Content uploaded by Dr. Satyam Kumar Agrawal
Author content
All content in this area was uploaded by Dr. Satyam Kumar Agrawal on May 14, 2014
Content may be subject to copyright.
This article was downloaded by: [Satyam Kumar Agrawal]
On: 14 October 2011, At: 02:55
Publisher: Routledge
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Nutrition and Cancer
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/hnuc20
Induction of Apoptosis in Human Promyelocytic
Leukemia HL60 Cells by an Extract From Erythrina
suberosa Stem Bark
Satyam Kumar Agrawal
a
, Madhunika Agrawal
a
, Parduman Raj Sharma
a
, Bishan Datt Gupta
b
, Saroj Arora
c
& Ajit Kumar Saxena
a
a
Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu, India
b
Natural Product Chemistry, Indian Institute of Integrative Medicine, Jammu, India
c
Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar,
India
Available online: 28 Jun 2011
To cite this article: Satyam Kumar Agrawal, Madhunika Agrawal, Parduman Raj Sharma, Bishan Datt Gupta, Saroj Arora &
Ajit Kumar Saxena (2011): Induction of Apoptosis in Human Promyelocytic Leukemia HL60 Cells by an Extract From Erythrina
suberosa Stem Bark, Nutrition and Cancer, 63:5, 802-813
To link to this article: http://dx.doi.org/10.1080/01635581.2011.573900
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to
anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should
be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,
proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in
connection with or arising out of the use of this material.
Nutrition and Cancer, 63(5), 802–813
Copyright
C
2011, Taylor & Francis Group, LLC
ISSN: 0163-5581 print / 1532-7914 online
DOI: 10.1080/01635581.2011.573900
Induction of Apoptosis in Human Promyelocytic Leukemia
HL60 Cells by an Extract From Erythrina suberosa Stem Bark
Satyam Kumar Agrawal, Madhunika Agrawal, and Parduman Raj Sharma
Cancer Pharmacology Division, Indian Institute of Integrative Medicine,
Jammu, India
Bishan Datt Gupta
Natural Product Chemistry, Indian Institute of Integrative Medicine, Jammu, India
Saroj Arora
Department of Botanical and Environmental Sciences, Guru Nanak Dev University,
Amritsar, India
Ajit Kumar Saxena
Cancer Pharmacology Division, Indian Institute of Integrative Medicine,
Jammu, India
In this study, the apoptosis-inducing effect of an alcoholic ex-
tract from Erythrina suberosa stem bark (ESB) was investigated
using human promyelocytic leukemia HL60 cells. Cell viability
was estimated by MTT assay. We found that the ESB inhibited
cell proliferation in a dose- and time-dependent manner. A series
of well-documented morphological changes, such as cell shrink-
age, condensation of nuclear chromatin, and nuclear fragmenta-
tion, were observed by fluorescence microscopy. The gold standard
scanning electron micrographs showed apoptotic bodies and for-
mation of blebs. Cell cycle analysis showed a significant increase
in Sub G
0
population of cells above 50 µg/ml. ESB treatment re-
sulted in a dose-dependent increase in annexin V positive cells.
Increase in intracellular ROS production up to sixfold was de-
tected in ESB-treated HL60 cells by DCFH-DA assay. Dissipation
of mitochondrial membrane potential of intact cells accompanied
by increase in cytosolic cytochrome c was observed, which was fol-
lowed by activation of caspase-9 and -3 but not caspase-8. DNA
fragmentation analysis revealed typical ladders as early as 18 h
indicative of caspase-3 role in the apoptotic pathway. The overall
results suggest that ESB induces mitochondria-mediated intrinsic
apoptotic pathway in HL60 cells and might have therapeutic value
against human leukemia.
Submitted 18 May 2010; accepted in final form 6 March 2011.
Address correspondence to Satyam Kumar Agrawal, Cancer
Pharmacology Division, Indian Institute of Integrative Medicine,
Canal Road, Jammu, India 180001. Phone: +91-9569662155. E-mail:
satyamka@gmail.com
INTRODUCTION
Natural products, especially plant-based products, have fre-
quently been examined as anticancer agents. There exists high
hope for effective treatment of different cancers by systematic
screening of a variety of natural products. Ayurveda, the an-
cient Indian science of health, is based on natural products,
including several phytochemicals for treatment of a variety of
diseases. Reports have appeared showing that plant extracts dis-
played antitumor/anticancer/antiproliferative effects on cultured
human cancer/tumor cell lines (1,2). As part of an ongoing in-
vestigation aimed at anticancer agents from plants, Erythrina
suberosa was studied using in vitro assays for both the cyto-
toxic and apoptotic activity as one approach pursued in this
study, seeking to identify medicinal agents capable of retarding
the cell cycle and/or activating the cellular apoptotic response
in cancerous cells.
Erythrina suberosa Roxb. belongs to the family Fabaceae.
Plants of this genus are known to have cytotoxic activity (3,4).
In India, Erythrina suberosa Roxb. has been used as a very
important medicinal plant for the treatment of various ailments.
Other parts of this plant that are widely used are the roots, leaves,
seeds, and bark. The main chemical constituents of Erythrina
suberosa Roxb. are waxes, sterols, lectins, and isoflavanoides
(5–7). The ethanol extract of the leaves has been reported to have
antitumor activity (8). As the antiproliferative and apoptosis-
inducing effects of Erythrina suberosa on human promyelocytic
leukemia HL60 cells have not been explored, this study was
undertaken to investigate whether Erythrina suberosa extracts
have antiproliferative effects in human promyelocytic leukemia
802
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
INDUCTION OF APOPTOSIS IN HL60 CELLS BY ERYTHRINA SUBEROSA STEM BARK 803
cells and to determine its mechanism of cell death in HL60
cells.
MATERIALS AND METHODS
Chemicals
RPMI-1640, fetal bovine serum (FBS), phosphate buffer
saline (PBS), trypsin, gentamycin, penicillin, adriamycin, camp-
tothecin, propidium iodide (PI), 3-(4,5-dimethylthiazol-2yl)-
2,5-biphenyl tetrazolium bromide (MTT), agarose, acridine or-
ange, hoechst 33258, ethidium bromide, proteinase-K, RNase
A, n-acetyl cysteine (NAC), dimethyl sulfoxide (DMSO), 2
,7
-
dichlorodihydrofluorescein diacetate (DCFH-DA), rhodamine
123 (Rh-123), annexin V-FITC kit, caspase-3, and caspase-8
colorimetric assay kits were purchased from Sigma Chemical
Co. (St. Louis, MO). Mitochondria/cytosol fractionation kit was
procured from Biovision (Mountain View, CA). Cytochrome c
ELISA kit was purchased from Calbiochem (Gibbstown, NJ).
