Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2012, Article ID 201414, 11 pages
Autophagic CellDeath as wellas Caspase-Dependent Apoptosis in
MinHo Han,1Won SupLee,2JingNan Lu,3Jeong Won Yun,3GonSupKim,4
Jin MyungJung,5Gi-YoungKim,6Su-Jae Lee,7Wun-Jae Kim,8andYung HyunChoi1
1Department of Biochemistry, Dongeui University College of Oriental Medicine and Department of Biomaterial Control (BK21
Program), Dongeui University Graduate School, 42 San, Yangjung-don, Busan 614-052, Republic of Korea
2Department of Internal Medicine, Institute of Health Sciences and Gyeongnam Regional Cancer Center,
Gyeongsang National University School of Medicine, 90 Chilam-dong, Jinju 660-702, Republic of Korea
3Department of Internal Medicine, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-702,
Republic of Korea
4Institute of Life Science and School of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea
5Department of Neurosurgery, Institute of Health Sciences, Gyeongsang National University School of Medicine,
Jinju 660-702, Republic of Korea
6Laboratory of Immunobiology, Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea
7Department of Chemistry, Hanyang University, Seoul 133-791, Republic of Korea
8Department of Urology, Chungbuk National University College of Medicine, Cheongju, Chungbuk 361-763, Republic of Korea
Correspondence should be addressed to Won Sup Lee, firstname.lastname@example.org and Yung Hyun Choi, email@example.com
Received 7 May 2011; Revised 24 June 2011; Accepted 24 June 2011
Academic Editor: Shrikant Anant
Copyright © 2012 Min Ho Han et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Tetraarsenic hexaoxide (As4O6) has been used in Korean folk remedy for the treatment of cancer since the late 1980s, and arsenic
trioxide(As2O3) iscurrently usedasa chemotherapeutic agent. However, evidence suggests thatAs4O6-inducedcell death pathway
was different from that of As2O3. Besides, the anticancer effects and mechanisms of As4O6are not fully understood. Therefore, we
was inhibited by As4O6treatment in a dose- and a time-dependent manner, and IC50for As4O6was less than 2μM. As4O6induced
caspase-dependent apoptosis and Beclin-1-induced autophagy, both of which were significantly attenuated by Bcl-2 augmentation
and N-acetylcysteine (NAC) treatment. This study suggests that As4O6should induce Beclin-1-induced autophagic cell death as
well as caspase-dependent apoptosis and that it might be a promising agent for the treatment of leukemia.
Arsenic trioxide (As2O3), a component of Chinese medicine,
has been successfully employed for the treatment of acute
promyelocytic leukemia (APL) [1, 2] and it has recently
been shown to have some efficacy against a certain type
of solid cancers [3, 4]. It is taken parenterally via an IV
drip. With regard to anticancer effects of As2O3, many
studies have shown that As2O3 is capable of inducing
programmed cell death. There are two types of programmed
cell death reported. One is apoptosis, type I programmed
cell death which is characterized by a highly stereotypical
series of morphological and biological changes, such as
cytoplasmic shrinkage, blebbing of the plasma membrane,
is autophagy, type II programmed cell death . Autophagy
with sequestering cytoplasmic organelles in a membrane
vacuole called autophagosome, which are double-membrane
cytoplasmic vesicles to engulf various cellular constituents,
and to fuse with lysosomes, where the sequestered cellular
constituents are degraded and recycled.
2Evidence-Based Complementary and Alternative Medicine
Tetraarsenic hexoxide (As4O6) has been used as a Korean
folk remedy for the management of cancer since the late
1980s because its toxicities were minimal compared to con-
ventional cytotoxic chemotherapy. However, the anticancer
effects of As4O6have not been investigated much although
the anticancer effects of arsenic trioxide (As2O3) have been
investigated in many leukemic cells [7–9]. A comparison
study of the anticancer effects between As2O3 and As4O6
demonstrated that As4O6was more effective in suppressing
human cancer cells in vitro and in vivo, and that As4O6-
induced cell death pathway was different from that of
As2O3. Upregulation of p53 and v-erb-b2 erythroblastic
leukemia viral oncogene homolog 2 (ERBB2) was noted in
As4O6-induced cell, but not in As4O6-induced cell death.
