased from M/s Amersham Biosciences, UK. Otherreagents used were AR grade and available locally.

2.2. Collection of plant material and isolation of essential oil (MC)

M. citriodora Cerv.ex Lag. was cultivated in the farm house of Indian Institute ofIntegrative Medicine (IIIM), CSIR, Jammu, India (Latitude 32�430N, Longitude74�540E, Altitude 34 m asl). The identification of species was done via leaf, flowerand tuber morphology by taxonomist in the Institute (IIIM). The voucher specimenof the plant has been deposited in the herbarium of IIIM, Jammu. The dried aerialparts of M. citriodora (300 g) were subjected to hydro-distillation for 4 h in a

clevenger type apparatus and distilled oil (MC) was dried over anhydrous Na2SO4.The three replicates of oil sample were stored at 4 �C until used for chemicalanalysis.

2.3. Chemical characterization of MC by GC and GC/MS

GC analysis was performed by using Varian GC-4000 gas chromatograph,equipped with flame ionization detector (FID). The analysis was carried using Var-ian Factor Four VF-5ms fused silica capillary column (30 m � 0.25 mm id, filmthickness 0.25 lM The injector and detector temperatures were 210 �C and280 �C, respectively. Nitrogen was used as the carrier gas at a flow rate of 1 ml/min; oven temperature programmed 50–240 �C at 5 �C/min; finally held isother-mally at 240 �C for 20 min. The identification of compounds was carried out bycomparison of their relative retention time with those of authentic standards.GC–MS analysis was carried out on a Varian GC–MS 4000. Temperature program-ming of the oven was from 50 �C to 240 �C at 5 �C/min rising rate. Helium was usedas the carrier gas at a flow rate of 1 ml/min. Mass spectra were recorded over 50–300 amu range at one scan per second with E.I. at 70 eV. The volatile constituentswere identified by calculation of their retention indices under temperature pro-grammed conditions for n-alkanes (C8–C20). Identification of individual com-pounds was made by comparison of their mass spectra with those of the internalreference mass spectra library (Wiley/NIST) or with authentic compounds and con-firmed by comparison of their retention indices with authentic compounds or withthose of reported in the literature database.

2.4. Identification of plant material by rbcL gene sequence

Genomic DNA of ten M. citriodora plants were extracted individually fromyoung leaf samples (0.2 g) (Ahmad et al., 2004). The leaves were washed in runningtap water and then dried in between filter paper before being used for DNA isola-tion. Estimation of DNA quantity and quality was done by a spectrophotometer(Nanovue, GE Healthcare Biosciences, UK) and agarose gel (0.5%) electrophoresis,respectively. The partial rbcL gene was amplified from the samples of M. citriodoraas described earlier (Savolainen et al., 2000). Amplified fragments of DNA werepurified with gel extraction kit (Qiagen, USA) and both strands were sequenced di-rectly, using the same primers, which were used for PCR, with a Big Dye TerminatorCycle Sequencing reaction kit (v. 3.1, Applied Biosystems, Tokyo, Japan) and anautomated sequencer (Model 310; Applied Biosystems). Resultant sequence(JX254905) was submitted to a gene bank and homology analysis of resultant se-quences was performed by using BLASTn tool of NCBI [http://www.ncbi.nlm.nih.-gov]. The Meg Align program of Laser Gene software (DNASTAR Inc., USA) wasused for multiple sequence alignment. Phylogenetic tree with bootstrap valueswas prepared to find the relative position of Monarda by using genebee Tools(www.genebee.msu.su). Chlorophytum borivilianum (EU311204) was used as outgroup member.

