Food and Chemical Toxicology 48 (2010) 336–344

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Food and Chemical Toxicology

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Cytotoxic and apoptotic activity of essential oil from Ocimum viride towardsCOLO 205 cells

Madhunika Sharma a, Satyam Kumar Agrawal a, P.R. Sharma a, B.S. Chadha c, M.K. Khosla b, A.K. Saxena a,*

a Oncology Group, Pharmacology Division, Indian Institute of Integrative Medicine, Jammu, J&K 180016, Indiab Botany Division, Indian Institute of Integrative Medicine, Jammu, J&K 180016, Indiac Department of Microbiology, Guru Nanak Dev University, Amritsar, Punjab 143005, India

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

Article history:Received 12 May 2009Accepted 16 October 2009

Keywords:Ocimum virideEssential oilApoptosisScanning electron microscopyCOLO 205

0278-6915/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.fct.2009.10.021

Abbreviations: 5-FU, 5-fluorouracil; DMSO, dimethdiamine tetraacetic acid; EO, essential oil from O. viriDWm, mitochondrial membrane potential; PBS, ppropidium iodide; Rh-123, Rhodamine-123; RPMIinstitute; SRB, sulforhodamine B; SDS, sodium dodeacetic acid.

* Corresponding author. Tel.: +91 9419131757; faxE-mail address: aksaxena2003@yahoo.com (A.K. S

We investigated the apoptosis inducing effect of essential oil (EO) from aerial parts of Ocimum viride inhuman colorectal adenocarcinoma cells (COLO 205 cell line). The COLO 205 cells were exposed to0.0125–0.1 ll/ml of EO for 24, 48 and 72 h. Growth inhibition was determined by sulphorhodamine B(SRB) assay. Double staining with acridine orange and ethidium bromide for nuclear changes was per-formed. Cell cycle analysis and change in mitochondrial membrane potential was quantified by flowcytometry. Subsequently, using annexin V/PI assay, the proportion of cells actively undergoing apoptosiswas determined. Changes in DNA were observed by DNA ladder assay. Eventually the surface morphologyof apoptotic cells was studied by scanning electron microscopy. EO is cytotoxic to COLO 205 cells in doseand time-dependent manner, as is evident by SRB assay. This observed cell death was due to apoptosis, asestablished by annexin V/PI assay, DNA ladder formation and scanning electron microscopy. Our resultsreveal that EO has apoptosis inducing effect against COLO 205 cells in vitro and is a promising candidatefor further anti-cancer study.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Colon cancer is rapidly rising in Asia. The incidence in manyAsian countries is in fact on par with the west. It is defined asany malignant neoplasm arising from the inner lining of the colo-nic epithelium. The occurrence of colon cancer is strongly relatedto age, with 90% of the cases arising in people who are 50 yearsor older (Rajamanickam and Agarwal, 2008). Most colorectal can-cers take approximately 8–10 years to develop from an adenoma-tous polyp into an invasive cancer. It is now the third mostcommon malignant disease in both men and women in Asia (Sung,2007). Conventional treatments such as surgical resection, radia-tion therapy and chemotherapy are not still satisfactory and pre-vention of this disease or at least stopping it at its inception isimportant (Kim et al., 2005). To relieve certain symptoms of cancerand to alleviate the side effects, which come with the use ofconventional treatments, the medical world has now turned to

ll rights reserved.

yl sulfoxide; EDTA, ethylene-de; FBS, foetal bovine serum;hosphate buffer saline; PI,

, Rosewell park memorialcyl sulphate; TCA, trichloro

: +91 191 2569019.axena).

complementary therapies for assistance. Ethno-traditional use ofplant-derived natural products such as essential oils has been amajor source for discovery of potential medicinal agents(Schwartsmann et al., 2000).

Essential oils are known for their antiseptic, antimicrobial, anal-gesic, anti-inflammatory properties. They are also used in embal-ment, preservation of foods and as sedative, spasmolytic andlocally anesthesic remedies. The application of essential oils inthe anti-cancer therapy may appear unconventional however theireasy availability, pleasant aroma and low or insignificant toxicitymake them more attractive candidates for the long term treatmentof various chronic ailments (Kumar et al., 2008). Therefore, thereexists high hope for effective treatment of different cancers by sys-tematic screening of a variety of essential oil based naturalproducts.

