Chemico-Biological Interactions 171 (2008) 332347

Available online at www.sciencedirect.com

An essential oil and its major constituent isointermedeol induce
apoptosis 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, India
b
Department of Biotechnology, Punjab University, Chandigarh 160014, India
Received 20 May 2007; received in revised form 5 October 2007; accepted 18 October 2007
Available online 24 October 2007
Abstract
An essential oil from a lemon grass variety of Cymbopogon exuosus (CFO) and its major chemical constituent sesquiterpene
isointermedeol (ISO) were investigated for their ability to induce apoptosis in human leukaemia HL-60 cells because dysregulation of
apoptosis is the hallmark of cancer cells. CFOand ISOinhibited 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, DNA
laddering, apoptotic bodies formation and an increase in hypo diploid sub-G0 DNA content during the early 6 h period of study.
This could be because of early surge in ROS formation with concurrent loss of mitochondrial membrane potential observed. Both
CFO and ISO activated apical death receptors TNFR1, DR4 and caspase-8 activity. Simultaneously, both increased the expression
of 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 apoptosis
pathways. The easy and abundant availability of the oil combined with its suggested mechanism of cytotoxicity make CFO highly
useful in the development of anti-cancer therapeutics.
2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cymbopogon exuosus; Essential oil; Isointermedeol; Apoptosis; HL-60 cells
1. Introduction
Cancer is the leading cause of death in the world next
to cardiovascular diseases. Cancer cells cleverly evade
self-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).
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 important
for the development of anti-cancer chemotherapeutics
[2]. Of several prescription drugs in use for cancer treat-
ment, almost 75%are derived fromplant species [3,4]. It
is surprising to note that essential oils, which are found
abundantly in nature, have never been exploited for their
anticancer potential, although they have found extensive
use in perfumery, aromatherapy, food and avors, etc.
since ages. Many essential oils or their constituents are
0009-2797/$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2007.10.003
A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347 333
knowntobe the potent antibacterial as well as anti-fungal
agents. The application of essential oils in the anti-cancer
therapy may appear unconventional however, their easy
availability, pleasant aroma and low or insignicant tox-
icity make them more attractive candidates for the long
termtreatment of various chronic ailments. In our efforts
towards the development of novel herbal products for
their anti-cancer potential, we report here for the rst
time, the pro-apoptotic effect of an essential oil and its
usefulness in the development of anticancer therapeutic
leads. The essential oil isolated form the lemon grass
Cymbopogon exuosus is characteristic for its isointer-
medeol presence, which constitutes almost 25% of its
contents. This plant is an East Indian perennial herb
belonging to the family poaceae. Essential oil derived
from this plant is used in various food and aroma indus-
try products. Also present in other diverse essential oils
are some common constituents found in Cymbopogon
exuosus oil (CFO) such as geraniol (20%), geranyl
acetate (12%), limonene (3.5%), -bisabolol (8.4%), all
of which individually have been reported for their cancer
cell cytoxicity [57]. Besides, this natural composition
of CFO also contains limonene known for its immunos-
timulatory activity [8], and borneol for analgesic and
anaesthetic activities [9]. This report is the rst of its
kind in providing insight into the basis of cytotoxicity of
CFO and its major constituent isointermedeol in cancer
cell. As such there is no report on the anti-cancer activ-
ity and molecular mechanism involved in the induction
of apoptosis by any other essential oil and therefore this
study provides green pasture for development of novel
anti-cancer therapeutics.
Apoptosis is a distinct form of cell death [10] that
is regulated by two major pathways. One involves the
execution through cell surface death receptors (TNFR1,
DR4 and CD95), recruiting Fas associated death domain
(FADD), caspase-8 auto activation [11] with down
stream up-regulation of caspase-3, -6 or -7. The sec-
ond pathway is mediated through mitochondria where
small molecules like cytochrome c are releasedtocytosol
through permeability transition pore or through channels
formed in the mitochondrial membrane by Bax leading
to activation of caspase-9 [12,13].
We report for the rst time that the essential oil as
well as its major chemical constituent isointermedeol
induce apoptosis in human leukaemia HL-60 cells and
therefore are potential candidates for the development
of novel anti-cancer therapeutics. The anti-proliferative
effect of CFO and ISO appear to be related to the down-
regulation of NF-B expression, caspases activation
mediated through both apical receptors and mitochon-
drial signaling pathways. Cytochrome c appeared to be
over expressed in mitochondria affecting simultaneous
release in the cytosol triggering apoptosis. Our studies
provide molecular mechanism of action of essential oil
and its major constituent ISO in the cytotoxicity of HL-
60 cells for the rst time, which may be found very useful
for further development for anti-cancer activity.
2. Materials and methods
2.1. Isolation of essential oil of Cymbopogon
exuosus (Nees ex Steud.) Wats [RRL, (J) CF HP]
by hydro-distillation
The Cymbopogon exuosus strain designated as RRL
(J) CF HP is a perennial densely tufted grass with no
signs of any disease and exhibits high survival under
adverse conditions. The grass is grown in our institute
farm. Freshly harvested aerial parts of the Cymbopogon
exuosus (500 g) were charged on a Clevenger type
hydro-distillation glass apparatus (10 L capacity) con-
taining 4 L of water and tted with a condenser. The
contents were heated to boiling temperature and the
hydro-distilled volatile part was collected and separated
(upper layer) from the aqueous portion (0.4% yield).
Triplicate distillation was performed in succession for
each sample of 500 g of herbage at leang stage. The
essential oil was dried over anhydrous sodium sulphate
and stored at 4

