Chemico-Biological Interactions 195 (2012) 133–143

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Chemico-Biological Interactions
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Induction of apoptosis and cell cycle arrest by Bis (2-ethylhexyl) phthalate

produced by marine Bacillus pumilus MB 40
A. Moushumi Priya, S. Jayachandran ⇑
Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry 605 014, India

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

Article history: Marine microorganisms represent a potential source for the production of biomedically useful com-
Received 5 August 2011 pounds active against inflammation, cancer, diabetes, etc. Marine Bacillus pumilus MB 40 (GenBank acces-
Received in revised form 3 November 2011 sion no. HQ705771) isolated from deep sea water column (1000 m depth) near Andaman and Nicobar
Accepted 10 November 2011
islands produced a bioactive lead, Bis (2-ethylhexyl) phthalate (BEHP) with a molecular formula of
Available online 3 December 2011
C6H4(CO2C8H17)2 and a molecular ion at m/z 391 (M+). Anti proliferative effect of the isolated compound
was examined by MTT assay in human erythroleukemic K562 cells and the IC50 of BEHP was found to be
Keywords:
21 lM. BEHP was able to induce apoptosis involving caspases pathway, besides regulating mitochondrial
Marine bacteria
Apoptosis
enzymes. Further, western blot analysis revealed the activation of caspases family proteins viz., caspase 8,
Caspases caspase-9 and caspase-3. An increase in the expression of Bax mRNA concomitant with a decrease in
Mitochondria mRNA of Bcl-2 in BEHP treated K562 cells was also observed. AO/EB staining of BEHP treated K562 cells
Cell cycle arrest further confirmed the progression of apoptosis as evidenced by morphological changes including nuclear
condensation, cell shrinkage, and formation of apoptotic bodies. Treatment of K562 cells with BEHP
induced the progressive accumulation of fragmented DNA in a time dependent manner. This pattern
appeared as a typical laddering distribution of DNA fragmentation due to intranucleosomal cleavage
associated with apoptosis. Based on flow cytometric analysis it has become evident that the compound
was also effective in arresting the cell cycle at a sub G0/G1 phase.
Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction downregulated the expression of pro inflammatory cytokines and

Cancer is recognized as the largest single cause of death in both
men and women in the developing as well as developed countries
in the world.
Over the last decade tremendous advances have been made in
understanding cancer biology and cancer genetics [1]. The decrease
in the rate of discovery of novel drugs from the terrestrial source
has necessitated the evaluation and establishment of a new source
of chemically diverse bioactive compounds [2]. The marine habitat
has enormous ecological diversity and represents an untapped
reservoir of novel bioactive substances. It is recognized that the
discovery of marine compounds as therapeutic agents is still in
its infancy and is expected that the ocean which remains virgin,
underexplored and unexploited will become an invaluable source
for tapping bioactive secondary metabolites possessing pharmaco-
dynamic properties, in the future.
In our earlier work we have shown that the ethyl acetate extract
of Staphylococcus arlettae and Planococcus maritimus isolated from
1000 m deep sea water column near Andaman and Nicobar islands
⇑ Corresponding author. Tel.: +91 413 2654428; fax: +91 413 2655715.
E-mail address: sr999jayachandran@gmail.com (S. Jayachandran).
mediators.
Apoptosis is an evolutionarily conserved and genetically regu-
lated process that plays an essential role in the development and
maintenance of cell homeostasis and is morphologically and bio-
chemically different from necrosis [3]. Hacker reported that cells
dying of apoptosis maintain membrane integrity until late in the
process, but display several morphological and biochemical altera-
tions including chromatin condensation, nuclear segmentation,
intranucleosomal DNA fragmentation, cytoplasmic vacuolization,
cell shrinkage and membrane blebbing with shedding of apoptotic
bodies [4]. Ability to manipulate the machinery of cell death is
considered an obvious goal of medical research and a control on
regulation of apoptosis might lead to new possibilities for cancer
treatment.
A central constituent of the apoptotic machinery is a family
of cysteine-containing, aspartate-specific protease, termed as
caspases [5]. Caspases exist in cells as inactive proenzymes, with
the active tetramer being formed by the removal of the prodomain
and cleavage between the large and small subunits [6]. Caspase
activity, either directly or indirectly, is responsible for the cleavage
of several intracellular proteins, which are typically proteolysed
during apoptosis [7]. It has been well described that two major

