Arsenic trioxide induces apoptosis and the formation of reactive oxygen species in rat glioma cells
Sun et al. Cellular & Molecular Biology Letters
Arsenic trioxide induces apoptosis and the formation of reactive oxygen species in rat glioma cells
Yuanyuan Sun 1
Chen Wang 0
Ligang Wang 0
Zhibo Dai 0
Kongbin Yang 0
0 Neurosurgery Department, First Affiliated Hospital, Harbin Medical University , Nangang District, Harbin 150000 , China
1 Nursing Support Center, First Affiliated Hospital, Harbin Medical University , Harbin 150000 , China
Background: Arsenic trioxide (As2O3) has a dramatic therapeutic effect on acute promyelocytic leukemia (APL) patients. It can also cause apoptosis in various tumor cells. This study investigated whether As2O3 has an antitumor effect on glioma and explored the underlying mechanism. Results: MTT and trypan blue assays showed that As2O3 remarkably inhibited growth of C6 and 9 L glioma cells. Cell viability decreased in glioma cells to a greater extent than in normal glia cells. The annexin V-FITC/PI and Hoechest/PI staining assays revealed a significant increase in apoptosis that correlated with the duration of As2O3 treatment and occurred in glioma cells to a greater extent than in normal glial cells. As2O3 treatment induced reactive oxygen species (ROS) production in C6 and 9 L cells in a time-dependent manner. Cells pretreated with the antioxidant N-acetylcysteine (NAC) showed significantly lower As2O3-induced ROS generation. As2O3 significantly inhibited the expression of the anti-apoptotic gene Bcl-2, and upregulated the proapoptotic gene Bax in both C6 and 9 L glioma cells in a time-dependent manner. Conclusions: As2O3 can significantly inhibit the growth of glioma cells and it can induce cell apoptosis in a time- and concentration-dependent manner. ROS were found to be responsible for apoptosis in glioma cells induced by As2O3. These results suggest As2O3 is a promising agent for the treatment of glioma.
Arsenic trioxide (As2O3); Reactive oxygen species (ROS); Glioma; Apoptosis
Despite commonly being known as a toxic metalloid, arsenic trioxide (As2O3) has
applications in traditional medicine in China. As early as the 1970s, a research group at
the First Affiliated Hospital of Harbin Medical University discovered that As2O3 can
induce remissions in up to 70% of acute promyelocytic leukemia (APL) patients [
The dramatic therapeutic effect of As2O3 on APL was achieved primarily through the
induction of cell differentiation and apoptosis [
]. At low concentrations, As2O3
promoted cell differentiation, while at concentrations above 0.5 μmol/l, it induced cell
As2O3 induced apoptosis not only in NB4 cells (an APL cell line) but also in various
other tumor cell lines [
]. The underlying mechanism remained unclear, but
inhibition of cell differentiation and growth and induction of apoptosis are speculated to be
the general mechanisms for tumor treatment  and As2O3 action [
research on As2O3 in APL showed that reactive oxygen species (ROS) play an important role
in the induction of apoptosis, and that APL cells are sensitive to the intracellular ROS
levels . However, there is still some discussion about whether ROS are involved in
As2O3 inhibition of the growth of tumor cells [
Due to the existence of the blood–brain barrier, it is hard for therapeutics drugs to affect
glioma cells. New therapeutics are required to overcome this challenge. Although it is still
unclear how As2O3 could cross the blood–brain barrier, several studies of As2O3 in
glioma indicate that it is a potential therapeutic agent for this type of cancer [
The effective concentrations of As2O3 applied in those studies were extremely high,
ranging from 4.0 μM to 5.0 mM [
]. High concentrations of As2O3 carry a major
health risk. Side effects include mild gastrointestinal discomfort, transient elevation of
liver enzymes, reversible neuropathy, hypokalemia, hyperglycemia and cardiac toxicity.