C. The separated oil (density 0.89) was

subjectedtogas chromatographyMSanalysis ona silica
capillary column (30 mm0.25 mm), which displayed
the presence of at least 40 peaks; the major constituents
(%) identied are geraniol (20.08), geranyl acetate
(12.20), -bisabolol (8.42) and isointermedeol (24.97).
The other minor constituents include citronellal (0.22),
citronellol (0.25), methyl isoeugenol (0.10), linalool
(1.70), borneol (1.90), camphor (0.07), camphene (1.33),
geranial (0.45), neral (0.43), caryophylleneoxide (0.36),
limonene (3.47), methyl ether eugenol (1.25), -
phellandrene (0.21), -pinene (0.63), -pinene (0.01),
piperitone (0.06), neryl acetate (3.44), sabinene (0.60),
allo-ocimene (0.06), myrcene (0.37), caryophyllene
(1.78), -humulene (0.26), (Z)-ocimene (1.78), (E)-
ocimene (0.03), geranyl butyrate (0.31), -farnesen
(0.38), elemicin (3.75), piperitol (1.66), carene-2 (1.02),
(2-E), farnesol (0.14), (Z)-2-p-menthenol (0.06).
2.2. Isolation of isointermedeol from C. exuosus
oil
The essential oil (2.0 g) obtained from Cymbopogon
exuosus was subjected to column chromatography over
silica gel (150 g, 60120 mesh). The elution of the col-
334 A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347
umn was carried with increasing ratio of ethyl acetate
in hexane (125%). The lower fractions thus obtained
were pooled together on the basis of TLC and rechro-
matographed over silica gel to separate the fraction
containing sesquiterpene isointermedeol, isolated as a
liquid that solidied after some time as white akes
(320 mg), mp 41

C and []
D
+1.2 (c, 2.0 MeOH). The
structure conrmation was completed by comparison of
1
H NMR,
13
CNMR 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-diphenyltetrazolium
bromide (MTT), staurosporine and camptothecin were
purchased from M/s Sigma chemicals Co., St. Louis.
Fetal bovine serum was obtained from M/s GIBCO
Invitrogen Corporation, USA. Other reagents used were
of analytical grade and available locally. AnnexinV-
FITC apoptosis detection kit, Mitochondrial Membrane
Sensor Kit and ApoAlert Glutathione detection kit were
obtained from M/s BD Biosciences while ApoAlert cas-
pases assay kits were from M/s B.D. Clontech. Mouse
anti-human antibodies to Bax (#SC20067), PARP-1
(#SC8007), Bcl-2 (SC7382), TNFR1 (#SC8436), actin
(#SC-8432), goat anti-human DR4 (#SC-6824), goat
anti-rabbit IgG-HRP (#SC2030) and goat anti-mouse
IgG-HRP (#SC2031) were from M/s Santa Cruz,
USA. Mouse anti-NF-B (#554184, clone G96-337)
and cytochrome c (#556433, clone 7H8.2C12) were
from M/s BD, Pharmingin. Anti-COX-IV used was
from M/s ApoAlert cell fractionation kit of Clontech,
CA (#630105). Electrophoresis reagents and protein
markers were fromM/s Bio-Rad, USAwhile Hyper lm
and ECL reagents from M/s Amersham Biosciences,
UK.
2.4. Cell culture, growth conditions and treatment
Human promyelocytic leukaemia cell line HL-60
cells were obtained from NCI, USA. The cells were
grown in RPMI-1640 medium containing 10% FCS,
100 units pencillin/100 g streptomycin per ml medium.
Cells were grown in CO
2
incubator (Thermocon Elec-
tron Corporation, USA) at 37