0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2011.11.005
134 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143
pathways, the death receptor pathway and the mitochondria-
dependent pathway, lead to the activation of caspases and
consequent apoptosis in mammalian cells [8,9].
In the course of our screening program of bioactive natural
products active against cancer we have isolated a battery of marine
bacteria at various unexplored sites in the Bay of Bengal near
Andaman and Nicobar islands. Bacillus pumilus MB 40 (GenBank
accession no. HQ705771) obtained from 1000 m deep water
columns produced Bis (2-ethylhexyl) phthalate (BEHP) that induced
apoptosis. To study the effect of BEHP human leukemic cells (K562)
were employed which are widely used in investigations related to
the evaluation of anti proliferative activity, differentiation and
apoptotic effect of potential active molecule. The sequence of the
events induced by BEHP in K562 cells involves activation of
caspases, upregulation of bax and down regulation of Bcl-2 and
release of cytochrome c. These effects are accompanied by cell cycle
arrest at the sub G0/G1 phase and induced DNA fragmentation and
chromatin condensation.
2. Methodology
2.1. Reagents
Zobell Marine Agar (ZMA), Zobell Marine Broth (ZMB),
Gentamycin, Amphotericin B, Penicillin, Streptomycin, 3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT),
Acridine orange, ethidium bromide, NaCl, MgCl2, Triton X-100,
SDS, Tris–Cl, Propidium Iodide and agarose were obtained from
HiMedia, India. Proteinase K, RNase A, RNAzol B, DEPC, MuLv
reverse transcriptase, Taq polymerase and phenol were obtained
from Genei, India. Ethyl acetate, Hexane, Methanol, Isopropanol
and Chloroform were obtained from Merck Solvents, Germany.
Caspase-8, caspase-9, and caspase-3 fluoremetric assay kits
obtained from Calbiochem, Merck, Germany. FBS, RPMI medium
supplemented with Glutamine, sodium orthovanadate, NP40,
protease inhibitor cocktail (EDTA free), dimethylsulfoxide (DMSO),
sodium bicarbonate (endotoxin-free) and Diethylpyrocarbonate
(DEPC) were obtained from Sigma, USA. All reagents were prepared
using deionized MilliQ water.
2.2. Isolation of marine bacteria
Marine bacteria were isolated essentially as per the method
described by Krishnaveni and Jayachandran [10]. Briefly, sea water
samples were collected from a 1000 m depth in the Bay of Bengal
near Andaman and Nicobar islands (13°050 00.1700 N; 92°420
42.4400 E) using Niskin water sampler and the collected water was
stored in sterile containers. Following serial dilution platting tech-
nique sea water samples were aseptically plated in ZMA and incu-
bated at 25 °C for 24 h. The bacteria were isolated based on colony
morphology and pigmentation. Among 109 bacterial strains
isolated one strain (B. pumilus MB 40) that showed the strongest
cytotoxic effect on K562 cells by MTT assay was chosen for further
studies.
2.3. Extraction, purification and structure elucidation of BEHP from B.
pumilus MB 40 [11]
For the production of secondary metabolites, the marine bacte-
rial isolate B. pumilus MB 40 was cultured in ten 3 l ZMB in 5 l
Hoffkin’s flasks. The flasks were incubated on a rotary shaker at
150 rpm for 5 days at 25 °C. To the culture, in a separating funnel
an equal volume of ethyl acetate was added and vigorously shaken
for 5 min. The organic (upper) layer was carefully separated and an
equal volume of fresh ethyl acetate was added and the extraction
into the organic layer was repeated twice. The pooled organic layer
was evaporated to dryness in a rota evaporator under reduced
pressure (Buchi, Germany). The residue was subjected to silica
gel open column chromatography and eluted with Hexane:ethyl
acetate (95:5). The fraction was further purified by reverse phase
HPLC (C18-MS column, 10  25 mm; linear gradient of methanol
in water, 80–100% in 65 min; flow rate, 0.5 ml/min; UV detection
at 211 nm) and the structure of pure compound was established
by NMR (1H and 13C), FTIR and GC–MS. The purified compound
was dissolved in DMSO for further use in biological studies.
2.4. Cell culture
Human erythroleukemic K562 cells were gifted from Prof. R.S.
Varma, IIT Madras, Chennai and the cells were grown in an RPMI
medium supplemented with Glutamine (100 U/ml), Streptomycin
(0.75 lg/ml), and Penicillin (120 U/ml), Amphotericin B (3 lg/ml),
Gentamycin (160 lg/ml), 10% FBS and were maintained at 37 °C
with a humidified atmosphere of 5% CO2. To avoid changes in cell
characteristics produced by extended cell culture periods, carci-
noma cells were used between passages 13 and 22. The cells were
split every 2–3 days to maintain exponential growth. The cell
number was assessed by the standard procedure of cell counting
using a hemocytometer.
2.5. Cell proliferation assay [12]
Cell survival rate was determined by using the 3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction
test. Exponentially growing K562 cells were seeded at
1  106 cells/ml in 96-well tissue culture plates with a volume of
200 ll per well. Cells were incubated with different concentrations
(0.4, 4, 20, 40, 60, 80, 100 lg/ml) of BEHP and incubated at 37 °C for
24, 48 and 72 h. At the end of incubation period, 20 ll of MTT stock