Caspase-6 and -9 colorimetric assay kits were purchased from
Biovision. Other reagents used were of cell culture grade and
procured locally.
Cell Line and Cell Culture
Human leukemia cell line HL60 was procured from the Na-
tional Cancer Institute (Frederick, MD). HL60 cells were rou-
tinely cultured in RPMI-1640, supplemented with 10% FBS,
100 units/ml penicillin, 100 µg/ml streptomycin (complete
medium) in a humidified incubator maintaining 5% CO
2
at
37
◦
C. For experimental purposes, HL60 were harvested by cen-
trifugation for 5 min at 500 g.
Preparation of E. suberosa Extract
Stem bark of Erythrina suberosa Roxb. (Family: Fabaceae;
common name: coral tree; local name: pangra) was identified
and collected locally in April 2005 by Dr. S. N. Sharma. A
specimen of the collection was submitted to the herbarium
of the Department of Botany, Indian Institute of Integrative
Medicine, Jammu, India (voucher specimen no. 20617). The
stem bark of E. suberosa was dried under shade and extracted
with 95% ethanol. The extract was filtered and concentrated us-
ing a rotavapor and evaporated to dryness. The yield of the ex-
tract was 5.325% of dry weight of collected plant material. The
E. suberosa stem bark ethanolic extract (ESB) was dissolved in
DMSO at a stock concentration of 20 mg/ml and stored at 4
◦
C.
This stock was used to derive a series of concentrations used in
this study.
HPLC Profile of Extract Preparation
Extract profile was generated using Waters HPLC, which
included a binary pump (Waters 515 HPLC pumps), inline vac-
uum degasser, thermostatic column compartment, UV detector
(Waters 2996 PDA, Milford, MA), and autosampler (Waters
717 plus autosampler). Samples were injected onto a Li chro-
spher RP-18 (250 mm × 4.0 mm, 5 µm, Merck, Darmstadt,
Germany) column with a mobile phase containing acetonitrile
(mobile phase A) and water (mobile phase B) with the follow-
ing gradient profile: In the first 15 min from 50–65% B, then a
linear rise to 100% of B in next 15 min, followed by 100–50%
of B in last 15 min, flow rate: 1 ml/min; detection wavelength:
280 nm; and injection volume: 10 µl.
Overall Cell Activity—MTT Assay
The MTT assay measures the metabolism of 3-(4,5-
dimethylthiazol-2yl)-2,5-biphenyl tetrazolium bromide to form
an insoluble formazan precipitate by mitochondrial dehydroge-
nases present only in viable cells. Cells were seeded in 96-well
microplates (4 × 10
4
cells/well in 100 µL of medium). ESB
was added to the cells in serial concentrations (25–150 µg/ml)
in quadruplets and incubated for 24, 48, and 72 h and along with
NAC for 48 h. Three h before the required incubation, 25 µLof
tetrazolium dye (MTT) solution (2.5 mg/ml in PBS) was added
to each well. After the completion of incubation, microplates
were centrifuged at 1,000 g for 10 min and the medium was re-
moved by aspiration. Finally formazan crystals were dissolved
in 100 µl DMSO and the absorbance at 540 nm was measured
using a 96-well microplate reader (Sunrise, Tecan, Switzerland).
Untreated cells under similar conditions were used as negative
control. MTT solution with DMSO (without medium and cells)
was used as a blank (9).
Analysis of Cell Death by Fluorescence Microscopy
Hoechst Staining
The morphology of HL60 cells exposed to ESB was ob-
served first under an inverted microscope. Controls and ESB-
treated (50 µg/ml; 18 h) cells were fixed on a glass slide in 4%
paraformaldehyde for 20 min, washed with PBS and stained
with Hoechst 33258 (1 mg/ml in PBS) for 10 min (10). Stained
cells were washed twice with PBS. The changes in nuclei were
observed with a Olympus IX70 (Tokyo, Japan) fluorescent mi-
croscope through a UV filter.
Acridine Orange/Ethidium Bromide Staining
The effect of the ESB on HL60 cells was also analyzed by
nuclear DNA staining. Cell suspensions were obtained after
harvesting and resuspending untreated and ESB-treated (50 and
100 µg/ml; 18 h) cells in complete RPMI-1640 medium at the
final concentration of 1 × 10
5
cells/ml. Fluorescent dyes, ethid-
ium bromide (100 µg/ml), and acridine orange (100 µg/ml)
were added and cells were incubated for 10 min in the dark. Cell
suspension (20 µl) was placed on a microscope slide and pho-
tographed under an inverted fluorescence microscope (Olympus
IX70) through a WB-filter using UV excitation (11).
Scanning Electron Microscopy
The samples for SEM were processed following standard
techniques (12). The cells were fixed in 2.5% glutaraldehyde
in 0.1 M cacodylate buffer (pH 7.3) at 4
◦
C. The cells were
postfixed in 1% OsO
4
in the same buffer at 4
◦
C. Cells were
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
804 S. K. AGRAWAL ET AL.
then dehydrated with ethanol/acetone, cleared in amyl acetate,
critical point-dried using CO
2
, and then coated with carbon and
gold. The processed cells were then examined by a Jeol 100CXII
electron microscope (Tokyo, Japan) with ASID at 40 KV.
Cell Cycle Analysis
HL60 cells in the exponential phase of growth were treated
with ESB (25–100 µg/ml) for 18 h, then harvested by centrifuga-
tion, washed twice with ice-cold PBS, and fixed by 70% ethanol
at −20
◦
C overnight. The fixed cells were then washed twice with
ice-cold PBS and stained with 25 µg/ml of propidium iodide in
the presence of 250 µg/ml RNase A for 30 min (13). Cell cycle
distribution was analyzed using BD-LSR flow cytometer. Data
for 10,000 cells per sample were collected and analyzed us-
ing CellQuestPro analysis software (BD Biosciences, Franklin
Lakes, NJ).
Annexin V/Propidium Iodide (PI) Flow
Cytometric Analysis
Phosphatidylserine exposed on the outside of the apoptotic
cells was determined by an annexin V-FITC apoptosis detection
kit (Sigma Chemical Co., St. Louis, MO), as per the instruc-
tions of the manufacturer. Briefly, following treatments with
ESB for 18 h, cells were harvested by low-speed centrifugation,
washed twice with ice-cold PBS, pelleted, and resuspended in
binding buffer at cell density 5 × 10
5
/ml. To 195 µl of cell
suspension, 5 µl of annexin V-FITC was added, which was
then conjugated and incubated in the dark for 10 min. Cells
were washed with PBS and resuspended in 190 µl binding
buffer and 10 µlofPI(20µg/ml) and incubated for further
15 min in the dark (14). Cells were then analyzed using a BD-
LSR flow cytometer.