C with 95% humidity

and 5% CO
2
gas environment. Cells were treated with
test materials dissolved in DMSO while the untreated
cultures received only the vehicle (DMSO, <0.2%, v/v).
2.5. Cell proliferation assay
Cell proliferation was determined using MTT as
described earlier [15]. HL-60 cells 2.5 10
4
/200 l
media in 96-well culture plates were treated with var-
ious concentrations of CFO and ISO for 48 h. The MTT
formazan crystals formed were dissolved in 200 l of
DMSO; OD measured at 570 nm (reference wave length
620 nm). Cell growth as percent viability was calculated
by comparing the absorbance of treated verses untreated
cells.
2.6. Flow cytometric analysis of apoptosis and
necrosis
HL-60 cells (1 10
6
/2 ml) were treated with CFO
and ISO at 30 g/ml for indicated time periods, cells
were collected, washed twice with PBSand suspended in
0.1 ml binding buffer provided with apoptosis detection
kit (BDBiosciences). Cells were stained with annexinV-
FITC antibody and propidium iodide as per instructions
of the manufacturer. Cells were scanned in FL-1 (FITC)
versus FL-2 (PI) channels on BD-LSR ow cytometer
[15] using quadrant statistics for apoptotic and necrotic
cell populations.
2.7. Hoechst 33258 staining of cells for nuclear
morphology
CFO and ISO treated cells (2 10
6
cells/4 ml) were
xed for Hoechst 33258 staining [15]. The slides were
observed for any nuclear morphological changes and
apoptotic bodies under inverted uorescence microscope
(Olympus IX70).
2.8. Measurement of mitochondrial membrane
potential (
m
)
Mitochondrial membrane potential (
m
) was mea-
sured by using a Mitochondrial Membrane Sensor Kit
containing JC-1 dye, as described by the manufac-
turer (BD Bioscience, CA). The cells after treatment
were washed and stained with the dye and analyzed
by Flow cytometry. The MitoSensor reagent aggre-
gates in the mitochondria of healthy cells producing
A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347 335
red uorescence observed in FL-2 channel while cells
with altered mitochondrial membrane potentials, the
MitoSensor reagent remains as monomers in the cyto-
plasm where it uoresced green and analyzed on FL-1
channel. The decrease in FL-2 uorescence also predicts
altered mitochondrial membrane potential. In fact JC-1
is more specic for mitochondrial membrane potential
and more consistent in its response to depolarization,
though we also employed another commonly used dye
Rh-123 where the decrease in FL-1 channel uorescence
depicts depolarization [15].
2.9. Caspase assays
Cells (2 10
6
/3 ml/well, 6-well plate) were incu-
bated with CFO and ISO for indicated time periods. At
the end of treatment cells were washed in PBS and pel-
lets lysed in cell lysis buffer. Activities of caspase-3, -9/6
and -8 in the cell lysates were determined uorometri-
cally, using BD ApoAlert caspase uorescent assay kits.
Caspase-3 and -8 employed uorochome conjugated
peptides DEVD-AFC and IETD-AFC as substrates,
respectively, while caspase-9 employed LEHD-AMC.
Release of AFC (7-amino-4-triuoromethyl coumarin)
and AMC (7-aminomethylcoumarin) were assayed
according to the instructions provided in the Manual
by the supplier. Specic inhibitors were used as nega-
tive control to determine whether uorescence intensity
changes were specic for the activity of caspases. The
peptide based inhibitors used were DEVD-CHO for
caspase-3, IETD-fmk for caspase-8 and LEHD-CHOfor
caspase-9/6.
2.10. DNA cell cycle analysis
Cells were treated for 24 h and collected at 160 g for
5 min in 5 ml polystyrene tube. Cell pellets were washed
once with PBS and xed in 70% ethanol for overnight at
4

C. Cells were again washed, suspended in 250 l of

PBS, incubated with RNaseA at 37

C and stained with

PI. The cells were analyzed for PI-DNA uorescence by
ow cytometerically as described earlier [15].
2.11. DNA agarose gel electrophoresis
DNA fragmentation typical of apoptosis was also
assessed by electrophoresis of extracted genomic DNA
from HL-60 cells. Briey, 2 10
6
cells after vari-
ous treatments were washed in PBS containing 10 mM
EDTA. The pellet was lysed in 250 l of lysis buffer
(100 mM NaCl, 5 mM EDTA, 10 mM TrisHCl, pH 8.0,
5% Triton X-100, 0.25% SDS) containing 400 g/ml
DNase-free RNase and incubated at 37

Cfor 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 of

phenol:chloroform:isoamyl alcohol (25:24:1) for 1 min
and centrifuged at 13000 g for 3 min. The aqueous
phase was further extracted with chloroform and cen-
trifuged. DNAwas precipitated fromaqueous phase with
3 volumes of chilled alcohol containing 0.3 M sodium
acetate at 4

Covernight. The precipitate was centrifuged

at 13000 g for 10 min. The DNA pellet was washed in
80% alcohol, dried, dissolved in 50 l TE buffer and
electrophoresed in 1.6% agarose gel at 50 V, stained
with ethidium bromide and visualized in Bio-Rad gel
documentation system.
2.12. Measurement of intracellular peroxides (ROS)
in HL-60 cells
The level of intracellular peroxides was determined
using dihydrorhodamine 123 (DHR123). DHR localizes
to mitochondria and uoresces when oxidized by ROS,
particularly peroxynitrite, to the positively charged Rho-
damine 123 derivative [16], which can be measured by
ow cytometery. HL-60 cells (2 10
6
/3 ml/well) after
various treatments in 6-well plate were incubated with
DHR123 (10 M) for 30 min. Cells were washed in PBS
and analyzed by ow cytometery.
2.13. Measurement of GSH contents in cells
Intracellular levels of GSH were estimated using
the BD ApoAlert
TM
Glutathione detection kit employ-
ing monochlorobimane (MCB) reagent. Briey, cells
after various treatments were lysed according to man-
ufacturers protocol. Cell lysates were incubated with
2 mM MCB for 3 h at 37

C. Reduced glutathione levels

were assayed uorometrically at 395 nm excitation and
480 nm emissions.
2.14. Preparation of cell lysates for western blots
analysis
Treated and untreated HL-60 cells were centrifuged at
400 g at 4

C, washed in PBSand cell pellets processed

for preparation of cytosolic, mitochondrial and whole
cell lysates as described [17].
2.15. Preparation of cytosolic and nuclear extracts
for NF-B immunoblot analysis
HL-60 cells (5 10
6
) were washed with ice-cold
phosphate-buffered saline after various treatments and
336 A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347
centrifuged. Cell pellets were homogenized in 200 l of
the buffer and processed for the preparation of cytosolic
and nuclear lysates [18].
2.16. Western blot analysis of PARP1, DR4,
TNFR1, NF-B, cytochrome c, COX-IV, Bax, Bcl-2
and actin proteins
The above lysates were resolved on SDS-PAGE anal-
ysis and electro-transferred to polyvinylidene diuoride
(PVDF) membranes (Bio-Rad) over night at 30 V, 4