solution (5 mg/ml) was added to each well and the plates were
further incubated for 4 h at 37 °C. The formazan crystals that
formed were dissolved by the addition of 100 ll of DMSO per
well. The soluble formazan product was spectrophotometrically
quantified using an ELISA reader at 575 nm. The results are based
on the cleavage of the tetrazolium salt by viable cells in the wells.
The following formula was used for the calculation:
Cell proliferation inhibited ð%Þ ¼ ½1  ðA value of the
experimental samples=A value
of the controlÞ  100%:
2.6. AO/EB staining [13]
Acridine orange is taken up both by viable and nonviable cells
and emits green fluorescence if intercalated into double stranded
nucleic acid or red fluorescence if bound to single stranded nucleic
acid. Ethidium bromide is taken up only by nonviable cells and
emits red fluorescence by intercalation into DNA. Cells were
treated with 21 lM of BEHP, incubated up to 24 h, harvested and
washed three times with sterile phosphate-buffered saline (PBS).
One microliter of the dye mixture (100 mg/ml AO and 100 mg/ml
EB in distilled water) was mixed with 9 ll of cell suspension
(1  106 cells/ml) on a clean microscope slide. The suspension
was immediately (fast uptake) examined by a fluorescence micro-
scope at 400 magnification.
2.7. DNA fragmentation assay [14]
K562 cells (1  106 cells/ml) were treated with BEHP at 21 lM
concentration for different time periods (0, 12 and 24 h). Cells were
 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143
harvested and washed thrice with PBS and lysed by incubating
with 500 ll of lysis buffer containing 100 mM NaCl, 20 mM EDTA,
50 mM Tris–Cl (pH 8.0), 0.5% SDS, and 1 mg/ml of proteinase K at
54 °C with gentle agitation for 2 h or until all solid material was
digested. DNA was extracted with phenol:chloroform and ethanol
precipitated. DNA was resuspended in 20 ll of the TE buffer
containing 50 lg/ml of RNase A, incubated for 30 min at 37 °C,
and analyzed on a 1.5% agarose gel for DNA fragmentation. The gels
were stained with 0.5 lg/ml of ethidium bromide and photo-
graphed under UV light.
2.8. Flow cytometric analysis
Flow cytometry was used to detect quantitatively the apoptotic
rate and the distribution of cell cycle as per the method of Liu et al.
[12]. Cells (1  106 cells/ml) were grown in 24 well plates with or
without BEHP for 24 h at 37 °C. Cells were harvested by centrifuga-
tion at 500g for 5 min, washed with ice cold PBS and fixed in 70%
ethanol at 4 °C overnight. After fixation, cells were washed twice
with 0.5 ml of Propidium Iodide (PI) buffer containing 0.1% Triton
X-100, 0.1% sodium citrate, 25 lg/ml of RNase A and 50 lg/ml of
PI and incubated in PI buffer for 10 min. The fluorescence emitted
by PI–DNA complex, was collected through an FL-2 filter (585 nm).
The percentages of cells in various phases of the cell cycle, namely
sub-G1, G1, S and G2/M, were assessed using an FACScan flow
cytometer equipped with Cell Quest software. Apoptotic rates were
determined by the percentage of hypoploid nuclei accumulated at
the peak of the sub G0 phase [15].
2.9. Caspases activity assay
The cleavage activity of Ile-Glu-Thr-Asp conjugated to
7-amino-4-trifluoromethylcoumarin (IETD-AFC), Leu-Glu-His-Asp
conjugated to 7-amino-4-trifluoromethylcoumarin (LEHD-AFC), and
Asp-Glu-Val-Asp conjugated to 7-amino-4-trifluoromethylcoumarin
(DEVD-AFC) was measured by using the caspase-8, caspase-9,
and caspase-3 fluoremetric assay kits (Calbiochem), respectively.
Briefly, cells at a density of 1  106 cells/ml were seeded in 12 well
plates. Exponentially growing cells were treated with BEHP at a
concentration of 21 lM in the presence or absence of caspase
specific inhibitors (Z-DEVD-CHO inhibitor for caspase-3, Z-IETD-CHO
inhibitor for caspase-8 and Z-LEHD-CHO inhibitor for caspase-9)
and incubated at 37 °C for 12 h. Cells were harvested by centrifuga-
tion and resuspended in 50 ll of cell lysis buffer. The protein
concentration was measured by using Bradford assay. Then
100 lg protein was diluted in 50 ll cell lysis buffer for each assay,
and 50 ll of 2 reaction buffer (containing 10 mM of DTT) was
added to each tube and incubated at 4 °C. Five microliters of fluo-
rogenic substrate specific for each caspase such as, DEVD-AFC for
caspase-3, IETD-AFC for caspase-8, LEHD-AFC for caspase-9 was
added into the tubes, respectively. After the samples were incubated
for 1.5 h at 37 °C, liberation of 7-amino-4-trifluoromethylcoumarin
was monitored by ELISA Microplate Reader using an excitation
wavelength of 380 nm and an emission wavelength of 450 nm.
2.10. RNA extraction and semi-quantitative reverse transcriptase-
polymerase chain reaction (RT-PCR) [16]
K562 cells at 1  106 cell/ml density were treated with BEHP at
IC50 value of 21 lM and incubated upto 24 h. Cells were harvested
and lysed in RNAzol B to obtain the total RNA, after extraction with
chloroform, and precipitation with isopropanol. The pellet was
washed with 70% ethanol and resuspended in 10 ll of DEPC
treated water. cDNA synthesis was carried out using synthetic
oligonucleotide primed transcription with 200 U of MuLv reverse
transcriptase. The cDNA was subjected to PCR with primers for
135