2.5. Cell culture and treatment

Human prostate cancer cell line PC-3, breast cancer cell line MDA-MB-231 andMCF-7, lung cancer cell line A549, normal breast epithelial cell line fR2 and promy-elocytic leukaemia cell line HL-60 were obtained from ECACC, UK. The cells weregrown in MEM/DMEM/RPMI-1640 medium containing 10% FCS, 100 lg/ml kana-mycin and streptomycin. Cells were grown in a CO2 incubator (Thermocon ElectronCorporation, USA) at 37 �C with 5% CO2 gas environment and 95% humidity. Cellsgrown in monolayer cultures were trypsinised with trypsin (0.1% w/v)/EDTA(1 mM) solution. Soon after cells were ready to detach, the trypsin/EDTA solutionwas removed. Cells were dispersed gently by pipetting in complete growth med-ium, centrifuged at 200xg, for 5 min. Cells were dispersed in complete medium inculture flasks and incubated in CO2 incubator. Cells grown in semi-confluent stage(approx. 70% confluent) were treated with test materials dissolved in DMSO whilethe untreated control cultures received only the vehicle (DMSO, < 0.2%).

2.6. Cell cytotoxicity assay

The cell cytotoxicity assay was performed by using MTT dye (Bhushan et al.,2007). Human leukaemia HL-60 cells (2 � 104/200 ll) seeded in 96-well cultureplates were treated with various concentrations of MC and thymol for 48 h. MTTdye was added 4 h prior to experiment termination (250 lg/ml). The MTT formazancrystals that formed were dissolved in 150 ll of DMSO and optical density mea-sured at 570 nm (reference wavelength 620 nm). Cell growth as percentage viabilitywas calculated by comparing the absorbance of treated verses untreated cells.

2.7. Cell cycle analysis

Cells (0.8x106/2 ml) were seeded in 12 well plates and treated 6 h later with MCand thymol at 30, 50 and 70 lg/ml for 24 h. Cells were collected at 400�g for 5 minin a 5 ml polystyrene tube. Cells were washed twice with PBS and fixed in 70% eth-anol for overnight at 4 �C. The following day cells were washed with PBS and incu-bated with RNase (200 lg/ml) at 37 �C for 1.5 h. Cells were stained with PI

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(propidium iodide) in the dark for 30 min and analyzed immediately in a Flowcytometer (FACS Calibur, BD) in list mode on 10,000 events for FL2-A vs. FL2-W.The Sub-G0 (apoptosis), G1, G2, and S phase cell fraction were analyzed by ModFitsoftware (Bhushan et al., 2013).

2.8. Loss of mitochondrial membrane potential

Mitochondrial membrane potential was analyzed by using Rhodamine-123 dye(Rh-123) that accumulate in the mitochondrial matrix due to electrochemical po-tential across the mitochondrial inner membrane. Cells were (0.8 � 106/2 ml)seeded in 12 well plates and treated with MC and thymol for 24 h at 30, 50 and70 lg/ml. The Rhodamine-123 dye was added 40 min prior to experiment termina-tion. Cells were collected at 400�g, washed with PBS twice and analyzed by flowcytometer at the FL-1 channel. Decrease in Rhodamine-123 fluorescence in MCand thymol treated cells represented the loss in mitochondrial membrane potentialwith respect to control.

2.9. Nuclear and cellular morphology

HL-60 cells (0.8 � 106/2 ml) seeded in 12 well plates and treated with MC andthymol for 24 h time period at 30, 50 and 70 lg/ml concentrations. The cellularmorphology was evaluated under an inverted phase contrast microscope. Cellswere collected at 400�g and washed with PBS and fixed overnight at 4 �C in a fixingsolution containing cold acetic acid: methanol (1:3 ratio, v/v). The following daycells were dispensed in 50 ll of fixing solution, spread on a clean cold slide anddried overnight at room temperature. Cells were stained with Hoechst 33258(5 lg/ml in 0.01 M citric acid and 0.45 M disodium phosphate containing 0.05%Tween-20) for 30 min at room temperature. Slides were washed with PBS and whilewet 40 ll of mounting fluid (PBS: glycerol, 1:1) were poured over the slide. Theslides were covered with a cover slip and sealed with nail-polish. Cells were ob-served under the microscope at 30� (Olympus IX70) for alteration in nuclear mor-phology (Bhushan et al., 2006).