C.
The membranes were blocked in blocking buffer (10 mM
TrisHCl, 150 mM NaCl, 0.1% Tween-20) containing
5% milk for 1 h and blotted with respective anti-human
primary antibodies for 2 h. Blots were washed in TBS
and incubated with horseradish peroxidase-conjugated
secondary antibody. Protein bands were detected using
enhanced chemiluminescences reagent, ECLkit (Amer-
sham Biosciences). The density of the bands was
arbitrarily quantied using Quantity One software of
Bio-Rad gel documentation system.
The protein contents were determined using Brad-
ford reagent (Bio-Rad protein assay kit) and aliquots
normalized to equal quantities before loading.
2.17. Statistical analysis
Data are presented as mean S.D. of the number
of experiments indicated. The comparisons were made
between control and treated cultures using unpaired stu-
dent t test and the difference was considered to be
statistically signicant if the P-value was <0.05.
3. Results
3.1. Cymbopogon exuosus oil (CFO) and ISO
inhibit cell proliferation
Both CFO and ISO were able to inhibit HL-60 cell
proliferation after 48 h with IC
50
values of approxi-
mately 30 and 20 g/ml, respectively. DMSO used as
delivery vehicle (<0.2%, v/v), did not affect the cell
growth when treated for the same time period (Fig. 1A).
Further, the effect of CFOandISOontime dependent cell
viability through 48 h was also examined at 30 g/ml;
both inhibited cell proliferation by about 27% at 6 h
(Fig. 1B).
3.2. CFO and ISO induce apoptosis
In order to understand the mechanism of cytotox-
icity of the essential oil and its major constituent
isointermedeol, we thought that the herbal composi-
tion might be inducing apoptosis in cancer cells. For
this purpose various biological end-points of apop-
tosis were analyzed during an early 6 h period of
exposure.
3.2.1. Induction of DNA fragmentation typical of
apoptosis
Both CFO and ISO induced concentration dependent
DNA laddering of 180200bp in HL-60 cells treated for
6 h. The minimal concentration inducing DNAfragmen-
tation was evident at 3 g/ml (Fig. 1C).
3.2.2. CFO and ISO alter nuclear morphology
observed by Hoechst staining
Hoechst 33258 stain selectively binds DNA and
allows monitoring of nuclear morphological changes
under uorescence microscopy. Both CFO and ISO at
30 g/ml induced nuclear condensation and blebbing in
HL-60 cells after 6 h of treatment (Fig. 1C). The pres-
ence of apoptotic bodies observed in cells treated with
both CFO and ISO conrmed that these herbal products
trigger cell demise by apoptosis.
3.2.3. CFO induces externalization of phosphatydyl
serine in HL-60 cells
AnnexinVbinding of phosphatydyl serine of exposed
cells is an early indicator of cells undergoing apoptosis.
Therefore, HL-60 cells were incubated with different
concentrations of CFO for 6 h, and the percentage of
cells undergoing apoptosis/necrosis was determined by
staining with annexinV-FITC and PI by ow cytome-
tery [15]. Both CFO and ISO produced comparable time
dependent increase in apoptotic cell population and at the
end of 6 h treatment more than 30% cells were apoptotic
(Fig. 2). It was interesting to note that neither CFO nor
ISO produced any necrosis and that cell death occurred
mainly by apoptosis.
3.2.4. CFO and ISO increase sub-G0 population
without blocking any cell cycle phase
Based on our above results, we used 30 g/ml as the
suitable dose of CFO and ISO for further interrogat-
ing in vitro mechanistic studies. Treatment of HL-60
cells with CFO and ISO observed a time dependent
increase in hypodiploid sub-G0 DNA fraction indicat-
ing apoptotic fraction due to DNA fragmentation. The
relative increase in this fraction was more with ISO
over CFO (Fig. 3). No initial blockage of G1, S and
G2/M cell cycle phases was elicited either by CFO or
ISO.
A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347 337
Fig. 1. CFO and ISO inhibit cell proliferation and induce apoptosis in HL-60 cells. (A) Cell proliferation: HL-60 cells (2.5 10
4
/well) grown in
96-well culture plate were incubated with indicated concentrations of CFO and ISO for 48 h. Cell proliferation was assessed by MTT reduction
assay 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 cell
proliferation: cells were incubated with CFO and ISO at 30 g/ml for different time periods. The treatment was staggered to incubate cells with
MTT at the same time. All other conditions were same as described in (A). (C) CFO and ISO induce DNA fragmentation. Fragmentation of genomic
DNA was studied in HL-60 cells exposed to the indicated concentrations of CFO and ISO for 6 h. Genomic DNA was isolated and electrophoresed
as described in Section 2. DNA from camptothecin (4 M) treated cells was used as positive control. The data are representative one of three similar
experiments. (D) Effect of CFO and ISO on the nuclear morphology of HL-60 cells. HL-60 cells were treated with 30 g/ml of CFO and ISO each
for 6 h and subsequently stained with Hoechst 33258 as described in Section 2. Cells were observed under uorescence microscopy (30). Both
CFO and ISO induced the formation of apoptotic bodies as indicated by arrows. Data are one of two similar experiments.
3.3. CFO and ISO bring about early surge of ROS
formation in HL-60 cells
A vast majority of cellular ROS produced in cells
originates from mitochondria during conditions that dis-
rupt mitochondrial electron transport [19]. Cells treated
with CFOand ISOat 30 g/ml for different time periods.
Cells were stained with DHR-123 [16] and FACscanned
in FL-1 channel of the ow cytometer for rhodamine-
123 positive cell population. Both CFO and ISO were
found to enhance oxidative stress during the rst 30 min,
followed by gradual decline over next 6 h of treatment.
The level of ROS generation was found to be marginally
higher in CFO treated cells compared to ISO treated
cells (Fig. 4A). The initial upsurge in ROS followed
by gradual decrease was also conrmed by another dye
DCFH-DA. The relative magnitude of the initial ROS
measured by this dye however was almost 4-fold lower
338 A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347
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 10
6