Bcl-2 (Forward: 50 -CAG CTG CAC CTG ACG CCC TT-30 , Reverse: 50 -GCC
TCC GTT ATC CTG GAT CC-30 ), Bax (Forward: 50 -TGC TTC AGG
GTT TCA TCC AG-30 , Reverse: 50 -GGC GGC AAT CAT CCT CTG-30 )
and GAPDH (Forward: 50 -CCA CCC ATG GCA AAT TCC ATG GCA-
30 , Reverse: 50 -TCT AGA CGG CAG GTC AGG TCC ACC-30 ). For PCR
1 ll of cDNA was added to the PCR mixture consisting of 1 ll
PCR buffer, 2.5 pmol dNTP, 5 pmol of forward and reverse primers,
0.8 U Taq polymerase and distilled water in a total volume of 10 ll.
The reaction mixture was subjected to amplification with initial
denaturation at 94 °C for 5 min, 94 °C for 1 min, primer annealing
at 52 °C for 1 min, extension at 72 °C for 1 min followed by a final
extension at 72 °C for 5 min for 30 cycles. PCR products were run
on 1.5% agarose gels, stained with ethidium bromide and docu-
mented. The density of the protein bands was quantitated by
scanning the bands on a gel documentation and analysis system.
2.11. Western blot analysis [17]
2.11.1. Caspases, Bax and Bcl-2
A modified version of Cavalieri et al. [17] was employed for esti-
mation of Caspases, Bax and Bcl-2. Exponentially growing cells
(1  106 cells/ml) were plated in six well plates and after the addi-
tion of BEHP at 21 lM were incubated for different time points
(0–24). Cells incubated for different periods of time were harvested
by centrifugation at 500g for 5 min, washed twice with PBS and
homogenized in 200 ll of lysis buffer containing 10 mM NaCl,
1.5 mM MgCl2, 10 mM Tris–HCl, 1 mM sodium orthovanadate,
0.3% NP40 and protease inhibitor cocktail (EDTA free). The lysates
thus obtained were centrifuged at 12,000g for 30 min at 4 °C. Fifty
micrograms of the total protein, as determined by Bradford’s assay,
was resolved into 10% SDS–PAGE and then transferred to nitro-
cellulose membrane using a semi dry electroblotter for 2 h at RT.
The membrane was blocked over night in 5% BSA. In all cases,
antibodies raised against active forms of caspase-8, caspase-9,
caspase-3, Bcl-2, Bax or b-actin (Calbiochem, Merck, Germany)
were incubated for 2 h with gentle agitation. After washing three
times with Tris buffered saline (TBS; 50 mM Tris/HCl, pH 7.5, and
0.15 M NaCl) containing 0.1% Triton X-100, the membrane was
incubated with alkaline phosphatase-conjugated secondary
antibodies for 2 h with agitation. The probed immunoblots were
visualized with the NBT/BCIP chromogenic substrate and docu-
mented. The optical density of the immunoblots was quantified
by densitometric scanning and data were analyzed by Biorad
QuantityOne software.
2.11.2. Cytochrome c [17]
Exponentially growing K562 cells treated with or without BEHP
were harvested by centrifugation at 500g for 5 min. The cells after
centrifugation were washed twice with ice cold PBS and centri-
fuged at 500g for 5 min. Cell pellet were suspended in 200 ll of
lysis buffer containing 10 mM NaCl, 1.5 mM MgCl2, 10 mM
Tris–HCl, 1 mM sodium orthovanadate, 0.3% NP40 and protease
inhibitor cocktail (EDTA free). In order to restore the isotonic envi-
ronment a solution containing 525 mM mannitol, 175 mM sucrose,
12.5 mM Tris–HCl, pH 7.5, 2.5 mM EDTA was added. Lysates were
centrifuged at 12,000g for 30 min at 4 °C. The cytosol thus obtained
was resolved into 10% SDS–PAGE and probed using an anti
cytochrome c antibody (Calbiochem, Merck, Germany) as per the
procedure described above.
2.12. Statistical analysis
The statistical difference between the groups was determined
by ANOVA and Tukey’s Studentized Range test. The level of
p < 0.05 was considered to be statistically significant.
136 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143
3. Results
3.1. Isolation, identification and characterization of BEHP from B.
pumilus MB 40
The crude ethyl acetate extract from B. pumilus MB 40 which
showed maximum cytotoxicity on K562 cells (data not shown)
was subjected to silica column chromatography. Based on TLC
profile, eluates were pooled together to obtain 11 different
fractions. All these fractions were again tested using MTT assay
and fraction 1 which showed maximum anti proliferative activity
was further purified by reverse phase HPLC. Based on the spectral
analysis (1H and 13C NMR, GC–MS and FTIR) the identity of the
pure compound was established as Bis (2-ethylhexyl) phthalate
(BEHP) (Fig. 1) with a molecular formula of C6H4(CO2C8H17)2 and
a molecular ion at m/z 391 (M+).
3.2. BEHP inhibits proliferation of K562 leukemic cell
Many bioactive leads from marine bacteria [11] and marine
animals [18] have been shown to inhibit proliferation of HT-29
and HeLa cells. To evaluate whether BEHP had the potential to
inhibit the growth of K562 leukemic cells, they were treated with
BEHP for different time periods and the progression of growth was
monitored by MTT assay (Fig. 2). BEHP was found to inhibit the
growth of K562 cells at all the time points tested. IC50 of BEHP
against K562 cells was found to be 21 lM at 24 h. However the
IC50 value of Doxorubicin, an anticancer drug commonly used to
treat leukemia and Hodgkin’s lymphoma as well as bladder and
breast cancers was found to be 17 lM at 24 h.
3.3. BEHP induces morphological changes in K562 cells
A morphological study of BEHP treated K562 cells was under-