In addition, As4O6 has been used orally, whereas As2O3
has been used as a parenteral drug. Oral agents are more
convenient to take than parenteral agents. Hence identifying
the molecular mechanisms involved in its anticancer effects
would allow us to contribute to developing a new oral agent.
Here, we investigated the mechanisms of anticancer effects of
As4O6in U937 human leukemic cells.
2.1. Cells and Reagents. U937 human leukemic cells from
the American type culture collection (Rockville, MD, USA)
were cultured in RPMI 1640 medium (Invitrogen Corp,
Carlsbad, CA, USA) supplemented with 10% (v/v) fetal
bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA),
1mM L-glutamine, 100U/mL penicillin, and 100μg/mL
streptomycin at 37◦C in a humidified atmosphere of 95%
air and 5% CO2. The Bcl-2 overexpressing U937 cells
were a generous gift from Dr T.K. Kwon (Department
of Immunology, Keimyung University School of Medicine,
Taegu, Republic of Korea) and were maintained in a medium
containing 0.7μg/mL geneticin (G418 sulfate). As4O6 was
obtained from Chonjisan institute (Seoul, Republic of
Korea). Antibodies against Bcl-2, Bax, Bad, Bcl-xL, XIAP,
procaspase 3, procaspase 8, and procaspase 9 were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Antibodies against poly (ADP-ribose) polymerase (PARP),
PLCγ-1, LC3, and Beclin-1 were purchased from PharMin-
gen (San Diego, CA, U.S.A.). Antibody against β-actin
was from Sigma (Beverly, MA). Peroxidase-labeled donkey
anti-rabbit and sheep anti-mouse immunoglobulin and an
enhanced chemiluminescence (ECL) kit were purchased
from Amersham (Arlington Heights, IL). Caspase activity
assay kits were purchased from R&D systems (Minneapolis,
MN, USA.). All other chemicals not specifically cited here
were purchased from Sigma Chemical Co. (St. Louis, MO).
All these solutions were stored at −20◦C. Stock solutions
of DAPI (100μg/mL) and propidium iodide (PI, 1mg/mL)
were prepared in phosphate-buffered saline (PBS).
2.2. Cell Viability Assays. For the cell viability assay, the
cells were seeded onto 24-well plates at a concentration
of 5 × 105cells/mL and then treated with the indicated
concentration of As4O6 for 24h. MTT (0.5mg/mL) was
subsequently added to each well. After 3h of additional
incubation, 100μL of a solution containing 10% SDS (pH
4.8) plus 0.01N HCl was added to dissolve the crystals. The
absorption values at 570nm were determined with an ELISA
2.3. Nuclear Staining. After treatment with the indicated
concentration of As4O6, the cells were harvested, washed
with phosphate-buffered saline (PBS), and fixed with 3.7%
paraformaldehyde in PBS for 10minutes at room temper-
ature. Fixed cells were washed with PBS and stained with
2.5μg/mL 4,6-diamidino-2-phenylindole (DAPI) solution
for 10min at room temperature. The cells were washed two
times with PBS and analyzed by a fluorescent microscope.
2.4. Flow Cytometry Assay. The cells were plated at a con-
centration of 2 × 105cells/well in six-well plates. Reduced
(sub-G1) DNA content was measured by PI staining. The
DNA content in each cell nucleus was determined with a
FACSCalibur flow cytometer (Becton-Dickinson, San Jose,
CA, U.S.A.). Two independent experiments were performed
2.5. Western Blotting. The cells were harvested and lysed,
protein assay (BioRad Lab., Hercules, CA, U.S.A.). The
proteins of the extracts were resolved by electrophoresis,
electrotransferred to a polyvinylidene difluoride membrane
(Millipore, Bedford, MA),and then the membranewasincu-
bated with the primary antibodies followed by a conjugated
secondary antibody to peroxidase. Blots were developed with
an ECL detection system.
2.6. Caspase Activity Assay. Caspase activity was determined
by a colorimetric assay according to the manufacturer’s
protocol in a kit for caspase activity. In brief, the cells
were lysed in the supplied lysis buffer. The supernatants
were collected and incubated with the supplied reaction
buffer containing dithiothreitol and substrates at 37◦C.