The genus Ocimum (Lamiaceae, formerly Labiatae) ranks amongthe most important aromatic medicinal plants from the time of oldcivilization. Being a polymorphic group of economically usefulherbs, it forms a rich source of many naturally occurring essentialoils and aroma chemicals that are of great value in perfumery,flavouring and pharmaceutical industry (Lans, 2006). Ocimumviride, the fever plant of Sierra Leone, is native of Africa and findsan important place in the indigenous system of medicine.Preparations from the whole plant and leaves of O. viride are usedas anticonvulsant, to stop diarrhoea, treatment of catarrh and

M. Sharma et al. / Food and Chemical Toxicology 48 (2010) 336–344 337

bronchitis (Gill, 1988). Hot aqueous extract of O. viride possess antibacterial properties (Isu, 2005). Hot leaves infusion is taken withmilk and sugar as febrifuge and diaphoretic. Leaf decoction is effec-tive against fever and cough. The plant has been introduced at In-dian Institute of Integrative Medicine (formerly RRL), Jammu, Indiaand a genetically improved species was produced in view of di-verse industrial uses and increasing demand of essential oils andtheir isolates. The EO derived from O. viride is a mixture of terpenesand one of the main compounds of this oil is a natural terpenoidthymol. Thymol exhibits multiple biological activities includinganti-inflammatory, immunomodulating, antioxidant, antibacterial,antifungal and free radical scavenging properties (Bukovska et al.,2007). Many terpenes are known to be use in the treatment or pro-phylaxis of cancer (Myers et al., 1997). Therefore we focused ourstudy on the cytotoxicity and apoptotic activities of this essentialoil on COLO 205 human colon adenocarcinoma cells.

2. Material and methods

2.1. Chemicals

RPMI-1640, trypsin, EDTA, gentamycin, penicillin, 5-fluorouracil (5-FU), doxo-rubicin, mitomycin-c, camptothecin, propidium iodide (PI), sulforhodamine B(SRB), phosphate buffer saline (PBS), agarose, ethidium bromide, sodium dodecylsulphate (SDS), proteinase-K, DNase-free RNase A, dimethyl sulfoxide (DMSO), Rho-damine-123 (Rh-123) and foetal bovine serum (FBS) were purchased from SigmaChemical Co., USA. Annexin V – FITC kit was purchased from Axxora, San Diego,CA. Other reagents and chemicals used were of analytical grade and purchasedlocally.

2.2. Plant material

Whole plant of O. viride at full flowering stage was harvested from herbal gar-den of Indian Institute of Integrative Medicine (IIIM), Jammu, India. A specimen ofthe plant was collected and submitted to the herbarium of the Department of Bot-any, IIIM, Jammu (voucher specimen No. 21568) as authenticated by Dr. B. K. Kapahiof Department of Botany, IIIM, Jammu.

2.3. Isolation of essential oil

The essential oil of O. viride (EO) was obtained by hydro-distillation of freshherb in Clevenger apparatus for 2 h 30 min. The oil sample was dried over sodiumsulphate and was used as basic material for the present study. Chemical composi-tion was determined by GC and GC–MS as reported previously (Khosla et al., 2000).

The GC analysis of essential oil was done on NUCON model 5765GC fitted with afused capillary column (30 m � 0.25 mm) coated with FFAP, injector 290 �C, columntemperature programmed from 90 �C (2 min.), 90–210�/7 �C/min, detector temper-ature 310 �C. The GC/MS analysis of oils was performed on QP-2000 Shimadzu mod-el fitted with a fused silica capillary column (20 m � 0.25 mm) coated with SF-30.The GC was run from 70� to 220� at a programmed rate of 40 �C/min using He asthe carrier gas. Components were identified by matching their peaks with publishedmass spectra in the literature (Stenhagen et al., 1974; Heller and Milne, 1978, 1980,1983).

2.4. Cell culture

Human colon adenocarcinoma cell line COLO 205, obtained from National Can-cer Institute, USA was grown at 37 �C in a humidified chamber with 5% CO2 asmonolayer adherent cultures in 75 cm2 tissue culture flasks. Cells were maintainedin RPMI-1640 Medium, supplemented with 10% FBS, 100 units/ml penicillin and100 lg/ml streptomycin (complete medium).