taken employing the AO/EB staining technique to obtain additional
information as to whether the cell death was a result of apoptosis.
Uniformly green live cells with normal morphology were seen in
control groups (Fig. 3A). Whereas BEHP treated K562 cells appear
green with nuclear margination and chromatin condensation at
12 h after treatment (Fig. 3B). Late apoptotic cells appeared orange
with fragmented nuclei (Karryorhexis) and typical apoptosis
blebbing at 24 h of incubation (Fig. 3C). Results of our study
suggested that BEHP was able to induce marked apoptotic
morphology in K562 cells.
3.4. Detection of BEHP induced apoptosis in K562 cells by agarose gel
electrophoresis
DNA fragmentation is a typical biochemical feature of apopto-
sis. To elucidate whether BEHP inhibits K562 cell proliferation
through the induction of apoptosis, we examined the cell death
by DNA fragmentation from 0 to 24 h. Time dependent cellular
fragmentation was studied by gel electrophoresis and a ladder like
pattern, a typical characteristic of DNA cleavage between nucleo-
somes, was visible at 24 h of incubation at 21 lM of BEHP (Fig. 4).
3.5. BEHP treatment induced the cell cycle arrest of K562 cells in sub
G1 phase
To quantify and assess the apoptotic rate of BEHP treated K562
cells the proportion of cells that had a DNA content of less than 2n
was analyzed by flow cytometry using Propidium Iodide (PI) stain-
ing. The profile of PI stained events clearly distinguished nuclei
with normal diploid DNA in control cells from nuclei with hypo-
diploid DNA found in treated cells. An analysis of cell distribution
in each phase of cell cycle (sub G1; G0/G1; S and G2/M) revealed a
significant increase of sub G1 (apoptotic) cells from 1.99% in con-
trol cells (Fig. 5A) to 33.74% after treating K562 cells with BEHP
for 24 h (Fig. 5B). Taken together these results suggest that BEHP
was capable of inducing apoptosis in K562 cells.
3.6. Activation of caspases induced by BEHP
It has been reported that the activation of the caspases cascade
is a key element in the apoptotic process [19]. It is well docu-
mented that the caspases family is at the heart of apoptotic
machinery, where these enzymes play key roles in the execution
of apoptosis [20]. To investigate whether BEHP induced apoptosis
is dependent upon caspases activation, we examined the enzy-
matic activity of caspase-8, caspase-9 and caspase-3 in K562 cells
treated with BEHP for 12 h in the presence of fluorogenic substrate
and specific inhibitors. BEHP markedly increased the activities of
caspase-8, caspase-9 and caspase-3 at a concentration of 21 lM
(Fig. 6) at 12 h post incubation; however, the increase in caspase
9 activity was more pronounced suggesting the possible involve-
ment of mitochondrial proteins in inducing apoptosis. Further
the specific inhibitors used, abrogated the induction of caspases
activity by BEHP as measured by the fluorescence intensity. This
strongly suggests that BEHP induces apoptosis in K562 cells in a
caspase-dependent manner.
3.7. Western blot analysis of caspase activation
Many workers have reported that caspase activation is the key
event in apoptosis [6,21]. Therefore, we felt it necessary to investi-
gate whether caspases activation is involved in apoptotic mecha-
nism in BEHP treated K562 cells. The results (Fig. 7A) revealed
the maximum expression of caspase-9 followed by caspase-3 and
caspase-8 at 12 h post incubation (Fig. 7B). Though caspase-3
remained unchanged, the expression of caspase-8 and caspase-9
activity recorded a decline after 24 h of incubation. The results of
our study show that apoptosis induction by BEHP in K562 cells is
mediated by caspases.
3.8. Up regulation of Bax and down regulation of Bcl-2 by BEHP in
K562 cells
Although caspases are known to play a central point in apoptotic
response, their activities are tightly regulated by a variety of intrin-
sic factors. Among these, Bcl-2 and Bax proteins have been shown
to play a dominant role [22]. To further analyze the possible mech-
anism underlying BEHP induced apoptosis, we tested the expres-
sion of mRNA of Bcl-2 (Fig. 8A) and Bax (Fig. 8B) in treated K562
cells. The increase in the expression of Bax mRNA was concomitant
with a decrease in mRNA of Bcl-2 in BEHP treated K562 cells upto
24 h. GAPDH served as an internal control (Fig. 8C). After having
determined the mRNA expression levels of Bax and Bcl-2 in K562
cells treated with BEHP, we wanted to determine whether Bax
and Bcl-2 proteins, which constitute a cascade of apoptosis
related proteins, are regulated by BEHP. In BEHP treated K562 cells
an increase in the expression of Bax (Fig. 9A) and a decrease in the
expression of Bcl-2 protein were observed (Fig. 9B). The finding of
our study suggests the involvement of Bcl-2 family proteins in the
apoptosis. The ratio of Bax/Bcl-2 dramatically increased with
increase in incubation period (Fig. 9C).
3.9. Cytochrome c release in K562 cells treated with BEHP
Cytochrome c release is known to be a key event during mito-
chondria dependent apoptosis. Therefore, to investigate whether
BEHP induced apoptosis in K562 cells involved the release of
 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143 137