Measurement of Intracellular ROS
HL60 cells were treated with ESB as previously de-
scribed. The production of reactive oxygen species (ROS) was
measured using membrane permeable dye 2
,7
-dichlorodi-
hydrofluorescein diacetate (DCFH-DA). The dye was added
to the cells cultured in plates at a final concentration of 5 µM,
and the plates were incubated at 37
◦
C for 1 h. Then the fluo-
rescence intensity was measured by a fluorescence plate reader
with excitation at 485 nm and emission at 530 nm (15).
Mitochondrial Membrane Potential (
mt
)
The change in mitochondria transmembrane potential was
analyzed using Rh-123 dye. Cells were harvested after treat-
ment and washed twice with PBS, followed by incubation with
5 µg/ml Rh-123 for 30 min at 37
◦
C in the dark (16). We fur-
ther determined mitochondrial membrane permeabilization in
HL60 cells treated in the presence and absence of an antioxi-
dant n-acetyl cysteine (NAC). For this, NAC (5mM) was added
to HL60 cells (1 × 10
6
/ml) 1 h before the treatment of ESB
(50 µg/ml) in a 6-well plate for 6 h. Cells were resuspended in
PBS. Rh-123 fluorescence was determined from 10,000 events,
analyzed in FL-1 channel on a BD-LSR flow cytometer.
Cytochrome c Release
Subcellular fractionation was done according to the man-
ufacturer’s protocol (Biovision, Mountain View, CA). Briefly,
treated cells (5 × 10
7
) were collected by centrifugation at 600 g
for 5 min at 4
◦
C and washed with 10 ml of ice-cold PBS. Pellets
were resuspended in 1.0 ml of 1× cytosol extraction buffer mix,
containing DTT and protease inhibitors, further incubated on
ice for 10 min and then centrifuged at 700 g for 10 min at 4
◦
C.
Supernatants were collected into a fresh Eppendorf tube and
centrifuged at 10,000 g for 30 min at 4
◦
C and used as cytosolic
fraction.
Cytochrome c estimation in the cytosolic fraction was done
according to the manufacturer’s protocol (Calbiochem, Gibb-
stown, NJ). Briefly, to each well provided with kit, 100 µlof
calibrator diluents RDP5 was added followed by the addition of
100 µl of cytosolic fraction in duplets (17). After incubation for
2 h, wells were washed with washing buffer. Then 200 µlofcy-
tochrome c conjugate was added to each well and incubated for
2 h followed by 30 min incubation with 200 µl of substrate. Fi-
nally, 50 µl of stop solution was added, and optical density was
recorded at 450 nm using a 96-well microplate reader (Sunrise,
Tecan, Switzerland).
Caspase Activity
Cell lysates were prepared according to the manufacturer’s
instructions. Briefly, cells were incubated for the indicated times
(6, 12, 18, and 24 h) and then harvested by centrifugation. Cell
pellets were resuspended in cold lysis buffer and placed on ice
for 10–15 min. Resuspension was centrifuged at 16,000 g for
15 min (for caspase-3 and -8) and 10,000 g for 1 min (for
caspase-6, -9). These supernatants were collected as cell lysates.
Cell lysates were incubated with colorimetric peptide sub-
strates, Ac-DEVD-pNA for caspase-3 and Ac-IETD-pNA for
caspase-8, Ac-VEID-pNA for caspase-6 and Ac-LEHD-pNA
for caspase-9 alone and with their respective inhibitors (for
measuring the nonspecific hydrolysis of the substrate) for 2 h
at 37
◦
C. The release of chromophore p-nitroanilide (pNA) was
measured at 405 nm using a 96-well microplate reader (Sunrise,
Tecan, Switzerland).
DNA Ladder Assay
Apoptosis was assessed by electrophoresis of extracted ge-
nomic DNA from treated HL60 cells as described previously,
with some modifications (18). Briefly, HL60 cells (1 × 10
6
)
were treated with different concentrations of ESB for 18 h or
at a fixed concentration of 75 µg/ml for varying times of ex-
posures (0–30 h), were centrifuged at 500 g for 10 min, and
were washed with PBS. The pellet was suspended in 250 µlof
lysis buffer (10 mM EDTA, 50 mM Tris–HCl, 0.5% SDS) for
15 min at 55
◦
C. Lysed cells were then digested with proteinase-
K (200 µg/ml) at 55
◦
C for 1 h and followed by incubation with
200 µg/ml DNase-free RNase at 55
◦
C for further 90 min. DNA
was extracted twice with 250 µl of phenol:chloroform:isoamyl
alcohol (25:24:1) for 1 min by gentle mixing and then
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
INDUCTION OF APOPTOSIS IN HL60 CELLS BY ERYTHRINA SUBEROSA STEM BARK 805
centrifuged at 10,000 g for 5 min. The aqueous phase was further
extracted with chloroform:isoamyl alcohol (24:1) and cen-
trifuged. DNA was precipitated from the aqueous phase with
0.1 volume of 2M NaCl and 2.5 volumes of chilled ethanol and
kept at −20
◦
C overnight. The precipitated DNA was centrifuged
at 10,000 g for 10 min, dissolved in tris-EDTA buffer (pH 8.0)
and electrophoresed in 1.5% agarose gel at 50 V for 1.5 h.
The gel was photographed using the BioRad gel documentation
system.
Statistical Analysis
All in vitro experiments were done in triplicate, and each
data point represents the average of at least 3 independent ex-
periments. The data are reported as the mean ± SD. The com-
parisons were made between controls and treated cultures using
unpaired Student’s t-tests, and the difference was considered to
be statistically significant if P < 0.05 (
∗
), highly significant if
P < 0.01 (
∗∗
), and extremely significant if P < 0.001 (
∗∗∗
).
RESULTS
Effect on Overall Cell Activity—MTT Assay
When the cells were treated with various concentrations of
ESB for 24, 48, and 72 h, we found that there was a marked
decrease in the viability of HL60 cells in a dose- and time-
dependent manner. The IC
50
was found to be 64.9, 45.9, and
39.6 µg/ml after 24, 48, and 72 h of ESB treatment, respectively.
The results are summarized in Fig. 1.
HPLC Fingerprint
Analyses of ESB in our bioassay suggest that it possesses
cytotoxicity, so we ascertained the chemical profile of ESB
using HPLC. Fig. 2 indicates the major compounds present
in ESB that may be responsible for the observed bioactivity.