The reaction was measured by determining the change in
absorbance at 405nm using the microplate reader .
2.7. Quantification of Acidic Vesicular Organelles (AVOs) with
Acridine Orange Staining. In acridine orange-stained cells,
the cytoplasm and nucleolus fluoresce bright green and
dim red, whereas acidic compartments fluoresce bright red.
Therefore, we stained the cells with acridine orange for
17min. Green (510–530nm) and red (650nm) fluorescence
emission from 1 × 104cells illuminated with blue (488nm)
excitation light was measured with a a FACSCalibur flow
cytometer (Becton-Dickinson, San Jose, CA, U.S.A.). Three
independent experiments were performed.
2.8. Statistics. Each experiment was performed in triplicate.
The results were expressed as means ± SD. Significant dif-
ferences were determined using the one-way analysis of
at least three treatment groups and Student’s t-test for two
Evidence-Based Complementary and Alternative Medicine3
group comparison. Statistical significance was defined as
P < 0.05.
3.1. Responses of U937 Human Leukemic Cells to As4O6. To
investigate the antitumor activity of As4O6, U937 cells were
treated with various concentrations of As4O6for 24h. The
cell growth was assessed by MTT assay. The MTT assay
treatment in a dose- and time-dependent manner, and the
50% inhibition of cell growth (IC50) was less than 2μM
(Figures 1(a) and 1(b)). The efficacy of As4O6was superior
to that of As2O3in terms of growth inhibition (Figure 1(a)).
3.2. Effects of As4O6 on Apoptosis. To determine whether
the decrease in viability of U937 cells was caused by the
induction of apoptosis, we assessed the changes in nuclear
morphology of As4O6-treated cells by DAPI staining. The
at a concentration of 2μM or higher. This is usually wit-
nessed in apoptosis (Figure 1(c)). To estimate the population
of the cell death, we measured cells with sub-G1 DNA
content by flow cytometry. A significant accumulation of
cells with sub-G1 DNA content was noted in a dose-
dependent (Figures 1(d) and 1(e)) and time-dependent
manner (Figures 1(f) and 1(g)).
3.3. Caspases Activation and Subsequent Cleavage of Their
Substrates by As4O6. We then assessed the effects of As4O6
on caspases and their substrates (PARP and PLCγ-1). As4O6
decreased the expression levels of procaspase-3, procaspase-
8, and procaspase-9 in a dose- and time-dependent manner.
With the decrease of procaspases, the cleavages of PARP
and PLCγ-1, the substrates of caspases, were found to be
progressed in a dose- and time-dependent manner (Figures
2(a) and 2(b)). These findings suggest that As4O6 may
induce apoptosis through caspase activation. To confirm and
quantify the proteolytic activation of caspases, we assessed
their activities using colorimetric assay kits. The caspase
activity assay also showed that As4O6increased proteolytic
activities of caspases in a dose- and time-dependent manner
(Figures 2(c) and 2(d)).
3.4. Effects of As4O6 on Bcl-2 Family Members and X-
Linked Inhibitor of Apoptosis (XIAP). To elucidate further
underlying mechanisms of As4O6-induced apoptosis, we
assessed the levels of Bax, Bcl-2, Bad, Bcl-xL, and XIAP,
which play a crucial role in apoptosis. Western blotting
revealed that As4O6induced an increase in the expressions of
Bax (proapoptotic protein) in a dose- and time-dependent
manner whereas the expression of Bcl-2, Bad, Bcl-xL,
and XIAP (antiapoptotic proteins) remained unchanged
or slightly reduced (Figure 3(a)). The induction of Bax
expression began to clearly be observed at 12 hours after
the treatment (Figure 3(b)). This finding suggested the
possibility that the mechanism of Bax induction was related
to the transcriptional activity. These findings suggested
that upregulation of Bax protein and increased Bax/Bcl-2
ratio should be an important mechanism of As4O6-induced
apoptosis in U937 cells.
3.5. Effects of As4O6 on Autophagy. Many studies have
demonstrated that As2O3 can induce cell death through
autophagy . During autophagy, LC3-1 is converted to
membrane-bound LC3-II that correlates with the extent of
autophagosome formation which characterizes autophagy.