Fig. 1. Structure of pure compound isolated from B. pumilus MB 40. The structure of the active compound was characterized by 1H (A) and 13C NMR (B) and molecular weight
by GC–MS (C) as Bis (2-ethylhexyl) phthalate (D), with a molecular formula of C6H4(CO2C8H17)2 and a molecular ion at m/z 391 (M+).
138 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143

Fig. 1 (continued)

Fig. 2. Dose–response analysis of BEHP isolated from B. pumilus MB 40 on inhibition of proliferation of human erythroleukemic K-562 cells. 1  106 cells/well were seeded in
96-well tissue culture plate followed by treatment with indicated concentrations of BEHP for 72 h. Inhibition of cell proliferation was determined by MTT reduction assay
after the specified incubation periods. Results are mean values ± SD of three independent experiments; ⁄p < 0.01 and ⁄⁄p < 0.05. 24 h (), 48 h (j) and 72 h (N).
 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143 139

Fig. 3. Detection of apoptosis in K562 cells by AO/EB staining. Cells were treated with BEHP (21 lM) for different time periods, harvested and stained with AO/EB stain. Green
color represents the viable cells, control (A), greenish yellow color represents early apoptotic cells at 12 h (B) and reddish orange color represents the late apoptotic cells at
24 h (C). Arrows in B indicate nuclear condensation. White arrow in C indicates fragmented nuclei and white dashed arrow indicates apoptosis blebbing. Magnification 400.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

cytochrome c from mitochondria, we prepared cytosolic fractions

from treated K562 cells and subjected them to an immunoblot
analysis (Fig. 10A). A time dependent release of cytochrome c in
cytosol was detected. Maximum cytochrome c release in treated
K562 cells was observed at 12 h after treatment; thereafter a
decrease in cytochrome c was observed (Fig. 10B).

4. Discussion

Now a days great emphasis is given for the isolation, identifica-

Fig. 4. DNA ladder assay showing BEHP induced apoptosis of K562 cells. Cells were
exposed to 21 lM of the compound for the indicated times. Cells were harvested by
centrifugation and the DNA was extracted. The extracted DNA was electrophoresed
on 1.5% agarose gel. Molecular weight marker – 1 kbp (A), control (B) and K562 cells
treated with BEHP for 0 h (C), 12 h (D) and 24 h (E).
tion and characterization of bioactive molecules produced by
micro organisms of marine origin possessing a broad spectrum of
activities e.g. anti microbial, anti neoplastic, anti inflammatory,
anti cancer, ichthyotoxic, etc. [23]. In our lab we have isolated,
identified and characterized many marine bacteria capable of pro-
ducing bioactive leads active against inflammation [10]. Further, in
our endeavor to continue to isolate bioleads active against cancer
we have created a marine bacterial culture repository, where,
marine bacteria from different ecological regimes, temperature
gradient, etc., are available.
The ability to induce tumor cell apoptosis is a desirable attri-
bute of a candidate drug which prima facie discriminates between
anticancer drugs and toxic compounds [24]. In this direction great
strides have been made in identifying compounds that influence
apoptosis and their mechanism of action [25,26]. In the present
study we investigated the cytotoxic effect of the marine bacterial
isolates on human leukemic cells. K562 cell line was used as a
model system of resistance to apoptosis induced by conventional
chemotherapeutics [27,28]. The crude ethyl acetate extract from