The identity of the active substance in ESB will require several
rounds of chromatographic separation prior to identification.
Analysis of Cell Death by Fluorescence Microscopy
ESB-treated HL60 cells were stained with Hoechst 33258
and acridine orange/ethidium bromide and analyzed under a
fluorescence microscope for changes in viable, early or late
apoptotic and necrotic cells.
Hoechst 33258 Staining
Hoechst 33258 stain selectively binds DNA and allows mon-
itoring of nuclear morphological changes under a fluorescence
microscope. We observed that the nuclei of untreated cells
stained blue with Hoechst exhibited loose chromatin and dis-
tributed throughout the cell. ESB at 50 µg/ml induced significant
nuclear fragmentation and condensation in HL60 cells after 18 h
of treatment. The majority of cells shrink and had a relatively
smaller size than untreated cells.
Acridine Orange/Ethidium Bromide Staining
To assess the type of cell death induced by ESB in HL60 cells,
we analyzed their morphological changes after double-staining
cells with acridine orange and ethidium bromide. These fluo-
rescent dyes emit different fluorescence and possess a different
ability to penetrate cells. Acridine orange is cell-permeable in
viable cells with intact membranes and fluoresces green after
intercalation into DNA. The second dye, ethidium bromide, is
excluded from viable cells and emits red fluorescence in the
presence of DNA in cells with an altered cell membrane.
The results obtained with AO/EtBr double-staining are rep-
resented in Fig. 3. The fluorescence in the chromatin was found
to be homogenous and no nuclear fragmentation was observed
in cells without any treatment (Fig. 3A). In contrast, cells treated
with ESB (50 µg/ml, 18 h; Fig. 3B) showed early apoptotic cells
FIG. 1. Cytotoxicity evaluation of Erythrina suberosa stem bark (ESB) against HL60 cells. 4 × 10
4
well grown in 96-well culture plate were incubated with
indicated concentrations of ESB for 24, 48, and 72 h and ESB along with 5 mM n-acetyl cysteine (NAC) for 48 h. Cell proliferation was assessed by MTT assay as
described earlier. Time- and dose-dependent effects of ESB on HL60 cell proliferation were observed. Bars denote ±SD, n = 4.
∗
Significant P < 0.05.
∗∗
Highly
significant P < 0.01.
∗∗∗
Extremely significant P < 0.001.
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
806 S. K. AGRAWAL ET AL.
FIG. 2. HPLC profiles of Erythrina suberosa stem bark (ESB). Retention times of major peaks are indicated.
FIG. 3. Morphological analysis of Erythrina suberosa stem bark (ESB)-treated HL60 cells after AO/EtBr dual staining. Morphological changes of HL60
following treatment without ESB (A), with low dose of 50 µg/ml (B), and with high dose of 100 µg/ml of ESB (C) for 18 h. Cells were stained with acridine
orange (100 µg/ml) and ethidium bromide (100 µg/ml). Samples were examined immediately under a fluorescent microscope using a NB filter with UV excitation.
Early apoptotic (arrows), late apoptotic (arrowheads) and necrotic (N) cells were observed after ESB treatment (B,C). Photographs are typical of 3 independent
experiments, each performed under identical conditions.
FIG. 4. Effect of Erythrina suberosa stem bark (ESB) on cell membrane in HL60 cells using scanning electron microscopy (SEM). A: Untreated HL60 cells
showed roughness (R) over the entire cell surface. B–C: HL60 cells treated with 50 µg/ml ESB for 18 h revealed smoothing of the cell surface (S); formation of
apoptotic bodies (arrows) and formation of pits (arrowheads) are evident due to apoptosis. Magnification: 3000×.
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
INDUCTION OF APOPTOSIS IN HL60 CELLS BY ERYTHRINA SUBEROSA STEM BARK 807
FIG. 5. Cell-cycle analysis of Erythrina suberosa stem bark (ESB)-treated
HL60 cells. Briefly, 1 × 10
6
cells were exposed to different doses (25–
100 µg/ml) of ESB for 18 h. Following incubation, the cells were harvested,
treated with RNase, stained with PI (25 µg/ml), and analyzed by a BD-LSR
flow cytometer. Sub-G
0
population indicative of DNA damage was analyzed
from the hypo diploid fraction (<2n DNA) of DNA cell cycle analysis. Data are
representative of 1 of 3 similar experiments.
(arrows) with visible nuclear fragmentation and blebbing. At a
higher dose (100 µg/ml, 18 h; Fig. 3C), there was a significant
increase in early apoptotic cells (arrows), late apoptotic cells
(arrowheads), and necrotic cells (N).
Ultra Structure Observations Under a Scanning
Electron Microscope
Under a scanning electron microscope, it was observed that
untreated HL60 cells were spherical in shape with a rough sur-
face (R) and numerous surface projections over the entire cell
surface (Fig. 4A). In cells exposed to 50 µg/ml ESB for 18 h,
surface projections disappeared from surface after treatment,
resulting in a smooth surface (S). Shrinkage in cell size and
the formation of numerous apoptotic bodies were evident that
appear to detach from the cell (Fig. 4B; arrows). In some cells,
formation of pits, due to detachment of apoptotic bodies, was
also observed (Fig. 4C; arrow heads). All these morphological
changes are characteristic of apoptosis (19).
ESB Increased Sub-G
0
Fraction of Cell Cycle
To investigate the effects of ESB on cell-cycle status, HL60
cells were treated with different concentrations of ESB for 18 h
and then analyzed for cell-cycle alteration by flow cytometry.
We observed that ESB caused a dose-dependent increase in hy-
podiploid sub-G
0
DNA fraction (<2n DNA), indicating apop-
tosis due to DNA fragmentation. The sub-G
0
fraction was 6%
in untreated cells, which gradually increased from 19% at 25
µg/ml to 46% after 100 µg/ml of ESB treatment. There was
hardly any significant effect after 18 h of treatment on G2/M
fraction, which indicated that ESB does not produce mitotic
block or delay in cell cycle (Fig. 5).
ESB-Induced Phosphatidylserine Externalization
The apoptosis-inducing effect of the ESB was also evalu-
ated by annexin V/PI binding. After treatment with ESB (25–
100 µg/ml) for 18 h, cells were labeled with these 2 dyes, which
were analyzed by flow cytometry. As displayed in Fig. 6, the
apoptotic cells were increasing in a concentration-dependent
manner. The early and late apoptotic fraction was 3.44% and
3.16%, respectively, in control cells, which increased to 26.69%
and 4.4% after 75 µg/ml and to 22.57% and 7.78% after
100 µg/ml of ESB treatment; it was 20.63% and 29.44% with
5 µg/ml treatment of camptothecin, a well-established positive
control, under similar conditions. PI positive cells also increased
marginally at higher concentrations.