/ml) were incubated with 30 g/ml of CFOand ISOfor indicated time periods and stained with annexinV-FITC/PI as described
in Section 2. Quadrant analysis of uorescence intensity of gated cells in FL-1 vs. FL-2 channels was from 10,000 events. Cells in the lower right
quadrant represented apoptosis while in the upper right quadrant indicated post-apoptotic necrosis. FACScan is representative one of three similar
experiments. (B) Quantitative presentation of apoptosis induced by CFO and ISO in HL-60 cells taken from the lower right quadrant of the given dot
plot analysis. Data are mean S.D. of three similar experiments. Other conditions were same as described in (A). Statistical signicance: *P-value
<0.001 compared to untreated control.
A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347 339
Fig. 3. CFO and ISO increased hypodiploid Sub-G0 cell population in HL-60 cells measured by ow cytometry. HL-60 cells (1 10
6
/ml) in culture
were treated with CFO and ISO each (30 g/ml) for indicated time periods. Cells were stained with PI to determine DNA uorescence by ow
cytometery as described in Section 2. Sub-G0 population indicative of DNA damage was analyzed from the hypo diploid fraction (<2n DNA) of
DNA cell cycle analysis. Data are representative one of three similar experiments.
(not shown) than observed with DHR-123. It is known
that DHR-123 is more specic for mitochondrial ROS
while DCFH-DA measures ROS largely localized in the
cytoplasm where DCF is mainly trapped. This further
suggested that mitochondria may be the primary target
of CFO toxicity, and prompted us to nd out whether
early surge in ROS may be involved in the apoptotic
signal triggered by CFO and ISO.
3.4. CFO induced apoptosis is not protected by
anti-oxidants
To know if the apoptosis induced by CFO and ISO is
because of early ROS generation, we treated HL-60 cells
with various antioxidants for 1 h prior to treatment with
CFO or ISO at 30 g/ml for 6 h. The studies revealed
that various antioxidants used did not have any inu-
ence on the extent of sub-G0 hypodiploid DNA fraction
in CFO and ISO treated HL-60 cells (Fig. 4B and C). On
the contrary prior treatment with ascorbate increased the
sub-G0 population in cells. These studies suggested that
any ROS formed are not protected by the anti-oxidants
during this early period of exposure and may thus not
be involved directly in the apoptotic death by CFO. Cor-
respondingly, there was hardly any potential decline in
GSH level (Fig. 5), while at this time most of the cells
suffered remarkable apoptotic features. GSH is impor-
tant for maintaining redox state in the cell and this was
signicantly decreased in cells treated with BSOused as
positive control.
3.5. CFO and ISO bring about early activation of
initiator and executioner caspases leading to PARP1
cleavage
Both CFO and ISO produced an early time depen-
dent increase in caspase-3 activity (Fig. 6A), the effects
being comparable and registered almost 4-fold increase
after 6 h treatment of cells. In the caspase-dependent
pathway 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 proteolytic
cleavage of downstream substrate PARP1, an end-point
of apoptosis (Fig. 6D). Caspase-3 activity is known to
be up-regulated through a variety of signaling cascades
340 A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347
Fig. 4. (A) Effect of CFO and ISO on the generation of ROS in HL-60 cells. HL-60 cells (1 10
6
/ml) were treated with 30 g/ml of CFO and
ISO each for indicated time periods. Cells were stained with DHR123 (5 M) and 10000 events acquired, and gated population analyzed using
BD-LSR ow cytometer. Other conditions were same as described in Section 2. Representative data of three independent experiments are shown. (B
and C) Effect of antioxidants on Sub-G0 DNA fraction of CFO and ISO treated HL-60 cells. HL-60 cells (1 10
6
/ml) were treated with 30 g/ml
of CFO (B) and ISO (C) in 12-well culture plates for 6 h. One hour prior to the treatment, cells were incubated with various antioxidants (Tiron,
1 mM; Trolox, 200 M; NAC, 5 mM; Ascorbate, 5 mM). After treatment cells were stained with PI for FACS analysis. Other conditions are same
as described in Section 2. Data represented are mean S.D. of three similar experiments taken from sub-G0 population of the histogram FLA-2 vs.
cell counts. *P<0.05; **P<0.01 for treated vs. control.
emanating from caspase-8 and -9 activation. We there-
fore, examined for any corresponding increase in the
activity of either caspase-8 or -9 following treatment
of HL-60 cells with CFO and ISO over a period of 6 h
(Fig. 6AC). HL-60 exposed to CFO and ISO displayed
higher than 2-fold caspase-8 activity at 6 h. Similarly the
activity of caspase-9 observed a time dependent increase
of about 2.5-fold (Fig. 6C). In general, ISO treated cells
expressed higher caspase-8 and -9 activities over CFO
treated cells.
A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347 341
Fig. 5. Effect of CFO and ISO on GSH content in HL-60 cells. HL-
60 cells (3 10
6
/well, 6-well plate) were treated with CFO and ISO
(30 g/ml) for indicated time periods. Cells were extracted for deter-
mination of reduced glutathione contents as described in Section 2.
Data are mean S.D. of three similar experiments.
3.6. CFO and ISO appeared to have similar effects
in regulating the expression of apical death
receptors DR4 and TNFR1 in HL-60 cells
Because of increased activity of caspase-8, it was
interesting to analyze the expression of TNFR1 and DR4
proteins in CFO and ISO treated cells. Death receptors
DR4 (TRAIL-R1) and TNFR1 can deliver a powerful
and rapid pro-apoptotic signal through death domain
(DD) mediating recruitment of FADD and formation
of so called death inducing signaling complex (DISC)
[21]. FADDin turn can activate procaspase-8 and deliver
the pro-apoptotic signal down stream by either directly
activating executioner caspases or indirectly by helping
release of cytochrome c. Both CFO and ISO produced a
signicant increase in the expression of both TNFR1 and
DR4 (TRAIL-R1) in a time dependent manner (Fig. 7A).
Expression of both the receptors was found to be higher
in ISO treated cells when compared to CFO. We also