Fig. 5. Cells cycle analysis of K562 cells treated with BEHP. Cell cycle progression was examined by flow cytometry. 1  106 cells were treated with 21 lM of BEHP and
incubated for 24 h. Cells were harvested and stained with PI as described in Section 2. The area labeled M1is Sub G0/G1 cell population; M2 is G1; M3 is S and M4 is G2/M.
Untreated control (A) and K562 cells treated with BEHP (B). The percentage of each phase was analyzed by Cell Quest software.
140 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143
Fig. 6. Activation of caspase 3, caspase 8 and caspase 9 by BEHP in K562 cells. The
activation of caspases after 12 h treatment of K562 cells without BEHP ( ), with
BEHP ( ) and with BEHP and inhibitor ( ) was determined by incubating 50 lg
total protein with an appropriate substrate as given in Section 2. Results are
represented as mean ± SD of three independent experiments; p < 0.05.
B. pumilus MB 40 exhibited inhibition of K562 cells in a dose and
time dependent manner (data not shown). However, for better
understanding of structure–function relationships of the pure
compound we have purified the crude ethyl acetate extract and
the purified compound was identified as Bis (2-ethylhexyl) phthalate.
A structural analog of BEHP, Dioctylphthalate obtained from
marine brown alga Sargassum wightii was found to exhibit anti
microbial activity [29]. Cells undergo death by two major mecha-
nisms: necrosis, in which primary damage to the metabolic or
membrane integrity of the cell occurs and apoptosis, which is an
internal suicide program contained in all cells [30]. Though several
Fig. 7. Western blot analysis of caspase 3, caspase 8 and caspase 9 in K562 cells treated with BEHP. Cells were treated with 21 lM of BEHP and resolved by SDS–PAGE.
Western blot analysis was performed using specific antibodies. b-Actin was used as internal control (A). The expression levels of the proteins were subsequently quantified by
densitometric analysis with that of control being 100% (B). Caspase 3 ( ), caspase 8 ( ) and caspase 9 ( ).
methods for detecting apoptosis are in vogue, no single assay can
be used alone with perfect specificity and sensitivity. Though
morphological criteria for apoptosis viz., condensation of chromatin,
cell shrinkage, as well as blebbing, are the oldest, they remain
probably the most specific of all assays. According to Gasiorowski
et al. and Savitskiy, staining of apoptotic cells with fluorescent dyes
such as AO and EB is considered the correct method for evaluating
the changed nuclear morphology [31,32]. Nuclear morphology
(perinuclear chromatin condensation, nuclear collapse and even-
tual fragmentation) has allowed us to distinguish several sub
populations of apoptotic cells from viable and necrotic cells.
Induction of DNA fragmentation from intact genomic DNA is
indicative of the process of apoptosis [11]. In our studies BEHP
was able to bring about fragmentation of DNA obtained from
K562 cells. By combining our three observations i.e., AO/EB stain-
ing, DNA fragmentation and flow cytometry, it has become evident
that BEHP is capable of triggering apoptosis in K562 cells.
Many anticancer agents arrest the cell cycle at G1, S or G2/M
phase and then induce apoptotic cell death [33,34]. Regulation of
cancer cell is one strategy in the development of anticancer drugs
[35]. As an estimate of apoptosis we calculated the percentage of
cells in sub G0/G1 phase. This G0/G1 peak represents a population
of cells with reduced DNA content because of DNA fragmentation.
The appearance of hypodiploid peak (sub G1 peak) in K562 cells
treated with BEHP is probably due to the presence of cells in apop-
tosis and apoptotic bodies with DNA content less than 2n. Taken
together the results of the present investigation confirm that BEHP
at an IC50 value of 21 lM induced apoptosis in K562 cells.
Caspases are a ubiquitous family of cysteine proteases that in-
clude both upstream (initiator) and downstream (effector) caspase
[36]. The initiator caspases, typically caspase-8 and caspase-9, are
activated by two alternative pathways. The first involves cell death
receptor mediated apoptosis through caspase-8 and the second in-
volves mitochondria mediated apoptosis through caspase-9 [37].
In both pathways the initiator caspase cleaves and activates down-
stream, effector caspases, such as caspase-3 [38]. This caspase
 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143 141

Fig. 8. RT PCR analysis of Bcl-2 and Bax at different time points in K562 cells treated with BEHP. K562 cells (1  106 cells/ml) grown in the RPMI medium were treated with
21 lM of BEHP and incubated upto 24 h. At 12 and 24 h post incubation the cells were harvested for RNA isolation and semi quantitative RT-PCR analysis was done for Bcl-2
(A) and Bax (B). Untreated cells were used as corresponding controls. The expression levels were subsequently quantified by densitometric analysis with that of control being
100% (C). The data represented are the mean ± SD of three independent experiments (p < 0.05). Bcl-2 ( ) and Bax ( ).