Effect of ESB on ROS Production
We quantified ROS levels in ESB-treated HL60 cells 1 h
after the addition of DCFH-DA. Intracellular ROS levels were
significantly increased to 2.6-fold after 50 µg/ml ESB treatment
for only 6 h. As the exposure of ESB was further increased from
12 to 24 h, gradual increase in ROS production was detected
(Fig. 7). These data suggest that ROS production may be the
cause of ESB-induced apoptosis in HL60 cells.
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
808 S. K. AGRAWAL ET AL.
FIG. 6. Flow cytometric analysis of Erythrina suberosa stem bark (ESB) induced apoptosis and postapoptotic necrosis in HL60 cells using annexin V-FITC/PI
double staining. Apoptosis induced by ESB is taken from the lower right quadrant of the given dot plot. Data are representative of 1 of 3 similar experiments.
Values represented as percentage.
Because ESB treatment led to the enhancement of ROS gen-
eration, it is possible that alterations in the cellular state could
play a role in ESB-induced apoptosis. To examine this, the an-
tioxidant agent NAC was used to counter the ESB-induced ROS
generation and consequent events. Our results clearly demon-
strated that ESB is able to generate a strong oxidative stress
in HL-60 cells, and pretreatment with NAC resulted in marked
protection against ROS generation (data not shown).
Mitochondrial Membrane Depolarization by ESB
Mitochondrial dysfunction has been shown to participate in
the induction of apoptosis and has been suggested to be central to
the apoptotic pathway (20). Rh-123 uptake into mitochondria is
driven by mitochondrial transmembrane potential (
mt
) that al-
lows the determination of cell population with active integrated
mitochondrial functions. Loss of
mt
leads to depolarization of
mitochondrial membranes leading to collapse of mitochondrial
functions ensuing cell death. HL60 cells exposed to ESB for 18
h were analyzed for Rh-123 uptake by flow cytometry. In the
untreated cells, >96% cells were functionally active with high
Rh-123 fluorescence (Fig. 8). At concentrations ≥50 µg/ml,
ESB caused significant mitochondrial damage. The decrease
of
mt
was only 12.9% at 25 µg/ml, which gradually in-
creased up to 80.1% at 100 µg/ml. Under similar test conditions,
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
INDUCTION OF APOPTOSIS IN HL60 CELLS BY ERYTHRINA SUBEROSA STEM BARK 809
FIG. 7. The production of reactive oxygen species in HL60 cells treated with
Erythrina suberosa stem bark (ESB) over a 24-h period. The production of ROS
was determined by the DCFH-DA assay fluorometrically. Y-axis shows fold
increase in DCF fluorescence. Error bars indicate ±SD.
∗
Significant P < 0.05.
∗∗
Highly significant P < 0.01.
5 µg/ml camptothecin could decrease
mt
to 60.7%. Further,
in the presence of 5mM NAC, there was only 7.3% decrease
of
mt
.
Increase in Cytosolic Cytochrome c
To elucidate whether ESB-induced apoptosis involved
release of cytochrome c from mitochondria, we prepared cy-
tosolic fractions from treated HL60 cells and subjected these
preparations to colorimetric assay. A dose-dependent release
of cytochrome c in cytosol was detected. After 18 h of ESB
treatment (25–100 µg/ml), cytosolic cytochrome c significantly
increased from 2.6- to 9.5-fold, as compared to control cells
(Fig. 9). This cytochrome c release correlated with the loss in
mitochondrial membrane potential observed in treated HL60
cells, suggesting that the induction of apoptosis was mediated
via mitochondrial pathway.
Caspase Activation Involved in ESB-Induced Apoptosis
We also monitored the enzymatic activity of caspase dur-
ing ESB-induced apoptosis at 50 µg/ml, using 4 colorimetric
peptide substrates as follows: Ac-DEVD-pNA, Ac-IETD-pNA,
Ac-VEID-pNA, and Ac-LEHD-pNA, which are specific sub-
strates for caspase-3, -8, -6, and -9, respectively. As illustrated
in Fig. 10, showing the time course of the activity of the various
caspases, ESB induced a rise in caspase-9 activity to an approx-
imately 1.8-fold increase after 6 h. This treatment correlated
with the gradual increase of cytosolic cytochrome c after the
addition of ESB. Furthermore, caspase-3 was time-dependently
activated by ESB from 2.3-fold at 6 h to approximately fivefold
at 24 h. Activation of caspase-8 in HL60 cells was increased
only 1.4-fold even after 24 h exposure to ESB. In contrast to the
significant increase in caspase-3 and -9, a negligible increase
in caspase-6 was observed. However, in the presence of 25 µM
caspase inhibitors, there was negligible change in caspase lev-
els. These results suggest that caspase activation participate in
ESB-induced apoptosis in HL60 cells.
Analysis of ESB-Induced DNA fragmentation
The endonucleolytic DNA cleavages were checked by
agarose gel electrophoresis. Efficient DNA fragmentation was
observed after 12 h at 75 µg/ml. With increase in concentra-
tion and time, a more intense pattern of DNA fragments were
detected. No fragmentation was observed with DNA extracted
from untreated cells. A prominent ladder was also observed
in cells treated with 5 µg/ml camptothecin for 6 h (Figs. 11A
and B).
DISCUSSION
The ability to induce tumor cell apoptosis is an important
property of a candidate anticancer drug, which discriminates
between anticancer drugs and toxic compounds. Much effort
has been directed toward the searching for compounds that in-
fluence apoptosis and understanding their mechanisms of action
(21). Recently, extracts prepared from a variety of plants were
demonstrated to possess the ability in triggering the apoptotic
pathway (13,22). The mechanisms of apoptosis induction are
complex and not fully known, but some key events are identi-
fied that appear essential for the cell to enter apoptosis (23,24).
The notion that apoptosis represents a critical element in cell
number control in physiological and pathological situations has
been well reviewed and its role in oncogenesis is now well
established (25).
Alcoholic extracts were normally used for anticancer screen-
ing because traditional practitioners believed that mostly the
polar compounds were responsible for the claimed anticancer
properties (26). Some recent studies on methanolic extract from
Coriolus versicolor showed cytotoxicity against B16 cells in a
dose-dependent manner (27). An aqueous-ethanol extract pre-
pared from C. versicolor also showed dose-dependent in vitro
cytotoxicity against HL60 cells (28). Grapeseed extract strongly
inhibits cell growth and induces cell cycle arrest and apoptosis
in human colon carcinoma cells (29). Extracts from Vitis rotun-
difolia and Vitis vinifera reduced the growth of MOLT-4 cells
at 48 h in a concentration-dependent manner (30). Aqueous ex-
tracts of Oldenlandia diffusa significantly inhibited the growth
of the HL60 cells (31).