analyzed the level of transcription factor NF-B in the
nucleus of both CFO and ISO treated cells. The nuclear
level of NF-B observed a time-related decline with
maximal effect being at 6 h of study while ISO exerted
almost similar effect. On the contrary, the transcription
factor was highly over expressed in the cytosol (Fig. 7A).
3.7. Translocation of Bax from cytosol to
mitochondria without affecting the expression of
Bcl-2 by CFO and ISO in HL-60 cells
Bax is a pro-apoptotic protein that exists as a sol-
uble monomer in cytosol or is loosely associated with
mitochondria. However, uponapoptotic stimulation, Bax
translocates to mitochondria where it forms oligomers
that are inserted into the outer mitochondrial membrane
[22], and forms pores leading to release of cytochrome
c from mitochondria to cytosol. Bcl-2 on the other hand
is an anti-apoptotic protein, which prevents the release
of cytochrome c from the mitochondria. In fact this is
the ratio rather than amount of pro-apoptotic and anti-
apoptotic proteins that determines whether apoptotic
signaling can proceed [23,24]. We therefore analyzed
the expression of Bax in the cytosolic and mitochon-
drial fractions by immunoblot analysis. Both CFO and
ISO decreased the cytosolic expression of Bax with cor-
responding rise in the mitochondria (Fig. 7B). It was
interesting to note that Bcl-2 expression was not changed
either by CFO or ISO (Fig. 7B).
3.8. Enhanced mitochondrial expression of
cytochrome c prevents early loss of mitochondrial
membrane potential
Our data on annexinV analysis, sub-G0 DNA frac-
tion and activation of caspases suggested that induction
of apoptosis by CFO and ISO in HL-60 cells is an early
event beginning within rst hour of treatment; we asked
if the perturbation of mitochondrial membrane poten-
tial is correlated with these events. We analyzed the
change in mitochondrial membrane potential
m
by
ow cytometry using the uorescent lipophilic cationic
probe JC-1 (5,5,6,6-tetrachloro-1,1,3,3 tetraethyl ben-
zimidazolcarbocyanine iodide), and found that CFO
treated cells observed a signicant loss of mitochondrial
membrane potential, maximum being at 3 h followed by
a gradual decrease through 6 h period of study while ISO,
which followed similar trend, relatively was less effec-
tive (Fig. 8A). The loss of
m
was also conrmed by
another dye rhodamine-123andthe results obtainedwere
comparable to JC-1 (Fig. 8B). On the contrary activa-
tion of caspase-9 requires cytochrome c that is released
through permeabilized outer mitochondrial membrane
or due to loss mitochondrial membrane potential
m,
when cytochrome c is an important component of elec-
tron transport chain [25]. In this concern we made two
important observations, 1) that both CFOand ISOenable
cells to over express cytochrome c in the mitochondria
and 2) that outer mitochondrial membrane simultane-
ouslyenabledthe release of cytochrome c intothe cytosol
without undergoing any signicant early loss of
m
(Fig. 9). Similarly enhanced mitochondrial expression
of cytochrome c was observed in Molt-4 and HeLa cell
lines (Data not shown).
In order to understand whether other proteins related
to mitochondrial electron transport chain such as Cox-IV
are also affected by the treatment with CFOand ISO, we
342 A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347
Fig. 6. (AC) CFO and ISO induce caspases activation in HL-60 cells. HL-60 cells (2 10
6
/3 ml) were exposed to CFO and ISO at 30 g/ml for
indicated time periods for assay of caspase-3 (A), caspase-8 (B) and caspase-9 (C) activities. The activities were determined uorimetrically in the
cell lysates of HL-60 cells using BD ApoAlert caspase uorescent assay kits. Specic peptide based inhibitors provided along with the assay kits
were used for negative control (data not shown) to determine whether uorescence intensity changes were specic for the activity of caspases as
described 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 induce
PARP1 cleavage. HL-60 cells were treated with CFO and ISO (30 g/ml) for indicated time periods. Equal amounts of total cell lysate proteins
were resolved on 10% SDS-PAGE, then transferred to PVDF membrane and probed with anti-PARP antibody. Anti-body to actin served as sample
loading control for protein level. Other conditions were same as described in Section 2. Western blot is representative from one of three similar
experiments.
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 complex
IV. This complex is one of the major sites for oxidative
phosphorylation [26]. We observed a down-regulation of
COX-IV with time in both CFO and ISO treated HL-60
cells (Fig. 9A). Suppression of COX-IVhas been related
to the inhibition of electron transport chain [27] and its
decline in mitochondria may sensitize cells to undergo
apoptosis [27].
4. Discussion
Much of the contemporary research in the develop-
ment of anticancer therapeutics from plants has been
focused on investigating the molecular mechanism by
which an agent induces cytotoxicity and apoptosis in
cancer cells. This study provides valuable insight into
the mechanism of action of the Cymbopogon exuosus
oil (CFO) and its major chemical constituent isointer-
medeol (ISO) that are able to trigger apoptosis in cancer
A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347 343
Fig. 7. (A) Immunoblot analysis of TNF-R1, DR-4 and NF-B. HL-60
cells treated with CFO and ISO (30 g/ml) for indicated time periods.
Total cell lysates were prepared for TNFR-1 and DR-4 while nuclear
extract for NF-B analysis. Protein samples 50 g were loaded on
SDS-PAGE gel for western blot analysis as described in Section 2.
Relative density of each band for NF-B indicates arbitrary units of
expression analyzed by Quantity One software of Bio-Rad gel doc-
umentation system. P-values: *P<0.05; **P<0.01; CFO and ISO
treated vs. untreated control cells. (B) CFOand ISOinduced transloca-
tion of Bax with no effect on Bcl-2. HL-60 cells were treated with CFO
and ISO (30 g/ml) for indicated time periods. Immunoblot analysis
of Bax, Bcl-2 was performed in designated sub-cellular lysate. Equal
amount of proteins were loaded and resolved on 15% SDS-PAGE.