For the autophagosome formation, Beclin-1 is important in
mammalian cells. Hence, we assessed the expression of LC-
3 (a marker for autophagy) and beclin-1 to check whether
As4O6-induced cell death is involved in type II programmed
cell death, autophgy. Western blotting revealed that As4O6
I) and increased the expressions of beclin-1 in a dose- and
time-dependent manner (Figures 4(a) and 4(b)). The level
of autophagosome formation corresponds with the ratio
of LC3-II/LC3-I. Moreover, we also obtained evidence for
As4O6-induced autophagy by measuring AVO formation
through acridine orange staining. As shown in Figures 4(c)
and 4(d),As4O6induced the accumulationof AVO in a dose-
and time-dependent manner.
3.6. Effects of Bcl-2 on As4O6-Induced Autophagy and Apop-
tosis. From the above, we found that As4O6 induced not
only apoptosis through Bax induction but also autophgy
through Beclin-1 induction. It has been suggested that the
autophagy can be induced by apoptotic insults through up-
regulation of Beclin-1. Bcl-2 is a well-known antiapoptotic
molecule, and the interaction between Bcl-2 and Beclin-1
is important in the induction of autophgy. Therefore, we
assessed Beclin-1 response to Bcl-2 overexpression and the
effects of Bcl-2 overexpression on As4O6-induced autophgy
and apoptosis by comparing those between U937/vector and
U937/Bcl-2 cells that constitutively express high levels of
Bcl-2. As shown in Figure 5(a), Bcl-2 overexpression led to
significantly suppress the apoptosis induced by As4O6. We
cells by DAPI staining. The DAPI staining showed that Bcl-2
apoptosis (Figure 5(b)). We also assessed the effects of Bcl-
2 overexpression on As4O6-induced autophagosome forma-
tion. It reduced the As4O6-As4O6-induced AVO formation
(Figure 5(c)). To confirm this finding at the molecular level,
we performed western blotting for the molecules involved in
As4O6-induced apoptosis and autophagy. It was observed on
Western blotting that the overexpression of Bcl-2 suppressed
the induction of Beclin-1 and LC3 conversion in response
to As4O6, with the suppression of As4O6-induced caspase-
3 activation and PARP cleavages (Figures 5(d) and 5(e)).
These findings suggested that the increased Bcl-2 should
induction by As4O6might be related to apoptosis induction.
4Evidence-Based Complementary and Alternative Medicine
Cell viability (%)
Apoptotic cells (%)
Apoptotic cells (%)
Cell viability (%)
Figure 1: Inhibition of cell growth and induction of apoptosis by As4O6in U937 cells. The growth inhibition and cytotoxicity As4O6are a
dose- and time-dependent manner. The efficacy of As4O6is superior to that of As2O3. The cells were seeded at the density of 5 × 104cells
per mL. The inhibition of cell growth was measured by MTT assay. (a) and (c) The cells were treated with the indicated concentrations
of As4O6and As2O3for 24hours. (b) and (f) The cells were treated with 3μM of As4O6for the indicated times. The growth inhibition
and cytotoxicity As4O6are exhibited in a time-dependent manner. (c) After fixation, the cells were stained with DAPI solution to observe
apoptotic bodies, which were more frequently seen in higher doses. Stained nuclei were then observed under fluorescent microscope using
a blue filter (Magnification, X 400). (d)–(g) To quantify the extent of As4O6-induced apoptosis, sub-G1 DNA content, which represents the
fractions undergoing apoptotic DNA degradation, was analyzed by flow cytometry. The data are shown as means ± SD of three independent
experiments.∗P < 0.05 between the treated and the untreated control groups.
Evidence-Based Complementary and Alternative Medicine5
Figure 2: Activation of caspases and cleavage of PARP during the As4O6-induced apoptosis in U937 cells. The activation of caspases and
cleavage of PARP by As4O6are a dose- and time-dependent. (a) and (c) The cells were incubated at the indicated concentrations of As4O6
for 24h. (b) and (d) The cells were treated with 3μM of As4O6for the indicated times. (a) and (b) Total cell lysates were resolved by SDS-
polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were probed with the anticaspase-3, anticaspase-8,
anticaspase-9, and anti-PARP antibodies. The proteins were visualized using an ECL detection system. β-Actin was used as an internal
control. (c) and (d) The cell lysates from the cells treated with As4O6were assayed for in vitro caspase-3, caspase-8, and caspase-9 activity
using DEVD-pNA, IETD-pNA, and LEHD-pNA, respectively, as substrates. The released fluorescent products were measured. Each bar
graph represents mean ± SD of three independent experiments.∗P < 0.05 between the treated and the untreated control groups.