2.10. Preparation of whole cell lysates

Cells (2 � 106/5 ml) were seeded in 60 mm culture plate and treated with MCand thymol at 30, 50 and 70 lg/ml concentrations were harvested and resuspendedin a RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 30 mMNa2HPO4, 5 mM EDTA, 0.1% SDS, 50 mM NaF, 0.5 mM NaVO4, 2 mM phenylmethyl-sulfonyl fluoride (PMSF), and 10% protease cocktail inhibitor). Cells were incubatedwith a RIPA buffer maintained on ice for 30 min, vortex and centrifuged at12,000 rpm for 15 min (Bhushan et al., 2007). Supernatants were collected and pro-teins were estimated using Bradford reagent.

Fig. 1. (A) Chemical characterization of essential oil (MC) of Monarda citriodora by meaPhylogenetic trees were constructed by using maximum parsimony (MP) and phylip metbased on rbcL region. The bootstrap values are shown on the branches (100 permutatio

2.11. Preparation of cytosolic and mitochondrial fractions

Cytosolic fractions were prepared by selective plasma membrane permeabiliza-tion with a digitonin buffer. Cells (0.8 � 106/2 ml) were seeded in 12 well plates andtreated with MC and thymol for 24 h. Cells were collected, washed with PBS twiceand then lysed for 1 min in a digitonin buffer (75 mM NaCl, 8 mM Na2HPO4, 1 mMNaH2PO4, 1 mM EDTA, and 350 lg/ml digitonin and 1%(v/v) eukaryotic proteaseinhibitor cocktail). The cell suspension was centrifuged at 12,000 rpm for 2 minand the supernatant collected as the cytosolic fraction. The pellet was resuspendedin a RIPA buffer for 30 min on ice, vortex and centrifuged at 12,000 rpm for 20 min.The supernatant was collected as the mitochondrial fraction and stored at �70 �C(Bhushan et al., 2007).

2.12. Western blotting

Equal amounts of protein (40–70 lg) were resolved on SDS-PAGE and wereelectro transferred to PVDF membranes for 2 h. Membranes were blocked with5% milk or 3% BSA in blocking buffer (10 mM, Tris-HCl, 150 mM NaCl, 1% Tween-20) for 1 h and blotted with their respective primary antibodies for 3 h. Membraneswere washed twice with blocking buffer for 5 min each and then incubated withtheir respective HRP linked secondary antibody for 1 h. Protein bands were detectedusing enhanced chemiluminescence’s reagent (ECL kit, Amersham Biosciences).

3. Results

3.1. The analysis of M. citriodora essential oil (MC)

The essential oil of M. citriodora (MC) was evaluated by GC &GC/MS. A total of 22 compounds were identified, representingapproximately 98.25% of the oil (Figs. 1A and B). The major constit-uents of the MC were thymol (82.29%), carvacrol (4.82%), b-myr-cene (3.45), Terpinen-4-ol (2.77%), and p-cymene (1.53%). Mostof the major compounds in this study were found to be similarto previous reports but with different percentages noted (Dormanand Deans, 2004). The percentage of thymol in our study was com-paratively higher (82.29%) in comparison to other reported plantspecies. This might be due to the variation in the location of plantcollection. Dorman and Deans, 2004 collected their plant materialfrom the commercial supplies in the UK, whereas plants in thisstudy were collected from Jammu, India. Therefore, the currentaccession of M. citriodora contains a rich source of thymol.

ns of GC/MS. (B) GC/MS chromatogram of Monarda citriodora essential oil (MC). (C)hods showing the relationship of Monarda citriodora with other members of lamialesns) Chlorophytum borivilianum was used as out group member.

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3.2. Identification of plant material by rbcL gene sequence

Direct sequence based molecular markers were used to charac-terize the M. citriodora. DNA fragments for the rbcL gene were ob-tained and as expected the length was approximately 701 bp. Thesequence analysis of plastid rbcL gene of M. citriodora showed 98–99% homology with the members of lamiales. A sequence fromgene bank EU311204 (C.borivilianum) was included in the analysisas out group member. A phylogenetic tree with bootstrap valueswas prepared for the members of lamiales (Fig. 1C).