Other conditions are described in Section 2. Density of each band for
Bax was calculated as described in (A). Data are mean S.D. of three
similar experiments. *P<0.05; **P<0.01 for treated vs. control.
cells. We report for the rst time that the essential oil
from this elite variety, present in abundance in nature,
has other useful important pharmacological actions too,
other than its requirement in food avor, perfumery and
aromatherapy. Our studies demonstrated that CFO is a
potential pro-apoptotic agent and hence can be devel-
oped into an important anti-cancer lead of therapeutic
potential. This is evidenced frommeasurement of several
biological end-points of the apoptosis such as annexinV
binding, appearance of apoptotic bodies, DNAfragmen-
tationandincrease insub-G0DNAfactioninHL-60cells
treated with CFOand ISOwithin a brief exposure of 1 h.
We also used ISO along with CFO as it dominates the
contents of this essential oil and that the pro-apoptotic
properties of the oil may be contributed largely because
of ISO, though there are several other constituents in this
oil responsible for cell cytotoxicity.
The essential oil produced oxidative stress for a very
short while, which did not appear to partake in cyto-
toxicity because the effect was short lived. Moreover
the increase in sub-G0 DNA fraction was not rescued
by several anti-oxidants used in the study suggesting
other signaling pathways activated by CFO or ISO
might be contributing to the onset of apoptosis. On the
contrary, use of anti-oxidants and in particular of ascor-
bate augmented the sub-G0 DNA fraction probably the
antioxidants diverted necrotic cells to apoptotic popu-
lation. Such an enhanced action of ascorbate on the
susceptibility of HL-60 cells to chemotherapeutic agents
has also been noticed recently by others [28].
Requirement of signaling pathways activated by
CFO or ISO appeared to involve both extrinsic and
intrinsic cascades as evidenced by strong activation of
both caspase-8 and -9, respectively. The activation of
caspase-8 has been associated with upstream increased
expression of apical death receptors TNFR1 and DR4
(TRAIL-R1), which observed time dependent increase
in both CFOand ISOtreated HL-60 cells, indicating that
activation of caspase-8 is related to the activated expres-
sion of TNFR1 and DR4 (TRAIL-R1). The activation
of these receptors might engage Fas associated death
domain (FADD) and death inducing signaling complex
(DISC) allowing the release of active form of caspase-8
[29].
The release of cytochrome c from mitochondria to
cytosol is essential for the formation of apoptosome and
consequent activation of caspase-9 [12]. Interestingly
both CFOand ISOaugmented the mitochondrial expres-
sion of cytochrome c in HL-60 cells. However, release of
cytochrome c into the cytosol is not accompanied by any
concomitant decrease in the mitochondria, suggesting
that over expression of cytochrome c precedes apoptotic
344 A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347
Fig. 8. CFO and ISO induced loss of mitochondrial membrane potential (
m
) in HL-60 cells. (A) Measurement of
m
using JC-1 dye: FACScan
analysis of
m
loss in HL-60 cells treated with 30 g/ml of CFOand ISOfor indicated time periods. Cells were stained with JC-1 dye and un-gated
population analyzed by Flow cytometry as described in Section 2. A decrease in FL-2 uorescence and a concurrent increase in FL-1 uorescence
are indicative of mitochondrial membrane depolarization as described in Section 2. Data are representative of one of three similar experiments. (B)
Measurement of
m
using Rhodamine-123 dye: HL-60 cells as in (A) were incubated for indicated time periods with 30 g/ml CFO. Cells were
stained with RH-123, 30 min before the termination, and the un-gated population analyzed for decrease in FL-1 uorescence. Cell population in the
lower left quadrant indicated mitochondrial depolarized cells. Other conditions were same as described in (A).
A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347 345
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) for
indicated time periods. Immunoblot analysis of Cyt-c and COX-IVwas
performed in designated sub-cellular lysate. Equal amount of proteins
were loaded and resolved on 15% SDS-PAGE. Other conditions are
described in Section 2.
death of cancer cells by CFO and ISO. Increased mito-
chondrial cytochrome c expressionbycertainanti-cancer
drugs [30] and as observed in this study may be a mech-
anism to compensate its immediate loss to the cytosol,
although release of cytochrome c is a point of no return
for cancer cell to trigger self-demise. This salient event is
highly prominent during the initial period of exposure of
cells to CFOand ISOperhaps as means of self-defense to
anti-cancer drugs as the cells were not exposed beyond
6 h in our studies.
It may also be mentioned that cytochrome c consti-
tutes an important link in the electron transport chain
which shuttles electrons between complex III and com-
plex IV of the respiratory chain, this movement of
electrons is accompanied by pumping out protons from
the inner membrane, resulting in a potential difference
across the mitochondrial membranes [31]. The respira-
tory protein subunit IV of COX is a part of cytochrome
c oxidase complex IV of electron transport chain and
down-regulation of COX-IV has been associated with
slow down or complete inhibition of electron transport
chain [27], leading to loss of
m
. Both CFO and ISO
were also able to bring about a signicant
m
loss with
gradual decrease of COX-IVexpression. The late effects
on COX-IV seemingly appeared less important consid-
ering that onset of apoptosis is a much advanced and an
early event that distinctly is associated with translocation
of cytochrome c. Thus both CFOand ISOproduce strong
stress in the mitochondrial functionality by affecting var-
ious components of electron transport chain particularly
cytochrome c. Unlike its function as a respiratory elec-
tron carrier, cytochrome c is also reported as a potent
antioxidant [32]. With rise in mitochondrial ROS levels,
cytochrome c detaches from inner mitochondrial mem-
brane and is capable of reducing O
2