3.7. Inhibition of As4O6-Induced Apoptosis and Autophagy
in U937 Cells by N-Acetylcysteine (NAC). A previous study
showed that As4O6 induced reactive oxygen species (ROS)
leading to loss of mitochondrial potential (MMP, ΔΨm)
. In addition, As2O3induced apoptosis in leukemic cell
lines via modulation of the glutathione (GSH) redox system
. NAC is an antioxidant that functions by donating
a cysteine to the de novo synthesis of GSH. To assess the
effects of NAC on As4O6-induced autophgy and apoptosis,
we analyzed the cells with sub-G1 DNA content and AVOs
using flow cytometry after As4O6 treatment and observed
changes in nuclear morphology of As4O6-treated cells by
DAPI staining. We found that NAC reduced the As4O6-
6Evidence-Based Complementary and Alternative Medicine
Figure 3: Regulation of Bcl-2 and IAP family proteins during As4O6-induced apoptosis. As4O6increases the expressions of Bax in a dose-
and time-dependent manner whereas the expressions of Bcl-2, Bad, Bcl-xL, and XIAP remain unchanged or slightly reduced. (a) The cells
were treated with the indicated concentrations of As4O6for 24h. (b) The cells were treated with 3μM of As4O6for the indicated times.
The cells treated with As4O6were lysed and equal amounts of proteins were then separated by SDS-polyacrylamide gels and transferred
to nitrocellulose membranes. The membranes were probed with the indicated antibodies and detected by an ECL detection system. To
confirm equal loading, the blot was stripped of the bound antibody and reprobed with the anti β-Actin antibody. The results are from one
representative experiment of at least two independent experiments that showed similar patterns.
cell death (Figures 6(a) and 6(b)). The DAPI staining
revealed that NAC reduced the frequency of condensed
and fragmented nuclei in the As4O6-treated U937 cells
To confirm this finding at the molecular level and
determine whether the Beclin-1-induction is associated
with ROS production, we performed western blotting for
the molecules involved in As4O6-induced apoptosis and
autophagy. Western blotting revealed that NAC suppressed
As4O6-induced Beclin-1 induction and LC3 conversion and
As4O6-induced caspase-3 activation and PARP cleavages
(Figures 6(d) and 6(e)). These findings suggested that the
As4O6-induced autophagy as well as apopotosis should be
related to ROS production. These findings suggested that
ROS production by As4O6 should be related to Beclin-1-
induced autophagy as well as apoptosis.
This study was designed to determine whether As4O6 has
anticancer properties in human leukemic cells and further
to investigate the underlying mechanisms as compared to
that of the anticancer effects of As2O3. Regarding the As4O6-
induced cell death, it has not been reported that autophagic
cell death is a critical mechanism for the effects. To gain
insights into the mechanisms for As4O6-induced cell death,
we investigated the both apoptosis and autophagy. Here, we
found that As4O6 did not only induce caspase-dependent
apoptotic cell death but also induce autophagic cell death.
Arsenic trioxide (As2O3) is well known to have anticancer
properties against leukemic cells as well as other cancer cells.
The reported mechanisms of As2O3-induced cell death vary
depending on the cell lines: caspase-dependent apoptosis
[16, 17], caspase-independent , and autophagic cell
death [13, 19]. Even in the studies on As2O3-induced cell
death of U937 cells, some studies reported that caspase-
dependent apoptosis is a major mechanism for the cell death
 and other studies suggested that autophagic cell death
is a critical mechanism for the antileukemic effects . In
other leukemic cell lines, arsenic trioxide did not only induce
apoptosis but also induced autophagic cell death in leukemia
cell lines via upregulation of Beclin-1 . The mechanism
although there is a report showing a significant difference
between As2O3- and As4O6-induced cell death .