3.3. MC and thymol inhibit cell proliferation of different cancer celllines

The cell cytotoxicity of MC and thymol was evaluated by thecolorimetric cell proliferation assay/MTT assay. In active cells, theMTT dye is reduced into purple color formazan crystals, whichare dissolved into DMSO and then absorbance at 570 nm is mea-sured. Cytotoxic compounds kill cancer cells and that results inlow formazan color production and absorbance. Thymol and MCshowed a concentration dependent decrease in cell viability ofHL-60, PC-3, A-549, MDAMB-231and MCF-7 cell lines. MC has fourtime high IC50 value in normal human breast epithelial fR2 cellsthan in HL-60 cells (Fig. 2). Thymol is more cytotoxic than MC indifferent cell lines except in human leukaemia HL-60 cells wherethe IC50 value of thymol was two times higher in comparison toMC (Fig. 2). Therefore, we were interested to investigate the mech-anism underlying this and other cell death assays in HL-60 cells.

3.4. Thymol and MC induce apoptotic bodies formation

Apoptotic bodies formation is a characteristic feature of cellsundergoing apoptosis (Elmore, 2007). These are membrane en-closed vesicles consisting of damaged organelles and DNA. Induc-

Fig. 2. Cytotoxic profile of MC and thymol in different types of human cancer and normaltreated with indicated concentrations of MC and thymol for 48 h. Cells were incubated wiin the Materials and Methods section. Data are Mean ± SD (n = 8 wells), and are represe

tion of apoptotic bodies by chemotherapuetic agents has alwaysbeen a preferred choice in developing chemotherapeutics (Zhanget al., 2007). Apoptotic bodies were analyzed by visualizing cellularand nuclear morphology. Phase contrast microscopy of MC andthymol treated cells showed cellular blebs and multiple apoptoticbodies, while the untreated cells were round and intact. Nuclearmorphology assessed by Hoechst staining revealed that MC andthymol induce dot-like apoptotic body formation in HL-60 cellsin a dose-dependent manner (Fig. 3). The nucleus of control cellswas round in shape without any apoptotic bodies. Therefore, themode of HL-60 cell death induced by both MC and thymol wasapoptosis.

3.5. Thymol and MC increase sub G0 DNA fraction in cell cycle phasedistribution in Human leukaemia HL-60 and breast cancer MCF-7 cells

MC is more active than thymol in terms of cytotoxicity to hu-man leukaemia HL-60 cells and thymol has lowest IC50 value inMCF-7 cells. So we investigated the mechanistic details of MCand thymol induced cell death in HL-60 and MCF-7 cells. Thymoland MC induced a concentration dependent increase in sub G0DNA fraction as analyzed by ModFit software, indicating apoptoticcell death (Fig. 4). MC treated HL-60 cells at 30 lg/ml showed 18%subG0 population while at the same concentration thymol showedonly 8%. It was clear that the apoptotic degree of MC was nearlytwo times higher in comparison to thymol in HL-60 cells, whereasit was opposite in MCF-7 cells (Fig. 4). The next step was to find outthe early events associated with apoptotic cell death.

3.6. MC and thymol induce loss of mitochondrial membrane potential

Aerobic glycolysis is the most common feature of cancer cellsand elimination of mitochondrial DNA reduces the tumourogenic-ity of cancer cells (Douglas, 2012). Mitochondrial potential loss

human breast epithelial fR2 cells. The cells were grown in 96-well culture plate andth MTT solution and optical density of formazan crystals was measured as describedntative of three similar experiments.

Fig. 3. Effect of MC and thymol on the cellular and nuclear morphology of HL-60 cells. Cells were treated with 30–70 lg/ml concentrations of MC and thymol for 24 h andsubsequently stained with Hoechst 33258 as described in the Materials & Methods section and visualized for nuclear morphology and apoptotic bodies formation.Simultaneously cells were also visualized under a phase contrast inverted microscope for any cellular abnormalities. Condensed nuclei and apoptotic bodies are indicated bythe white and red arrows, respectively. Data are representative of one of three similar experiments and the magnification of the pictures used was 30� on Olympus 1� 70inverted microscopes.