to molecular
oxygen. ROS stress generated in mitochondria during
rst hour of treatment with CFO followed by decline at
3 or 6 h of treatment may be attributed to increased mito-
chondrial expression of cytochrome c, which by virtue of
its strong antioxidant activity possibly caused quenching
of ROS to rescue cells from DNA damage. Activation of
the intrinsic and extrinsic signaling pathways may thus
be important for apoptotic death, as various antioxidants
used in the study could not prevent DNA fragmentation
induced by CFO or ISO.
Many apoptotic molecules relocate subcellularly in
cells undergoing apoptosis [33] while pro-apoptotic pro-
tein Bax plays an important role. Bax is implicated in the
formation of pores in the outer mitochondrial membrane
after translocation from cytosol allowing subsequent
release of cytochrome c and apoptosis activating fac-
tors [34] leading to caspase-9 activation. We observed
decline in the cytosolic level of Bax with corresponding
increase in mitochondria in both CFO and ISO treated
cells. The level of Bcl-2 however, remained unchanged
in the treated cells. But this is the ratio of Bax/Bcl-2
rather than the level that appears to decide the direction
of apoptotic signal. Our results indicate that the ratio of
Bax/Bcl-2 in CFO and ISO treated cells to be in favor
of apoptosis. Up-regulation of caspase-8 and -9 lead to
activation of downstream executioner caspase-3 and its
cleavage of PARP1 is consequential events culminat-
ing into apoptosis; both CFO and ISO were distinctly
effective in these actions.
NF-B family of transcription factor plays a central
role in the regulation of apoptosis, oncogenesis, inam-
matory and immune responses and is activated by a wide
range 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 nuclear
NF-B dimer p65/rel. The decrease could be related to
the inhibition of activation and therefore poor transloca-
tion to nucleus; it would be our interest to understand
further the effect of CFO and other constituents on NF-
B regulated genes expression in cancer cells. NF-B
in the cytosol normally remains in complex form with
its inhibitory regulatory proteins, which upon activa-
tion by various stimuli is released to nucleus. The over
expression observed in cytosol in these studies suggested
that the protein is in complexation with the inhibitory
regulatory proteins restraining its availability for tran-
scription activation of several genes. This may be the
reasonthat decreasedexpressionof this transcriptionfac-
tor is observed in the nucleus. It may be mentioned that
both CFOand ISOfollowed similar pathways of produc-
ing cytotoxicity in cancer cells. Our studies demonstrate
that the essential oil and its major constituents may nd
346 A. Kumar et al. / Chemico-Biological Interactions 171 (2008) 332347
useful applicationinthe development of anti-cancer ther-
apeutics because of its stability, abundant availability
combined with safety, and oral LD50 >2000 mg/kg b.wt.
mice (not shown).
In conclusion, apoptosis induced by Cymbopogon
exuosus oil (CFO) and isointermedeol (ISO) utilize a
wide range of molecular targets that include both api-
cal receptors and mitochondrial dependent pathways
besides molecules like NF-B involved in cell prolifer-
ation and differentiation. Being an herbal composition,
which already is being used in aromatherapy perfumery
and neutraceuticals, CFO and isointermedeol provide
great potentials to be developed into anticancer thera-
peutics.
Acknowledgements
Thanks are due to the Council of Scientic and Indus-
trial Research, India, for nancial support for senior
research fellowships to Ajay Kumar and Fayaz Malik.
We greatly appreciate the help of Dr. Sarang Bani of our
institute in the use of Flow Cytometery and analysis of
results.
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