In the present study, we initially evaluated ESB against a
panel of human cancer cell lines from different tissue origin viz.
HL60 and Molt-4 (leukemia), A-549 (lung), Colo-205 and SW-
620 (colon), DU-145 and PC-3 (prostate), IGR-OV-1 (ovary),
MCF-7 (breast), Hep-2 (liver), and IMR-32 (neuroblastoma)
for its cytotoxic potential. We found a differential effect of ESB
with IC
50
values between 30–100 µg/ml, after 48-h treatment,
depending on the cell lines used. Furthermore, the activity of
ESB was not significant against WI-38 (lung) and CV-1 (mon-
key kidney) normal cell lines (data not shown). In HL60 cells,
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
810 S. K. AGRAWAL ET AL.
FIG. 8. Analysis of Erythrina suberosa stem bark (ESB)-induced alterations in mitochondrial membrane potential (
mt
). Exponentially growing cells were
treated with the indicated concentration of ESB for 18 h. For the assessment of loss in
mt
, cells were incubated with the fluorophore Rh-123 (5 µg/ml) for
30 min at 37
◦
C in the dark. The unbound dye was removed by washing the cells with chilled phosphate buffer saline (PBS) and analyzed by flow cytometry.
Graphs show the percentages of Rh-123 negative cells.
we observed promising cytotoxicity. The present study elab-
orates the effect of ESB on human promyelocytic leukemia
HL60 cells. We showed that ESB exerted a significant prolifer-
ation inhibitory activity against HL60 cells in a dose- and time-
dependent manner. The cytotoxic activities of this plant may
be due to the presence of pterocarpans, isoflavones, flavanones,
and chalcones that occurred in genus Erythrina (3,32,33). Fur-
ther cellular and biochemical analysis indicated that the pro-
liferation of inhibitory activity of ESB was related to the in-
duction of apoptosis. Several sensitive methods for detecting
apoptosis have been developed. Staining of apoptotic cells with
fluorescent dyes such as Hoechst 33258, AO, and EtBr is con-
sidered the correct method for evaluating the changed nuclear
morphology (13,34). ESB-induced morphological alterations,
such as chromatin condensation and nuclear fragmentation,
was revealed by fluorescence microscopic analysis predomi-
nantly in a dose-dependent manner. Cytoplasmic blebbing, the
formation of a number of membrane-bounded apoptotic bod-
ies that have a diverse appearance, particularly in regard to
their size, were studied by scanning electron microscopy. The
development of cytofluorimetric approaches to monitor these
multiple cell alterations permits a precise and reliable quan-
tification of apoptosis. Apoptotic cells exhibit some morpho-
logical modifications that are readily detected according to
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
INDUCTION OF APOPTOSIS IN HL60 CELLS BY ERYTHRINA SUBEROSA STEM BARK 811
FIG. 9. Effect of Erythrina suberosa stem bark (ESB) on cytochrome c release.
HL60 cells were exposed to ESB (25–100 µg/ml) for 18 h. At the end of
the exposure time, release of cytochrome c into cytosol was detected using a
cytochrome c ELISA kit. Each bar represents ±SD.
∗
Significant P < 0.05.
∗∗
Highly significant P < 0.01.
their light scatter properties (FSC/ SCC) by flow cytometry
(35).
There are several methods that can be used to quantify apop-
tosis. Use of a fluorochrome, such as PI, that is capable of
FIG. 10. Caspase-3, -8, -9, and -6 activity after 6 h, 12 h, 18 h, and 24 h
of incubation with 50 µg/ml of Erythrina suberosa stem bark (ESB) in HL60
cells. The activity was calculated as fold-increase of untreated cells. Error bars
indicate ±SD.
∗∗
Highly significant P < 0.01.
∗∗∗
Extremely significant P <
0.001.
FIG. 11. Erythrina suberosa stem bark (ESB) induced DNA fragmentation.
Fragmentation of genomic DNA was studied in HL60 cells exposed to ESB in
a concentration- and time-dependent manner. Genomic DNA was isolated and
electrophoresed as described earlier. Data are representative of 1 of 3 similar
experiments. A: Lane 1 = control, Lane 2 = 5 µg/ml, Lane 3 = 25 µg/ml,
Lane 4 = 50 µg/ml, Lane 5 = 75 µg/ml, Lane 6 = 100 µg/ml, and Lane 7 =
Camptothecin: 5 µMfor6h.B:Lane1= control, Lane 2 = 4h,Lane3= 8h,
Lane 4 = 12 h, Lane 5 = 16 h, Lane 6 = 20 h, Lane 7 = 24 h, and Lane 8 =
30 h. A prominent DNA ladder observed after 12 h at 75 µg/ml and camp-
tothecin.
binding and labeling DNA makes it possible to obtain a rapid
and precise evaluation of cellular DNA content by flow cy-
tometric analysis and subsequent identification of hypodiploid
cells, which generally appear in the sub-G
0
/G
1
peak region in
the histogram (36). The proportion of hypodiploid cells in total
cell population represents the intensity of apoptosis-inducing
activity of the tested sample (37). The DNA content of cells
treated with ESB was obtained as a flow cytometric histogram
and showed significant increase in sub-G
0
/G
1
population.
One of the earliest events of apoptosis is the loss of
plasma membrane polarity, accompanied by translocation of
phosphatidylserine (PS) from the inner to outer membrane
leaflets, thereby exposing PS to the external environment. The
phospholipid-binding protein annexin V has a high affinity for
PS and can bind to cells with fluorescently labeled annexin V
correlates with loss of membrane polarity during apoptosis but
precedes the complete loss of membrane integrity that accom-
panies later stages of cell death, resulting from either apoptosis
or necrosis. In contrast, PI can only enter cells after loss of
membrane integrity. Thus, dual staining with annexin V and PI
allows clear discrimination between affected cells (annexin V
negative, PI negative), early apoptotic cells (annexin V positive,
PI negative), and the late apoptotic or necrotic cells (annexin V
positive, PI positive) (14). HL60 cells treated with ESB showed
annexin V positive cells at lower concentrations and both an-
nexin V and PI positive cells at higher concentrations which
revealed that there is postapoptotic necrosis.