2.5. Cytotoxicity assay

Stock solution of 20 ll/ml of EO was prepared in 2% DMSO in complete med-ium. The stock solution was serially diluted with complete medium supplementedwith 50 lg/ml gentamycin to obtain solutions of desired concentrations. In vitrocytotoxicity against COLO 205 cell line was determined using sulphorhodamineB assay as described previously (Skehan et al., 1990). Briefly, COLO 205 cells wereharvested in log phase using trypsin (0.05% trypsin, 0.02% EDTA, in PBS), countedusing a haemocytometer and 1.5 � 104 cells/well in an aliquot of 100 ll wereseeded in 96 wells cell culture plates. The cells were incubated for 24 h (at 37 �Cin an atmosphere of 5% CO2 and 95% relative humidity in a CO2 incubator) to ad-here. Test materials (100 ll/well) were added after 24 h incubation to the wellscontaining cells. Cells were also incubated with 10 lM mitomycin-c, 1 lM doxoru-

bicin and 20 & 100 lM 5-FU which were used as positive control (Ikebukuro et al.,2000). Suitable controls with equivalent concentration of DMSO were also in-cluded. The plates were further incubated for 24, 48 and 72 h in a CO2 incubatorafter addition of test material. After incubation cells were fixed by gently layeringtrichloroacetic acid (50 ll/well, 50% w/v) on top of the medium in all the wells andincubated at 4 �C for 1 h. The plates were washed five times with distilled waterand air-dried. Cell growth was measured by staining with sulforhodamine B dye(0.4% w/v in 1% acetic acid, 100 ll/well). The unbound dye was washed 3–5 timeswith 1% acetic acid and plates were air dried. The adsorbed dye was dissolved inTris-Buffer (100 ll/ well, 0.01 M, pH 10.4) and plates were gently shaken for10 min on a mechanical shaker. The optical density (OD) was recorded using a96 well plate reader (Sunrise, Tecan, Austria) at 540 nm. Growth inhibition wascalculated by following formula.

% Growth inhibition ¼ 100� ODðtest sampleÞ � ODðblankÞ=ODðcontrolÞ � ODðblankÞ� �

� 100

2.6. Fluorescence microscopy

Morphological observation by fluorescence microscopy was done as describedearlier (Kania et al., 2007). Briefly, COLO 205 (5 � 105 cells/ml) cells were grownin six well plate and allowed to adhere overnight, treated with 0.05 ll/ml and0.1 ll/ml EO and further incubated for 24 h. After incubation, control and EO trea-ted cells were removed from the six well plate by trypsinization, centrifuged andsuspended in PBS at the final concentration of 1 � 105 cells/ml. Fluorescent dyes,ethidium bromide (100 lg/ml) and acridine orange (100 lg/ml) were added andthe cells were incubated for 10 min at room temperature in dark. Cells were ob-served and photographed under an inverted fluorescence microscope (Olympus1X70 magnification 30�, NB filter) using UV excitation.

2.7. Cell cycle phase distribution

Cell cycle phase distribution was done as described earlier (Park et al., 2007).Briefly, COLO 205 cells (1 � 106/ml) in exponential phase of growth were seededin six wells plate and allowed to adhere for 24 h. The old medium was replacedby fresh medium without and with EO in different concentrations and further incu-bated for 24 h. Cells were trypsnised, centrifuged and washed with PBS. Cells werefixed in 70% ethanol, washed with PBS and then incubated with PI (25 lg/ml) withsimultaneous treatment of RNase at 37 �C for 30 min. The percentages of cells hav-ing the sub-G0 population were measured using BD-LSR flow cytometer equippedwith electronic doublet discrimination capability using blue (488 nm) excitationfrom argon laser. Data were collected in list mode on 10,000 events for FL2-A vs.FL2-W.