Fig. 4. DNA cell cycle analyses of HL-60 and MCF-7 cells exposed to MC and thymol.Cells (0.8 � 106/2 ml/12 well culture plate) were treated with indicated concentra-tions of MC and thymol for 24 h. After treatment cells were stained with propidiumiodide, PI (10 lg/ml) to determine DNA fluorescence and cell cycle phase distribu-tion as described in the Materials and Methods section. The data were analyzed byModfit software (Verity Software House Inc., Topsham, ME) for the proportions ofdifferent cell cycle phases. The fraction of cells in apoptosis, G1, S and G2 phasesanalyzed from FL2- A vs. cell counts are shown in percentage. Data are represen-tative of one of three similar experiments.

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triggered by thymol and MC in HL-60 cells was analyzed by study-ing the uptake of Rhodamine-123 dye (Rh-123). Damaged cellshave low uptake of the Rh-123 dye and hence low fluorescence.We have excludes the non viable cells by applying the gate on via-ble cells during flowcytometer data analysis. MC treated HL-60cells showed 38% mitochondrial potential loss at 30 lg/ml,whereas thymol showed only 18% potential loss (Fig. 5). Therefore,MC was twice more effective as thymol in triggering mitochondrialpotential loss.

3.7. Mitochondrial dysfunction triggered by MC and thymol isassociated with translocation of mitochondrial apoptotic proteins

Mitochondria play a key role in activation of apoptotic or nonapoptotic cell death. Bcl-2 member family proteins translocatefrom mitochondria to cytosol during apoptotic induction. BothMC and thymol significantly inhibit the expression of anti-apopto-tic protein Bcl-2 and Bcl-xL (Fig. 6A). We explored the effect of MCand thymol on translocation of cytochrome c and Bax in HL-60cells. Cytochrome c expression increased in the cytosol and de-creased in the mitochondria in a concentration dependent mannerin MC and thymol-treated HL-60 cells while the expression of Baxincreased in the mitochondria and decreased in the cytosol(Fig. 6A). MC and thymol drastically reduced the Bcl-2/bax ratioin HL-60 cells from 5 to 0.1 (Fig. 6B). As evident by the resultsabove, the mitochondrial potential dysfunction of MC was signifi-cantly higher than thymol.

Fig. 5. MC and thymol induced loss of mitochondrial membrane potential (Dwmt) in HL-60 cells. Cells (0.8 � 106/2 ml/12 well culture plate) were incubated with theindicated doses of MC and thymol for 24 h. Thereafter, cells were stained with Rhodamine-123 (200 nM), added 40 min prior to experiment termination and analyzed in FL-1channel of flow cytometer by excluding the non viable cells via gate application. Data are representative of one of three similar experiments.

Fig. 6. Influence of MC and thymol on the expression of important proteins involved in the initiation of apoptosis. (A) HL-60 cells (2 � 106/5 ml/60 mm plate) were treatedwith 30–70 lg/ml concentrations of MC and thymol for 24 h. Protein lysates were prepared and electrophoreses as described in the Materials and Methods section. b-actinwas used as an internal control to represent the same amount of proteins applied for SDS-PAGE. Specific antibodies were used for detection of the indicated proteins indesignated cell lysates. Data are representative of one of three similar experiments. (B) Influence of MC and thymol on the Bcl-2/Bax ratio in HL-60 cells. The relative densityof each band was measured as arbitrary units by Quantity One software of Bio-RAD gel documentation system.