Apoptosis is the process of programmed cell death that
can be executed through extrinsic pathway and intrinsic
pathway. Either pathway is involved in mitochondrial outer
tosis . The mitochondrial outer membrane permeabi-
lization is controlled by several factors, such as the Bcl-2 and
IAP protein family. The Bcl-2 family consists of proapoptotic
factors (e.g., Bax, Bad, etc.) and antiapoptotic factors (e.g.,
Bcl-2, Bcl-xL, etc.). The Bax/Bcl-2 ratio is known as a key
factor in triggering the apoptotic process. We found that
caspase-dependent apoptosis was one of mechanisms for
Evidence-Based Complementary and Alternative Medicine7
AVO formation (%)
AVO formation (%)
Figure 4: Effects of As4O6on the autophagy in U937 cells. AVO formation by As4O6is dose- and time-dependent. (a) and (c) The cells
were treated with the indicated concentrations of As4O6for 24h. (b) and (d) The cells were treated with 3μM of As4O6for the indicated
times. (a) and (c) The cells treated with As4O6were lysed and equal amounts of proteins were then separated by SDS-polyacrylamide gels
and transferred to nitrocellulose membranes. The membranes were probed with the indicated antibodies and detected by an ECL detection
system. To confirm equal loading, the blot was stripped of the bound antibody and reprobed with the anti β-Actin antibody. (b) and (d)
The cells treated with As4O6were stained with 5μg/mL acridine orange for 17min and collected in phenol red-free growth medium. Green
(510–530nm) and red (650nm) fluorescence emission illuminated with blue (488nm) excitation light was measured with a FACSCalibur
the antileukemic effects of As4O6through the induction of
Bax protein. At first we were puzzled at this result (Bax
induction by As4O6) in p53-deficient U937 cells because
tumor suppressor p53 plays the central role in regulating
Bax protein, a proapoptotic protein. However, the previous
report that Bax protein can be induced in U937 cells through
the transaction of p73 gene can explain our results .
This study also suggested that the Beclin-1-induced
autophagic cell death could be another mechanism for
As4O6-induced cell death. This finding showing As4O6-
induced autophagy in As4O6-induced cell death is also
Recently it has been reported that arsenic trioxide induces
a Beclin-1-independent autophagic cell death in ovarian
cancer cells . This finding suggested that mechanisms of
As2O3-induced cell death should vary depending on the cell
lines; so it is not unknown whether our results are applicable
to other cancer cells. Therefore, we are going to investigate
the mechanism for As4O6-induced cell death in other solid
cancer cells. Our results were derived from a single leukemic
cell line; so it is difficult to generalize this finding to all
leukemic cells. However, those indicated that As4O6-induced
Beclin-1 induction which led to autophagy can be another
mechanism for its antileukemic effects on U937 cells.
Another limitation is that we have not verified yet
whether Beclin-1-induced autophagy is a critical mechanism
for As4O6-induced cell death or a mechanism to rescue
cancer cells from toxic damage. Now that the autophagic
cell death is mainly a morphologic definition (i.e., cell death
associated with autophagosomes/autolysosomes), there is
still no definite evidence that a specific mechanism for
autophagic death actually exists. Nonetheless, it is quite
conceivable that the autophagy induced by As4O6 could
eventually destroy a cell because it has been reported
8Evidence-Based Complementary and Alternative Medicine
Apoptotic cells (%)
AVO formation (%)
Relative caspase-3 activity
the induction of Beclin-1 and LC3 conversion in response to As4O6as well as As4O6-induced caspase-3 activation and PARP cleavages (a)
U937/vector or U937/Bcl-2 cells were treated with 3μM of As4O6for 24h. Sub-G1 DNA content was analyzed by flow cytometry. (b) To
confirm apoptosis, the cells were stained with DAPI solution after fixation. Stained nuclei were then observed under fluorescent microscope
in phenol red-free growth medium. Green (510–530nm) and red (650nm) fluorescence emission illuminated with blue (488 nm) excitation
light was measured with a FACSCalibur (Becton Dickinson). (d) The cells were lysed and equal amounts of proteins were then separated
by SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with the indicated antibodies and
detected by an ECL detection system. (e) The cell lysates from the cells treated with As4O6were assayed for in vitro caspase-3activity using
DEVD-pNA. The released fluorescent products were measured. The data are shown as means ± SD of three independent experiments.∗P <
0.05 between the groups treated with and without As4O6,†P < 0.05 between the U937/vector and U937/Bcl-2 cells.