Prolongation of the life quality has been detected in as many as 38% of patients treated
with As2O3 [
]. In this study, we investigated the anti-tumor effect of a low
concentration range (0–8 μmol/l) of As2O3 in the glioma cell lines C6 and 9 L, assessed
changes to non-tumor (glial) cells, and explored the underlying mechanism by studying
As2O3 was obtained from Yida. Stock solutions were prepared in phosphate buffered
saline (PBS) to exclude any unknown influence from other solvents. Working solutions
were diluted in RPMI-1640 medium (Gibco) and Dulbecco’s modified Eagle’s medium
(DMEM; Gibco), supplemented with 10% heat-inactivated fetal calf serum (FCS).
Rat C6 and 9 L glioma cells were obtained from Harbin Medical Neurosurgical Institute
and were respectively cultured in 10% RPMI-1640 medium and 10% DMEM, in both
cases supplemented with 10% FCS. Primary glial cells were isolated from new suckling
Wistar mice within 24 h of birth using the method of McCarthy and de Vellis [
cell concentration was adjusted to 5 × 105 cells/ml in 15% DMEM. The fourth generation
(after about 20 days of culture) was used. The cells were maintained at 37 °C, 95% air and
5% CO2 in a humidified incubator (Heraeus).
Determination of cell viability
To test cell viability, cell suspensions of 2 × 105 cells/ml were mixed with 0.4% trypan
blue. After 5–10 min, dye exclusion was examined for viable cells under a light
microscope. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
bromide assay was also used to determine the number of viable cells after exposure to
As2O3. 200 μl cell suspensions (4 × 104 cells/ml) were seeded in 96-well plates. Serially
diluted As2O3 was added at final concentrations of 0 (control), 0.5, 1.0, 3.0, 5.0, 6.0, 7.0
and 8.0 μmol/l. Each experiment was performed in quadruplicate and repeated at least
three times. After 24, 48 and 72 h, the MTT products were quantified and the results
were presented as the percentage of viable cells and normalized to the level of controls.
The optimal concentration was determined as 5.0 μmol/l and used to treat the rat C6
and 9 L cells.
Measurement of apoptosis
After cultured for 24, 48 and 72 h, cell apoptosis was assessed using propidium iodide
(PI) and annexin-V conjugated to fluorescein isothiocyanate (FITC) according to the
manufacturer’s instructions (BD Biosciences). Briefly, cells with or without As2O3 were
incubated with FITC-conjugated annexin-V. Then, the cells were collected, washed and
centrifuged at 200 g for 10 min. The cell pellet was gently resuspended in 200 μl PI
and incubated in the dark for 30 min at room temperature. Apoptosis was then
assessed using flow cytometry.
Cell apoptosis and necrosis were further examined by staining with Hoechst 33,342
(HOE) and PI, respectively. Cells were plated into 96-well plates and treated with
5.0 μmol/l As2O3 for 24, 48 and 72 h. Cells (5 × 106 cells/ml) were incubated for 15 min at
37 °C with HOE (10 μg/ml in PBS), centrifuged, washed in PBS, and resuspended at
density of 1 × 107 cells/ml. PI (50 μg/ml in PBS) was added before observation. Cells were
examined using a light microscope (Olympus) equipped with a fluorescent light source and a
UV-2A filter cube with an excitation wavelength of 330–380 nm and a barrier filter of
420 nm. All experiments were repeated at least three times.
Measurement of ROS levels
The generation of ROS was measured as previously described [
]. Briefly, cell suspensions
(2 × 106 cells/ml) were exposed to As2O3 at 5.0 μmol/l for 24, 48 and 72 h. To evaluate the
major organelles that governed the ROS-mediated stress in glioma cells, C6 and 9 L cells
were pretreated with 5 nM antioxidant N-acetylcysteine (NAC) for 2 h, and were exposed
to As2O3 at 5.0 μmol/l for 24 h [
]. After exposure, cells were incubated in 10 μM of
2′,7′-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes) at 37 °C for 30 min.