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3.8. Thymol and MC induced extrinsic and intrinsic apoptoticsignalling in human leukaemia HL-60 cells

We further explored the mechanistic insights of thymol and MCapoptotic effects in human leukaemia HL-60 cells. Both MC andthymol induced the cleavage of both procaspase-8 and 9 in HL-60 cells in a concentration-dependent manner (Fig. 6A). The cleav-age of procaspase-8 and 9 in MC treated cells started at 30 lg/mlwhile thymol-treated cells showed this effect at 50 lg/ml. Boththese extrinsic and intrinsic apoptotic events merged on caspase-3 activation, which resulted in the PARP cleavage and activation.MC showed a more pronounced effect on caspase-3 and PARPcleavage than thymol at 30 lg/ml concentrations in HL-60 cells(Fig. 6A). These results showed that MC had a more pronounced ef-fect in the caspases activation than thymol in human leukaemiaHL-60 cells as well as inducing apoptosis by both extrinsic andintrinsic activation pathways.

3.9. Inhibition of PI3K/Akt/mTOR pathway by MC and thymol

PI3 K/Akt/mTORpathway is responsible for the proliferation ofthe majority of cancers. Constitutive activation of PI3K/Akt path-way is required for survival of myleiod leukemia cells (Xu et al.,2003). The catalytic subunit of type I PI3K, p110a is mutated inthe majority of cancers. MC and thymol inhibited the expressionof p110-a in a concentration dependent manner in HL-60 cells.MC significantly inhibited the expression of p110a even at 30and 50 lg/ml, whereas thymol showed the same effect on 70 lg/ml concentration (Fig. 7). MC increased the expression of tumor-suppressor gene PTEN in a concentration-dependent manner whilethymol induced the expression at 50–70 lg/ml concentration(Fig. 7). Thymol and MC inhibited the expression of Akt and its acti-vated form (p-Akt, Serine 473) in a dose-dependent manner(Fig. 7). Akt is a serine threonine kinase, which affects downstreamsignalling, that is involved in cell proliferation and growth. Thenutritional sensor of the cell, mTOR is downstream to Akt(Wullschleger et al., 2006). It is mutated in various types of cancersand targeting mTOR is also a hot area in target-based anticancer

Fig. 7. MC and thymol attenuates PI3 K/AKT/mTOR signaling in HL-60 and MCF-7 cells. Cthymol for 24 h, after completion of the treatment time, cells were collected at 400 g, wawere separated by SDS-PAGE and were transferred to PVDF membranes. Western blots osection. Specific antibodies were used for the detection of the proteins of interest and b-aexperiments.

drug discovery. Thymol and MC inhibited the expression of mTORand its active phosphorylated form at serine-2448 in a concentra-tion-dependent manner (Fig. 7). Two downstream targets of mTOR,p70S6 K and eIF4E were also inhibited by MC and thymol at 30 and50 lg/ml concentrations (Fig. 7). mTOR plays a pivotal role in angi-ogenesis initiated by cancer cells during hypoxic conditions viavascular endothelial growth factor (VEGF) signalling. We foundthat both MC and thymol significantly inhibit the VEGFR-2 expres-sion in human leukaemia cells at 50 and 70 lg/ml concentrations(Fig. 7).Overall the degree of inhibition of the PI3-K/Akt/mTORpathway by MC was significantly higher than thymol in HL-60 cellswhereas in MCF-7 cells it was contrary (Fig. 7).

4. Discussion

Cancer is preceded only by cardiovascular and infectious dis-eases as the leading causes of death worldwide. Its cure is still achallenge for the medical world. Leukaemia represented the sixthleading cause of death in the United States in 2013 and can affectpeople at any age (Siegel et al., 2013). Natural products have al-ways been preferred as a source of novel human therapeutics fordecades, and as a result more than 75% of the drugs in the marketare from natural products. Now the aromatherapy or the use ofessential oils in the cure of various diseases has become an attrac-tive choice in recent years. Essential oils are used as antiviral, anti-bacterial or antifungal agents but reports regarding their antitumoractivity have also been published in recent years (Kumar et al.,2008; Cha et al., 2009; Torres et al., 2011; Bakkali et al., 2008).Due to their low toxicity, aromatic nature and abundant availabil-ity it may be an attractive candidate for the treatment of differentcancers. In this work, we tried to explore the comparative analysisof MC, an essential oil of M. citriodora and its major component,thymol in induction of apoptosis and inhibition of PI3K/Akt/mTORpathway in human leukaemia HL-60 cells. We are reporting for thefirst time the mechanism of action of apoptosis via influencing thePI3K/Akt/mTOR signalling cascade in human leukaemia HL-60 cellsby MC. Our study demonstrated that MC and thymol inhibited the