The impairment of mitochondrial function has been consid-
ered to be a key event in the ROS-mediated apoptotic pathway
(38). ROS accumulation has been proposed to be involved in
ESB-induced cell death. ROS levels were determined in HL60
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
812 S. K. AGRAWAL ET AL.
cells after ESB treatment using a peroxide sensitive fluores-
cent probe, DCFH-DA, and a six-fold increase was evidenced
in a period of 24 h. The integrity of mitochondrial membranes
of ESB-treated cells was examined by measuring their ability
to retain Rh-123, a fluorescent dye used to indicate the loss
of mitochondrial transmembrane potential (39). A significant
decrease of MMP was observed in HL60 cells after treatment
with 100 µg/ml ESB for 18 h. Mitochondrial outer membrane
permeabilization (MOMP) results in the release of cytochrome
c from the mitochondria into the cytosol, triggering caspase
activation and subsequent apoptosis (40). Further investigation
indicated that caspases are involved since caspase-9 and -3 were
activated. We also found that Ac-DEVD-CHO, a caspase-3 in-
hibitor, achieved near complete inhibition as well, suggesting
that caspase-3 is the major executioner caspase in ESB-induced
HL60 apoptosis. In general, both the mitochondria-initiated in-
trinsic pathway and the death receptor-triggered extrinsic path-
way can lead to caspase-3 activation (41,42). In our system,
caspase-9 was significantly activated, which implicated the mi-
tochondrial involvement since caspase-9 is the initiator caspase
for the mitochondria-mediated intrinsic apoptotic pathway. Fur-
thermore, as there was no significant activation of caspase-8,
the receptor-mediated pathway may have not been triggered.
One of the most commonly used techniques for confirma-
tion of apoptosis is identification of DNA ladders (43). A well-
documented characteristic of apoptosis is the fragmentation of
DNA into multimers of approximately 200 base pairs. To elu-
cidate whether ESB decreased cell survival by the induction of
DNA fragmentation, genomic DNA was isolated from cells ex-
posed to different concentrations of ESB from HL60 cells and
electrophoresed. The ESB treatment of HL60 cells indicated
internucleosomal DNA breakdown, leading to DNA fragmenta-
tion as expected for apoptotic cells.
In conclusion, our results indicate that an increase of ROS
is the initial essential event that results in the dissipation of
mitochondrial membrane potential, releasing cytochrome c fol-
lowed by activation of caspase-9 and -3 that commit the cells to
the mitochondria-mediated intrinsic apoptotic pathway in ESB-
treated HL60 cells. The present results suggest that ESB could
be a potential candidate for developing anticancer drugs for the
treatment of human leukemia cancer.
ACKNOWLEDGMENTS
Satyam Kumar Agrawal and Madhunika Agrawal would like
to thank Council for Scientific and Industrial Research (CSIR),
New Delhi, India, for Senior Research Fellowships. All authors
declare no personal or financial conflict of interest.
REFERENCES
1. Sandhya T, Lathika KM, Pandey BN, and Mishra KP: Potential of tradi-
tional ayurvedic formulation, Triphala, as a novel anticancer drug. Cancer
Lett 231, 206–214, 2006.
2. Sandhya T and Mishra KP: Cytotoxic response of breast cancer cell lines,
MCF 7 and T47D to triphala and its modification by antioxidants. Cancer
Lett 238, 304–313, 2006.
3. Nkengfack AE, Azebaze AG, Waffo AK, Fomum ZT, Meyer M, et al.: Cy-
totoxic isoflavones from Erythrina indica. Phytochemistry 58, 1113–1120,
2001.
4. Balachandran P and Govindarajan R: Cancer—an ayurvedic perspective.
Pharmacol Res 51, 19–30, 2005.
5. Singh H, Chawla AS, Rowe JW, and Toda JK: Waxes and sterols of Eryth-
rina suberosa bark. Phytochemistry 9, 1673–1675, 1970.
6. Bharracharyya L, Ghosh A, and Sen A: A comparative study on lectins
from four Erythrina species. Phytochemistry 25, 2117–2122, 1986.
7. Tanaka H, Etoh H, Watanabe N, Shimizu H, Ahmad M, et al.: Erysubins
C-F, four isoflavonoids from Erythrina suberosa var. glabrescences. Phyto-
chemistry 56, 769–773, 2001.
8. Dhar ML, Dhar MM, Mehrothra BN, Dhawanan BN, and Ray C: Screening
of Indian plants for biological activity: I. Indian J Exp Biol 6, 232–247,
1968.
9. Sa F, Gao JL, Fung KP, Zheng Y, Lee SMY, et al.: Antiproliferative and
pro-apoptotic effect of Smilax glabra Roxb. extract on hepatoma cell lines.
Chem Biol Interact 171, 1–14, 2008.
10. Hu H, Ahn NS, Yang X, Lee YS, and Kang KS: Ganoderma lucidum extract
induces cell cycle arrest and apoptosis in MCF-7 human breast cancer cell.
Int J Cancer 102, 250–253, 2002.
11. Kania K, Wasowska KM, Osiecka R, and Jozwiak Z: Analysis of
aclarubicin-induced cell death in human fibroblasts. Cell Biol Int 31,
1049–1056, 2007.
12. Rello S, Stockert JC, Moreno V, Gamez A, Pacheco M, et al.: Morpho-
logical criteria to distinguish cell death induced by apoptotic and necrotic
treatments. Apoptosis 10, 201–208, 2005.
13. Jo EH, Kim SH, Ra JC, Kim SR, Cho SD, et al.: Chemopreventive proper-
ties of the ethanol extract of Chinese licorice (Glycyrrhiza uralensis) root:
induction of apoptosis and G1 cell cycle arrest in MCF-7 human breast
cancer cells. Cancer Lett 230, 239–247, 2005.
14. Yang L, Wu S, Zhang Q, Liu F, and Wu P: 23,24-Dihydrocucurbitacin
B induces G2/M cell-cycle arrest and mitochondria-dependent apopto-
sis in human breast cancer cells (Bcap37). Cancer Lett 256, 267–278,
2007.
15. Carvalho DD, Costa FTM, Duran N, and Haun M: Cytotoxic activity of
violacein in human colon cancer cells. Toxicol In Vitro 20, 1514–1521,
2006.
16. Li H, Wang LJ, Qiu GF, Yu JQ, Liang SC, et al.: Apoptosis of HeLA cells
induced by extract from Cremanthodium humile. Food Chem Toxicol 45,
2040–2046, 2007.
17. Gockel HR, Lugering A, Heidemann J, Schmidt M, Domschke W, et al.:
Thalidomide induces apoptosis in human monocytes by using a cytochrome
c-dependent pathway. J Immunol 172, 5103–5109, 2004.