2.8. DNA ladder assay

Apoptosis was assessed by electrophoresis of extracted genomic DNA from trea-ted COLO 205 cells as described previously with some modifications (Tong et al.,2004). Briefly, COLO 205 cells (1 � 106/ml) after treatment with different concen-trations (0.025–0.1 ll/ml) for 24 h, were centrifuged at 1500 rpm for 10 min, andwashed with PBS. The resultant pellet was suspended in 250 ll of lysis buffer(10 mM EDTA, 50 mM Tris–HCl, 0.5% SDS) for 15 min at 55 �C. Lysed cells were thendigested with proteinase-K (500 lg/ml) at 55 �C for 1 h and followed by incubationwith 200 lg/ml DNase-free RNase at 55 �C for 90 min. The DNA was extracted twicewith 250 ll of phenol:chloroform:isoamyl alcohol (25:24:1) for 1 min and centri-fuged at 12,000 rpm for 5 min. The aqueous phase was further extracted with chlo-roform:isoamyl alcohol (24:1) and centrifuged. DNA was precipitated from aqueousphase with 0.1 volume of 2 M NaCl and 2.5 volumes of chilled ethanol and kept at�20 �C overnight. The precipitated DNA was centrifuged at 12,000 rpm for 10 minand dissolved in Tris-EDTA buffer (pH 8.0) and electrophoresed in 1.5% agarosegel at 50 V for 1.5 h. The gel was photographed using Bio-Rad Gel documentationsystem.

2.9. Annexin V binding

The extent of apoptosis was determined using FITC-labelled annexin V by flowcytometry (Vermes et al., 1995). Cells (1 � 106/ml) were incubated with the desig-nated doses of EO (0.0125–0.1 ll/ml) and camptothecin (1 lM) for 24 h, washedand stained with annexin V-FITC antibody and PI as per the instructions given bythe manufacturer. Stained cells were analyzed with a BD-LSR flow cytometer usingquadrant statistics for apoptotic and necrotic cell populations.

2.10. To measure mitochondrial membrane potential

Mitochondrial membrane potential was assessed using Rhodamine-123.Briefly, COLO 205 (1 � 106/ml) cells were incubated with the designated dosesof EO and 1 lM of camptothecin for 24 h. Cells were washed twice with PBSand incubated with complete medium containing Rhodamine-123 (5 lg/ml) at37 �C for 30 min. Cells were resuspended in PBS. Rh 123 staining intensity was

Conc./ Time dependant cytotoxicity against COLO 205

90100

ion

338 M. Sharma et al. / Food and Chemical Toxicology 48 (2010) 336–344

determined from 10,000 events, analyzed in FL-1 channel on flow cytometer.Intensity of Rh 123 is directly related to mitochondrial membrane potential (Wuet al., 2004).

01020304050607080

0 0.0125 0.025 0.05 0.075 0.1

Concenteration (µl/ml)

% G

row

th In

hibi

t

24 hrs 48 hrs 72 hrs

Fig. 1. Cytotoxicity evaluation of EO against COLO 205 cells. 1.5 � 104/well grownin 96-well culture plate were incubated with indicated concentrations of EO for 24,48, 72 h. Cell proliferation was assessed by SRB assay as described earlier. Time anddose dependent effect of EO on COLO 205 cell proliferation was observed.

2.11. Scanning electron microscopy

Changes of the cell membrane were visualized by scanning electron microscopy(SEM). COLO 205 cells were grown on plastic cover slips for scanning electronmicroscopy in a six well plate and allowed to adhere for 24 h in a CO2 incubatorat 37 �C with 5% CO2. After 24 h the growth medium was replaced with fresh med-ium containing 0.1 ll/ml of EO and further incubated for 18 h. The samples for SEMwere prepared following standard techniques (Rello et al., 2005). Briefly, treatedand untreated cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer(pH 7.3) at 4 �C for 2 h. Washed with washing buffer and cells were post fixed in1% OsO4 in the same buffer at 4 �C for 2 h, followed by dehydration with ascendingconcentrations of ethanol/acetone. The preparation was cleared in amyl acetate andcritical point-dried using CO2, followed by coating with carbon and gold. The sam-ples were then examined and photographed by JEOL 100CXII electron microscopewith ASID at 40 KV.

Mean ± S.D., n = 16.

2.12. Statistical analysis

All in vitro experiments were done in triplicates and each data point representsthe average of at least three independent experiments. All data are reported as themean ± SD. The comparisons were made between control and treated cultures usingunpaired student ‘t’ test and the difference was considered to be statistically signif-icant if the P-value was <0.001.