Fig. 9. Western blot analysis of Bcl-2 and Bax. Exponentially growing K562 cells were treated with 21 lM of BEHP for various time intervals, cell lysates were prepared and
protein levels of Bcl-2 and Bax were determined by western blot analysis (A). b-Actin was used as control, quantitative expression of Bax ( ) and Bcl-2 ( ) was determined
by densitomeric scanning (B). Bax/Bcl-2 ratio at different intervals was also plotted (C). Values are the mean ± SD of three independent experiments (p < 0.05).
142 A. Moushumi Priya, S. Jayachandran / Chemico-Biological Interactions 195 (2012) 133–143

Fig. 10. Western blot analysis of cytochrome c released from mitochondria to the cytosol in K562 cells treated with BEHP. K562 cells (1  106) were treated with the
compound for different periods of time the released cytosolic cytochrome c resolved by SDS–PAGE and Western blot analysis was performed using a specific antibody for
cytochrome c (A). Quantitative estimation of cytochrome c ( ) in cytosolic fraction of K562 cells treated with BEHP was done by densitometric scan (B). Untreated control
was taken as 100%. Values are the mean ± SD of three independent experiments (p < 0.05).

cascade ultimately leads to proteolytic cleavage and induces a
broad range of morphological changes of apoptosis. Our results
indicate that caspase-8, caspase-9 and caspase-3 activities were
rapidly elevated after BEHP treatment, and the kinetic activation
of caspase-3 was associated with the activation of caspase-8 and
caspase-9. It deserves further investigation on whether BEHP
induces other caspase independent pathways also. Western blot
analysis has revealed that BEHP was able to induce cytochrome c
release from the mitochondria in K562 cells. The data from our
study suggest that mitochondrial pathway as well as death
receptor signaling pathway is involved in the apoptosis induced
by BEHP in K562 cells.
These findings further suggest that apoptosis induction in K562
cells could be associated with caspases dependent cascade that
involves the activation of mitochondrial pathway, initiated by the
inhibition of Bcl-2 and activation of Bax. The release of cytochrome
c is deemed to be a process regulated tightly by Bcl-2 family
proteins that consist of anti-apoptotic and pro apoptotic members
[22]. Vander Heiden and Thompson opined that the ratio of anti
apoptotic Bcl-2 to pro-apoptotic Bax, at least in part, determines
the susceptibility of the cell to a death signal [39]. Our data clearly
demonstrated that treatment of K562 cells with BEHP resulted in a
time dependent increase in the levels of Bax with a concomitant
decrease in Bcl-2 levels and an increase in Bax:Bcl-2 ratio. Ren
et al. have shown that there are many regulatory molecules
involved in an upstream pathway, by which the expression of
many apoptosis regulatory genes such as Bcl-2 and Bax are
regulated [40]. Miyashita et al. reported that wild-type p53 can
down-regulate the expression of Bcl-2 and up-regulate that of
Bax, altering the balance of the couple genes in favor of apoptosis
[41]. A time dependent cytochrome c release in cytosolic fractions
was observed in K562 cells treated with BEHP. This suggested that
an increase in Bax expression levels leads to the release of
cytochrome c which augmented the apoptotic induction in K562
cells by BEHP. We also observed a decrease in Bcl-2 mRNA levels
after 12 h of treatment correlating with our earlier studies
demonstrating the role of Bcl-2 in apoptosis [42]. A decrease in
the Bcl-2 expression could result in homodimerisation of Bax in
the cytosol which when translocated to the mitochondrial
membrane might activate the release of cytochrome c [43]. The
release of cytochrome c is an effect of the translocation of Bax to
mitochondria leading to loss of mitochondrial membrane potential
[44]. Further, Lowe et al. reported that the key element in the mito-
chondrial pathway is the efflux of cytochrome c from mitochondria
to cytosol, where it subsequently forms a complex (apoptosome)
with caspase 9 leading to the activation of caspase-3 [45].
In conclusion, we have shown that BEHP from the marine
bacterium B. pumilus MB 40, is capable of inducing apoptosis in
leukemic K562 cells by the activation of caspase-8, caspase-9 and
caspase-3, release of cytochrome c from mitochondria, decrease
in Bcl-2 level with a concomitant increase of the Bax level. Addi-
tionally, BEHP was shown to arrest cell cycle at sub G0/G1 phase.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
This research was supported by research grants from the
University Grants Commission, New Delhi (F.No. 34-256/2008
(SR) dated 03/01/2009) and the Department of Biotechnology,
New Delhi (BT/PR-10486/BCE/08/657/2008 dated 27/08/2008).
M.P. is a recipient of Senior Research Fellowship from CSIR, New
Delhi. The authors are grateful to Mr. N. Ravi, Vessel Manager,
National Institute of Ocean Technology, Chennai, for arranging a
vessel, for sample collection from deep sea. Authors are grateful
to Mr. Karthik, Department of Chemistry for help rendered in
structure elucidation of purified compound and Ms. P. Lalitha,
Department of Biotechnology for technical assistance.
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