18. Tong X, Lin S, Fujii M, and Hou DX: Echinocystic acid induces apoptosis
in HL-60 cells through mitochondria-mediated death pathway. Cancer Lett
212, 21–32, 2004.
19. Zhang S, Liu J, Saafi EL, and Cooper GJS: Induction of apoptosis by human
amylin in RINm5F islet β-cells is associated with enhanced expression of
p53 and p21
WA F 1 / CI P 1
. FEBS Lett 455, 315–320, 1999.
20. Ly JD, Grubb DR, and Lawen A: The mitochondrial membrane potential
(ψm) in apoptosis; an update. Apoptosis 8, 115–128, 2003.
21. Ren G, Zhao YP, Yang L, and Fu C: Antiproliferative effect of cli-
tocine from the mushroom Leucopaxillus giganteus on human cervical
cancer HeLa cells by inducing apoptosis. Cancer Lett 262, 190–200,
2008.
22. Wu SJ, Ng LT, Chen CH, Lin DL, Wang SS, et al.: Antihepatoma activity
of Physalis angulata and P. peruviana extracts and their effects on apoptosis
in human Hep G2 cells. Life Sci 74, 2061–2073, 2004.
23. Segundo C, Medina F, Rodriguez C, Palencia RM, Cobian FL, et al.:
Surface molecule loss and bleb formation by human germinal center b cells
undergoing apoptosis: role of apoptotic blebs in monocyte chemotaxis.
Blood 94, 1012–1020, 1999.
24. Andersson B, Janson V, Motlagh PB, Henriksson R, and Grankvist K:
Induction of apoptosis by intracellular potassium ion depletion: using the
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
INDUCTION OF APOPTOSIS IN HL60 CELLS BY ERYTHRINA SUBEROSA STEM BARK 813
fluorescent dye PBFI in a 96-well plate method in cultured lung cancer
cells. Toxicol In Vitro 20, 986–994, 2006.
25. Hall PA: Assessing apoptosis: a critical survey. Endocr Relat Cancer 6,
3–8, 1999.
26. Tan ML, Sulaiman SF, Najimuddin N, Samian MR, and Muhammad TST:
Methanolic extract of Pereskia bleo (Kunth) DC. (Cactaceae) induces apop-
tosis in breast carcinoma, T47-D cell line. J Ethnopharmacol 96, 287–294,
2005.
27. Harhaji L, Mijatovic S, Ivanic DM, Stojanovic I, Momcilovic M, et al.:
Antitumor effect of Coriolus versicolor methanol extract against mouse
B16 melanoma cells: in vitro and in vivo study. Food Chem Toxicol 46,
1825–1833, 2008.
28. Lau CBS, Ho CY, Kim CF, Leung KN, Fung KP, et al.: Cytotoxic activities
of Coriolus versicolor (Yunzhi) extract on human leukemia and lymphoma
cells by induction of apoptosis. Life Sci 75, 797–808, 2004.
29. Kaur M, Mandair R, Agarwal R, and Agarwal C: Grape seed extract induces
cell cycle arrest and apoptosis in human colon carcinoma cells. Nutr Cancer
60, 2–11, 2008.
30. Talcott SUM, Percival SS, and Talcott ST: Extracts from red muscadine and
cabernet sauvignon wines induce cell death in MOLT-4 human leukemia
cells. Food Chem 108, 824–832, 2008.
31. Willimott S, Barker J, Jones LA, and Opara EI: Apoptotic effect of Olden-
landia diffusa on the leukaemic cell line HL60 and human lymphocytes. J
Ethnopharmacol 114, 290–299, 2007.
32. Rukachaisirikul T, Saekee A, Tharibun C, Watkuolham S, and Suksamrarn
A: Biological activities of the chemical constituents of Erythrina stricta and
Erythrina subumbrans. Arch Pharm Res 30, 1398–1403, 2007.
33. Nguyen PH, Le TV, Thuong PT, Dao TT, Ndinteh DT, et al.: Cytotoxic and
PTP1B inhibitory activities from Erythrina abyssinica. Bioorg Med Chem
Lett 19, 6745–6749, 2009.
34. Baskic D, Popovic S, Ristic P, and Arsenijevic NN: Analysis of
cycloheximide-induced apoptosis in human leukocytes: fluorescence mi-
croscopy using annexin V/propidium iodide versus acridin orange/ethidium
bromide. Cell Biol Int 30, 924–932, 2006.
35. Sharma M, Agrawal SK, Sharma PR, Chadha BS, Khosla MK,
et al.: Cytotoxic and apoptotic activity of essential oil from Ocimum
viride towards COLO 205 cells. Food Chem Toxicol 48, 336–344,
2010.
36. Riccardi C and Nicoletti I: Cell and developmental biology analysis of apop-
tosis by propidium iodide staining and flow cytometry. Nature Protocols 1,
1458–1461, 2006.
37. Li WX, Cui CB, Cai B, and Yao XS: Labdane-type diterpenes as new cell
cycle inhibitors and apoptosis inducers from Vitex trifolia L. JAsianNat
Prod Res 7, 95–105, 2005.
38. Wang R, Li C, Song D, Zhao G, Zhao L, et al.: Ethacrynic acid butyl-ester
induces apoptosis in leukemia cells through a hydrogen peroxide-mediated
pathway independent of glutathione S-transferase P1–1 inhibition. Cancer
Res 67, 7856–7864, 2007.
39. Kim KC, Kim JS, Son JK, and Kim IG: Enhanced induction of mito-
chondrial damage and apoptosis in human leukemia HL-60 cells by the
Ganoderma lucidum and Duchesnea chrysantha extracts. Cancer Lett 246,
210–217, 2007.
40. Arnoult D: Mitochondrial fragmentation in apoptosis. Trends Cell Biol 17,
6–12, 2007.
41. Li J, Xia X, Ke Y, Nie H, Smith MA, et al.: Trichosanthin induced
apoptosis in HL-60 cells via mitochondrial and endoplasmic reticu-
lum stress signaling pathways. Biochim Biophys Acta 1770, 1169–1180,
2007.
42. Jeong JC, Jang SW, Kim TH, Kwon CH, and Kim YK: Mulberry fruit
(Moris fructus) extracts induce human glioma cell death in vitro through
ROS-dependent mitochondrial pathway and inhibits glioma tumor growth
in vivo. Nutr Cancer
62, 402–412, 2010.
43. Wyllie AH: Glucocorticoid-induced thymocyte apoptosis is associated with
endogenous endonuclease activation. Nature 284, 555–556, 1980.
Downloaded by [Satyam Kumar Agrawal] at 02:55 14 October 2011