3. Results

3.1. Chemical composition of the essential oils

The EO obtained by hydro-distillation from whole plant of O.viride was qualitatively and quantitatively analysed by GC–MS.The major constituents of essential oil are thymol, b-caryophylleneoxide, a-pinene, geranyl acetate and 4-terpineol which constitutes95% of the essential oil. Data is shown in Table 1.

3.2. Cytotoxicity of EO on COLO 205 cancer cells

The effects of EO on COLO 205 cells as assessed by the colori-metric SRB assay with different concentrations (0.0125–0.1 ll/ml) are shown in Fig. 1. A dose and time dependent inhibition byEO was observed with IC50 values of 0.070, 0.058 and 0.033 at24, 48, and 72 h, respectively. Data are mean values ± S.D. of threeindependent experiments and calculated by Instat GraphPad soft-ware. DMSO (0.02%, v/v), did not affect the cell growth when trea-ted for the same time periods. Mitomycin-c (10 lM), doxorubicin(1 lM) and 5-FU (20 and 100 lM) which were used as positivecontrol showed 54%, 39%, 40% and 75% growth inhibition, respec-tively, after 48 h treatment. The cytotoxicity of EO was also deter-mined using normal monkey kidney cell line (CV-1) and nosignificant cytotoxicity was observed at 0.1 ll/ml concentration(data not included).

Table 1Main components (%) of the essential oil from the whole plant of Ocimum viride.

Components Percentage

a-Pinene 2.23Limonene 0.18Phellendrene 0.934-Terpineol 1.86Carvone 0.74b-Caryophyllene 0.74Thymol 80.44Geranyl acetate 1.86b-Caryophyllene oxide 9.12

3.3. Morphological analysis by fluorescence microscopy

When COLO 205 cells were treated with EO (0.05 and 0.1 ll/ml)for 24 h, the morphological features of apoptotic cells, such as cellsurface protuberances and nuclear fragments were identified byAO/EtBr staining by fluorescence microscope. Untreated cells hadlarge green nucleus with red–orange cytoplasm (live cells). Cellstreated with 0.05 ll/ml showed live cells and cells with irregulargreen (early apoptotic) and/or orange (late apoptotic) stained frag-mented nuclei with condensed chromatin. Cells treated with0.1 ll/ml showed apoptotic and a majority of uniformly orange-stained cell nuclei (necrotic). The results obtained with AO/EtBrdouble staining are represented in Fig. 2.

3.4. Cell cycle analysis

To further determine and confirm whether or not the growthinhibitory activity of EO was related to induction of apoptosis, sub-diploid (character of apoptosis) fraction was measured by flowcytometry. After the cells were exposed to 0.025–0.1 ll/ml EO for24 h exhibited continuous increase in sub-G0 fraction comprisingboth apoptotic and debris population, implying together the extentof cell death (Fig. 3). The damage was more apparent with higherEO concentration over the period of study. The results indicatedthat, compared with the control, 0.05 ll/ml and 0.1 ll/ml EO treat-ment increased the population of sub-G0 phase from 8.44% to20.55% and 77.06%, respectively. There was hardly any significanteffect after 24 h of treatment on G2/M fraction, which indicatedthat EO does not cause mitotic arrest.

3.5. DNA fragmentation assay

To elucidate whether EO decreases cell survival by the induc-tion of DNA fragmentation, genomic DNA was isolated from cellsexposed to different concentrations of EO from COLO 205 cellsand electrophoresed. DNA fragmentation was observed in dosedependant manner. The efficient induction for apoptosis was ob-served at 0.075 ll/ml and 0.1 ll/ml EO treatment for 24 h by for-mation of a distinct DNA ladder. DNA isolated from untreatedcontrol did not show DNA ladder (Fig. 4).

3.6. Phosphatidylserine externalization by EO

COLO 205 cells incubated with different doses of EO for 24 hand the extent of apoptosis vs. necrosis was analyzed from thequadrant analysis of the cell population stained with annexin

Fig. 3. Cell-cycle analysis of EO treated COLO 205 cells. Briefly, 1 � 106 cells were exposed to different doses (0.025–0.1 ll/ml) of EO for 24 h. Following incubation the cellswere harvested, treated with RNase, stained with PI (25 lg/ml) and analyzed by BD-LSR flow-cytometer. Sub-G0 population indicative of DNA damage was analyzed from thehypo diploid fraction (<2n DNA) of DNA cell cycle analysis. Data are representative one of three similar experiments.