ells (2 � 106/5 ml/60 mm plate) were incubated with the indicated doses of MC andshed once with PBS and cell lysates were prepared by using a RIPA buffer. Proteins

f the above indicated proteins were done as described in the Materials and Methodsctin was used as an internal control. Data are representative of one of three similar

A.S. Pathania et al. / Food and Chemical Toxicology 62 (2013) 246–254 253

cell viability of various cancer cell lines like PC-3, MDA-MB-231,MCF-7, A549 and HL-60 and found to be non toxic in normal breastepithelial fR2 cells (Fig. 1). The cyrotoxic profile of thymol washigher than MC in all the cell lines mentioned above except in hu-man leukaemia HL-60 cells. Therefore, we were interested in find-ing out the reason behind this. So we explored the pro-apoptoticeffect of MC in comparison to thymol in HL-60 cells. We exploredthe apoptosis by means of various biological endpoints like appear-ance of apoptotic bodies and increase in the subG0 DNA fraction.Then we studied possible early events, which may be associatedwith apoptosis and evaluated the extrinsic and intrinsic pathwaysof apoptosis and other major supportive pathways that may belinked like PI3K/Akt/mTOR signalling cascade.

Mitochondria and Bcl-2 family of proteins play a pivotal role inthe induction of apoptosis (Douglas, 2012). Bcl-2 associated pro-teins have both pro-apoptotic and anti-apoptotic effects in cancercells. These proteins regulate mitochondrial outer membrane po-tential and control the release of many apoptotic factors originat-ing in the mitochondria. Both MC and thymol decreased theexpression of mitochondrial associated anti-apoptotic proteinsBcl-2 and Bcl-xL in a concentration-dependent manner (Fig. 6).The down regulation of these two antiapoptotic proteins changesthe symmetry of mitochondria and activates the mitochondrialpermeability transition pores (PTP) and induces the loss of mito-chondrial membrane potential (Wmt). Loss of mitochondrial mem-brane potential is an early apoptotic event and damagedmitochondria relay many signals to downstream elements that ini-tiate intrinsic apoptotic death-signalling in cells. MC-treated HL-60cells show twice higher mitochondrial membrane potential lossthan thymol-treated cells (Fig. 3). The loss of mitochondrial mem-brane potential further triggers the apoptotic machinery via re-lease of pro-apoptotic factors such as cytochrome c and Bax.During apoptosis, Bax is translocated to the mitochondria and cre-ates pores in the mitochondrial outer membrane which results inan increase in mitochondrial permeability (Nechushtan et al.,2001). Both MC and thymol significantly decrease the level ofmitochondrial antiapoptotic proteins like Bcl-2, Bcl-xL and in-crease the expression of pro-apoptotic proteins Bax with concur-rent fall in Bcl-2/Bax ratio (Figs. 6A and B).The lethality of MC onmitochondrial proteins dysfunction was much higher than thymolin HL-60 cells, which we had proven in several previous experi-ments (Figs. 5 and 6).

MC and thymol induce loss of mitochondrial potential, there-fore we studied whether it was augmented with release of cyto-chrome c into the cytosol. During an intrinsic apoptotic stimuluscytochrome c is released from mitochondria into the cytosol thatresulted in the formation of an apoptosome complex, which acti-vate caspase-9, caspase-3 and PARP-1 cleavage (Jiang and wang,2004). The role of caspase in apoptosis is well established and ac-tive caspase-8 or caspase-9 activatevarious effector caspasesincluding caspase-3, -6, and -7, which results in the enhancedexpression of protease activity in the cell and cleavage of variousproteins like PARP-1 (Strasser et al., 2000; Rastogi et al., 2009).MC activates caspase-8 and 9 at 30 lg/ml concentration whereasthymol showed the same effect at 50 lg/ml concentration(Fig. 6A). Hence MC is more effective in inducing an apoptotic re-sponse as compared to thymol. As both MC and thymol activateboth caspase-8 and caspase-9, therefore, we can conclude that theyinduce apoptosis via both intrinsic and extrinsic activation path-ways in HL-60 cells.