that autophagic cell death is a major mechanism for the
as well as arsenic compounds [13, 21].
not suppress Bcl-2 expression in this study, but we tested
the effects of augmented Bcl-2 on apoptosis and autophagy
as well as apoptosis induced by As4O6. We observed that
augmented Bcl-2 significantly suppressed the autophagic cell
death as well as apoptotic cell death induced by As4O6. This
finding is consistent with the previous study [27–29].
In aerobic organisms ROS is produced in the mitochon-
dria via the electron transport chain during energy pro-
duction. Under normal circumstances, reductive enzymes
such as catalase and superoxide dismutase can defend
cells from the ROS damage, but if ROS is produced
high enough to cause severe cellular damage, a cell may
undergo programmed cell death [20, 30]. We observed
that NAC suppressed As4O6-induced autophagy as well as
As4O6-induced apoptosis. This finding suggested that ROS
production should be greatly involved in As4O6-induced
autophagy as well as As4O6-induced apoptosis. Although the
possibility that Beclin-1-induced autophagy can be a process
to rescue cancer cells from As4O6-induced apoptosis could
not be excluded, our finding suggested that ROS induced by
As4O6should lead to Beclin-1-induced autophagy.
In conclusion, we have demonstrated that As4O6-
induced cell death is carried on through Beclin-1-induced
and that the ROS production by As4O6 plays important
roles in triggering both Beclin-1-induced autophagic cell
Evidence-Based Complementary and Alternative Medicine9
Apoptotic cells (%)
AVO formation (%)
Relative caspase-3 activity
Figure 6: Inhibition of As4O6-induced apoptosis and autophagy in U937 cells by N-acetylcysteine (NAC). NAC reduces the As4O6-induced
autophagosome formation as well as As4O6-induced cell death. (a) U937 cells were treated with NAC (10mM) 30min before As4O6(3μM)
for 24h. The cells treated with As4O6 were stained with 5μg/mL acridine orange for 17min and collected in phenol red-free growth
medium. Green (510–530nm) and red (650nm) fluorescence emission illuminated with blue (488nm) excitation light was measured with a
FACSCalibur (Becton Dickinson). (b) Sub-G1 DNA content was analyzed by flow cytometry. (c) To confirm apoptosis, the cells were stained
with DAPI solution after fixation. Stained nuclei were then observed under fluorescent microscope using a blue filter (Magnification, X 400).
(d) The cells were lysed and equal amount of the lysate was separated by SDS-polyacrylamide gels and then transferred to nitrocellulose
membranes. The membranes were probed with the indicated antibodies and detected by an ECL detection system. To confirm equal loading,
the blot was stripped of the bound antibody and reprobed with the anti β-Actin antibody. (e) The cell lysates from the cells treated with
As4O6were assayed for in vitro caspase-3activity using DEVD-pNA. The released fluorescent products were measured. The data are shown
as means ± SD of three independent experiments.∗P < 0.05 between the groups treated with and without As4O6,†P < 0.05 between the
groups treated with and without NAC.
10Evidence-Based Complementary and Alternative Medicine
death and caspase-dependent apoptosis. This study pro-
vides evidence that As4O6-induced cell death is related to
Beclin-1-induced autophagy as well as caspase-dependent
ment of leukemia similar to As2O3.
This study was supported by grants from the National
R&D Program for Cancer Control, Ministry of Health
& Welfare, Republic of Korea (0820050), and from Basic
Science Research Program through the National Research
Foundation of Republic of Korea (NRF), the Ministry of
Education, Science, and Technology (2010–0001730). The
authors thank Professor Hicks Timothy R. for helping the
revision and proofreading of this manuscript.
 Z. X. Shen, G. Q. Chen, J. H. Ni et al., “Use of arsenic trioxide
(As2O3) in the treatment of acute promyelocytic leukemia
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