Fig. 2. Morphological analysis of EO treated COLO 205 cells after AO/EtBr dual staining. Morphological changes of COLO 205 following treatment without EO (A) with lowdose: 0.05 ll/ml (B) and high dose: 0.1 ll/ml of EO (C) for 24 h. Adherent cells were trypsinized and stained with acridine orange (100 lg/ml) and ethidium bromide (100 lg/ml). Samples were examined immediately under fluorescent microscope using NB filter with UV excitation. Early apoptotic (arrows), late apoptotic (arrow-heads) andnecrotic (N) cells were observed after EO treatment (B, C). Photographs are typical of three independent experiments, each performed under identical conditions.

M. Sharma et al. / Food and Chemical Toxicology 48 (2010) 336–344 339

V-FITC/PI. Cells in the lower right quadrant represented apoptosisand in the upper right quadrant represented necrosis or post-apop-totic necrosis (Bhushan et al., 2006). Effect of EO was most prom-

inent when COLO 205 cells treated at 0.075 ll/ml, an increase inthe total apoptotic cells to 26.51% as compared to that of control8.67% was observed. Elevation of exposure concentration to

Fig. 4. EO induced DNA fragmentation. Fragmentation of genomic DNA was studiedin COLO 205 cells exposed to the indicated concentrations of EO for 24 h. GenomicDNA was isolated and electrophoresed as described earlier. Data are representativeone of three similar experiments. Lane 1 – control, 2 – 0.025 ll/ml, 3 – 0.05 ll/ml, 4– 0.075 ll/ml and 5 – 0.1 ll/ml. A prominent DNA ladder was observed at 0.075 and0.1 ll/ml treatments.

340 M. Sharma et al. / Food and Chemical Toxicology 48 (2010) 336–344

0.1 ll/ml increases the population of necrotic cells (upper leftquadrant) from 1.05% to 45.06% and a slight increase of post-apop-totic population from 0.08% to 1.88% (Fig. 5; Table 2). COLO 205cells were also exposed to standard drug camptothecin (1 lM) un-der similar conditions for 24 h, which induced 26.42% apoptosis(Table 2). Thus apoptosis is likely to be the primary mode of celldeath induced by EO in COLO 205 cells.

3.7. Loss in mitochondrial membrane potential (DWm)

The disruption of mitochondrial integrity is one of the earlyevents leading to apoptosis. To assess whether the EO affects thefunction of mitochondria, potential changes in mitochondrialmembrane were analyzed by employing Rh-123. As observed inFig. 6, a drop in the mitochondrial membrane potential was ob-

served. EO caused concentration dependent mitochondrial damageand hence the decrease of mitochondrial membrane potential.There was a significant decrease of DWm from 5.10% in control to8.79–57.29%, in treatments ranging from 0.025–0.1 ll/ml after24 h. Camptothecin (1 lM, 24 h), under similar conditions, showed31.43% decrease of DWm. The results illustrate that EO can induceDWm dissipation in a time-dependent manner.

3.8. Alteration of the cell morphology by EO: scanning electronmicroscopy

The morphological changes of COLO 205 cells after exposure toEO (0.1 ll/ml) were determined via scanning electron microscopy.Untreated COLO 205 cells have rough surface with several cellularprocesses. EO treated COLO 205 cells showed a very high degree ofmembrane blebbing as compared to the control cells. There wasalso a uniform loss of cellular processes, smoothening of cell sur-face and formation of numerous apoptotic bodies (Fig. 7).

4. Discussion

In recent years, considerable attention has been focused onidentifying naturally occurring substances capable of inhibiting,retarding, or reversing the process of multistage carcinogenesis.Plant essential oils as antioxidants are believed to reduce the riskof cancer (Bennani et al., 2007). Because of the great number ofconstituents, essential oils seem to have no specific cellular tar-gets. As typical lipophiles, they pass through the cytoplasmicmembrane, disrupt the structure of their different layers of poly-saccharides, fatty acids and phospholipids and permeabilize them.Cytotoxicity appears to include such membrane damage (Bakkaliet al., 2008). Eugenol from Eugenia caryophyllata inhibits the pro-liferation of cancerous cells. Essential oil extracted from Thaimedicinal plants has antiproliferation activity against humanmouth epidermal carcinoma (KB) and murine leukemia (P388)cell lines. Myrica gale essential oil has an anticancerogenic activityon the lung and colon cancer cell lines. Geraniol inhibits DNA syn-thesis and reduces the volume of colon tumours (Bakkali et al.,2008). EO inhibited the cell proliferation and was cytotoxic in aconcentration and time-dependent manner in COLO 205 humancolorectal adenocarcinoma cells. EO did not produce significantcytotoxic effect towards normal cells, suggesting that the anti-cancer activity of EO might be specific to many other types of tu-mour cells such as leukemia, hepatoma, cervix, breast etc. (Wuet al., 2004).