Next, we explored the effect of MC and thymol on other majorsupportive pathways linked to apoptosis like PI3K/Akt/mTOR sig-nalling cascade. We are reporting for the first time that MC andthymol inhibit the PI3K/Akt/mTOR signalling in human leukaemiaHL-60 cells. The discovery of novel PI3K/AKT/mTOR inhibitors ishighly intriguing because of the significant altered expression of

these proteins in a variety of tumors, which make them an attrac-tive target for anti-cancer therapy. In recent years, extensive effortshave been made to discover inhibitors of the PI3K/AKT/mTOR path-way for the treatment of cancers and several of these inhibitorssuch as viz. NVP-BEZ-235, GSK-690693, PKI-587, XL-765 and PI-103 are being evaluated in clinical trials. The PI3K/AKT/mTORpathway regulates several cellular functions that are also criticalfor tumorigenesis such as cell proliferation, cell metabolism, angi-ogenesis, cell cycle progression, apoptosis and autophagy (Hen-nessy et al., 2005; Courtney et al., 2010). PI3 Kinases transmitextracellular signals into cells by generating phospholipids, whichcause the phosphorylation and activation of Akt, this relays signalsto mTOR which acts as a sensor for nutrient availability and is in-volved in cell growth (Wullschleger et al., 2006). mTOR is a serine/threonine kinase that stands in a central position on the cross roadfor various cell signal pathways (Ras, PI3K/Akt, VEGF, HIF) (Hayand Sonenberg, 2004). Over activation of mTOR downstreamp70S6K and eIF4E is frequently associated with activation of hy-poxia inducing factor (HIF) which regulates tumor genesis, angio-genesis and tumor growth through VEGF (Semenza, 2003). Thevascular endothelial growth factor (VEGF) which is a ligand forVEGFR1 and R2 is the most potent angiogenic factor to date andplays a major role in tumour and hypoxia induced angiogenesis(McDonald and Choyke, 2003). VEGFR2 mediates the majority ofthe VEGF downstream effects in angiogenesis and it is necessaryfor tumor angiogenesis and macroscopic solid tumor growth. MCand thymol inhibited the expression of p110a, Akt, p-Akt (S-473), mTOR, p-mTOR, p70S6K, eIF4E, VEGFR-2 and increased theexpression of tumour suppressor gene PTEN (Fig. 7). As noted ear-lier in other cell death events the degree of inhibition of the PI3K/Akt/mTOR pathway by MC was higher than thymol in HL-60 cells,whereas in MCF-7 cells it was contrary.

In conclusion, the current study highlights the importance ofessential oil in the discovery and development of novel anticanceragent. The essential oil (MC) of M. citriodora is a rich source of thy-mol (82%) as compared to other Monarda species and we arereporting here for the first time that MC and its major constituentthymol inhibit the PI3K/Akt/mTOR pathway and induce apoptosisvia both extrinsic and intrinsic pathways in human leukaemiaHL-60 cells. MC was found to be more active than thymol in termsof cytotoxicity, induction of apoptosis and disruption of the PI3K/Akt/mTOR pathway. The likely reason behind it may be the synergyof the thymol with the rest of the constituents present in theessential oil (MC). Hence the potential for essential oil (MC) ex-tracted from M. citriodora may be highly encouraging and furtherstudies are certainly warranted by the scientific and medical com-munities for its utilization as a possible future chemotherapeuticagent.

5. Conflict of interest

All authors declare that there are no conflicts of interest in thisstudy.

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