The effect showed by the essential oil was significantly higherthan those showed by thymol, the major constituent of oil (datanot showed). It indicated that, besides thymol, other cytotoxiccompounds present in the oil composition (as b-caryophyllene,b-caryophyllene oxide and geranyl acetate) might be synergisti-cally acting to show the results observed.

Recently interest has been focused on the manipulation ofapoptotic processes in the treatment and prevention of cancer (Yiet al., 2003). An essential oil from a lemon grass variety of Cymbo-pogon flexuosus has been shown to induce apoptosis in human leu-kemia HL-60 cells (Kumar et al., 2008). Apoptosis (programmedcell death) has been well characterized and is thought to proceedthrough a series of conserved steps that involves genetically con-trolled morphological and biochemical events (Schultz and Har-rington, 2003). Early steps include chromatin condensation,nuclear membrane blebbing and cytoplasmic shrinkage. Late stepsinclude loss of adherence, degradation of DNA, condensed cyto-plasm and formation of apoptotic bodies. (Gerchenson and Rotello,1992; Compton, 1992). Apoptotic changes are best seen by electronmicroscopy but can be observed at the light microscopic level

Fig. 5. Flow cytometric analysis of EO induced apoptosis and necrosis in COLO 205 cells using annexin V-FITC/PI double staining. Apoptosis induced by EO is taken from thelower right quadrant of the given dot plot. Data are representative from one of three similar experiments. Quantitative presentation of apoptosis induced by EO in COLO 205cells (Table 1). Values represented as percentage.

Table 2Quadrant analysis of the cell population stained with annexin V-FITC/PI in COLO 205cells.

Cells (%) Control EO (ll/ml) Camptothecin

0.025 0.050 0.075 0.100 1 lM

Live 90.20 81.75 67.80 64.38 32.65 37.59AV 8.67 11.55 22.30 26.51 20.41 26.42PI 1.05 6.41 9.50 8.52 45.06 29.95AV + PI 0.08 0.29 0.40 0.59 1.88 6.04

M. Sharma et al. / Food and Chemical Toxicology 48 (2010) 336–344 341

using nucleic acid-binding dyes, such as acridine orange, ethidiumbromide or propidium iodide (Coles et al., 1993; Hall et al., 1994).

EO induced morphological alterations typical of the apoptoticprocess, membrane blebbing, and chromatin condensation etc.Fluorescence microscopy was done by staining the COLO 205 cellswith AO/EtBr dual stain. The cells were divided into four categoriesas follows: living cells (green nucleus with red orange cytoplasm);early apoptotic (green irregularly nuclei with condensed chroma-

tin); late apoptotic (orange-stained nuclei with chromatin conden-sation or fragmentation) and necrotic cells (uniformly orange-stained cell nuclei) (Kania et al., 2007). Thus, the morphologicalalterations induced by the EO tested were compatible with thepresence of an increasing number of apoptotic cells.

One of the most commonly used techniques for confirmation ofapoptosis is identification of DNA ladders (Wyllie, 1980). Apoptosistypically involves intra-nucleosomal chromatin cleavage by endo-nucleases in multiples of 180 bp leading to DNA fragments produc-ing thereby a typical DNA laddering (Lecoeur et al., 1997). Toelucidate whether EO decreases cell survival by the induction ofDNA fragmentation, genomic DNA was isolated from cells exposedto different concentrations of EO from COLO 205 cells and electro-phoresed. The EO treatment of COLO 205 colon cancer cells indi-cated internucleosomal DNA breakdown, leading to DNAfragmentation as expected for apoptotic cells.

The development of cytofluorimetric approaches to monitorthese multiple cell alterations permits a precise and reliable quan-tification of apoptosis (Petit et al., 1995). Apoptotic cells exhibit

Fig. 6. Analysis of EO induced alterations in mitochondrial membrane potential (DWm). Exponentially growing cells were treated with the indicated concentration of EO for24 h. For the assessment of loss in DWm, trypsinized cells were incubated with the fluorophore Rh-123 (5 lg/ml) for 30 min at 37 �C in dark. The unbound dye was removedby washing the cells with chilled PBS and analyzed by flow cytometry. Graphs show the percentages of Rhodamine-123 negative cells.

342 M. Sharma et al. / Food and Chemical Toxicology 48 (2010) 336–344

some morphological modifications that are readily detected byflow cytometry according to their light scatter properties (FSC/SCC) (Wyllie, 1980; Lecoeur et al., 1997). To further determineand confirm whether or not the growth inhibitory activity of EOwas related to induction of apoptosis, subdiploid (character ofapoptosis) fraction was detected by flow cytometry. The presenceof the sub-G0 peak in the histogram of the DNA distribution indi-cated that a population of cells was dying by apoptosis.

Phosphatidylserine occurs exclusively on the inner leaflet of theplasma membrane in viable cells but becomes exposed on the out-er leaflet usually during the early stages of apoptosis and beforethe loss of membrane integrity. The loss of lipid asymmetry andexposure of phosphatidylserine can be detected using fluoro-chrome-conjugated annexin V (a Ca2+ dependent phospholipidsbinding protein), but because phosphatidylserine exposure also oc-curs during necrosis, simultaneous staining for membrane perme-ability (e.g., with PI) enables the two states to be distinguished(Bhushan et al., 2006). So the flow cytometric analysis of annexinV/PI-labelled cells was done. Healthy cells were negative for bothPI and annexin V. Apoptotic cells were PI negative and annexin Vpositive. Late apoptotic and necrotic cells were positive both forPI and annexin V. COLO 205 cells treated with the EO showed con-

centration dependant increase in apoptotic cells which eventuallylead to necrosis at higher concentration.

Mitochondrial function was indirectly assessed by variation inmitochondrial transmembrane potential using Rh-123, a cell per-meable cationic dye, which preferentially partitions into mito-chondria due to the highly negative mitochondrial membranepotential (DWm). Depolarization of DWm results in the loss ofRh-123 from the mitochondria and a decrease in intracellular fluo-rescence. Mitochondrial depolarization was assessed by flowcytometry using the Rh-123 dye, showing that this parameterwas also altered by treatment with EO, which indicated apoptosisinduction by mitochondrial pathway.

The phenomenon of apoptosis is defined by a series of morpho-logical changes and has been extensively reviewed. The classicalfeatures are best seen by electron microscopy (Wyllie, 1980; Kerret al., 1972). Cytoplasmic blebs appear on the cell surface andthe cell detaches from its neighbours. Eventually, the cells them-selves fragment, with the formation of a number of membranebounded apoptotic bodies which have a diverse appearance, partic-ularly in regard to their size. They are generally oval or round inshape. EO treated COLO 205 cells showed all the prominent fea-tures of apoptosis.

Fig. 7. Effect of EO on cell membrane in COLO 205 cells using scanning electron microscopy (SEM). Untreated COLO 205 cells showed numerous surface projections (arrow)and roughness (R) over the entire cell surface (A, B, C). COLO 205 cells treated with 0.1 ll/ml EO for 18 h revealed smoothening of cell surface (S) and disappearance of surfaceprojections (D, E). Apoptotic bodies (arrow-heads) are evident due to apoptosis (E, F). Magnification: A, D – 2000�; B, E – 4000�; C, F – 6000�.

M. Sharma et al. / Food and Chemical Toxicology 48 (2010) 336–344 343

5. Conclusion

In conclusion EO exhibited a cytotoxic effect on human coloncancer cells COLO 205, causes DNA damage, increase mitochondrialmembrane permeability and eventually induce apoptosis. Themechanistic link between these events remains to be determined.However, the present data point to the importance of EO as a po-tential candidate for colon cancer treatment.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgements

Madhunika Sharma and Satyam Kumar Agrawal would like tothank Council for Scientific and Industrial Research (CSIR), NewDelhi, India, for Senior Research Fellowships.

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