The cells were harvested and washed with cold PBS three times. Then, ROS levels were
determined through fluorescence-activated cell sorting.
Measurement of apoptotic proteins
Levels of apoptosis-related proteins (Bcl-2, Bax and Fas) were analyzed using Western blot
as previously described [
]. Briefly, cells were lysed at 4 °C via RIPA. Proteins were
separated using 10% SDS-PAGE, transferred to nitrocellulose membranes and incubated with
primary antibodies against Bcl-2, Bax, Fas and actin (1:100, Santa Cruz Biotechnology).
Then, the membranes were incubated with horseradish peroxidase-conjugated secondary
antibodies, and detected using an enhanced chemiluminescence (ECL) kit (Beyotime).
All quantitative data measurements were performed in triplicate and the results are
presented as means ± standard deviation. One-way analysis of variance (ANOVA) was
performed. The post hoc tests were Dunnett’s tests. Probability values (p) less than 0.05 were
considered statistically significant.
As2O3 decreased cell viability in C6 and 9 L glioma cells
The cytotoxicity of As2O3 in C6 and 9 L cells was assessed using the MTT and trypan
blue assays. As2O3 was applied at 0.5, 1.0, 3.0, 5.0, 6.0, 7.0 and 8.0 μmol/l, and the
inhibitory rates were determined after 24, 48 and 72 h (Fig. 1). The MTT assay showed
that the As2O3-induced inhibitory rates for C6 and 9 L cells were dose and time
dependent (Fig. 1a). The inhibitory effects of As2O3 on C6 and 9 L cells were
significantly stronger than on normal glial cells. For example, the inhibition rate for normal
glial cells exposed to 5.0 μmol/l As2O3 was less than 10% of that for glioma cells,
suggesting that As2O3 inhibited the growth of glioma cells but not normal glial cells in
range of 0–8 μmol/l. The calculated IC50 values for C6 and 9 L cells were respectively
5.0 and 5.6 μmol/l As2O3, so 5.0 μmol/l As2O3 was used in the following experiments.
The trypan blue assay showed that 5.0 μmol/l As2O3 significantly decreased cell
viabilities in C6 and 9 L in a time-dependent manner (Fig. 1b). Although the cell viability
in normal glial cells was also significantly decreased, the change was smaller than for
glioma cells, suggesting a greater inhibitory role in glioma than in glial cells.
As2O3 induced apoptosis in C6 and 9 L glioma cells
An annexin V-FITC/PI assay was used to assess cell apoptosis of glioma cells after
exposure to 5 μM As2O3. The numbers of early (annexin V+/PI–) and late (annexin V
+/PI+) apoptotic cells were calculated. In both C6 and 9 L glioma cells, apoptosis (seen
as both early and late apoptotic cells) was significantly induced by 5 μM As2O3 in a
time-dependent manner (Fig. 2a and b). The maximal percentages of apoptotic cells in
both cell lines were reached at 72 h (14.35% of C6 cells, and 13.13% of 9 L cells; Fig. 2).
However, the apoptotic rate for glial cells was only 3.59% (Fig. 2c and d). For cells
without As2O3 treatment, the apoptosis rate was close to 0 (data not shown).
The apoptosis in glial and glioma cells after exposure to 5 μM of As2O3 for 72 h was
further confirmed with HOE/PI double staining (Fig. 3). Cell uptake of PI indicated
necrosis. Cells with clear nuclear condensation but no PI uptake indicated apoptosis.
After exposure to As2O3 for 72 h, C6 and 9 L cells showed increases in both necrosis
and apoptosis. The level of apoptosis and necrosis induced by As2O3 was higher in C6
and 9 L cells than in glial cells.
Production of ROS in C6 and 9 L glioma cells exposed to As2O3
The extent of cellular oxidative stress in living cells was estimated by monitoring ROS
generation using the fluorescent dye DCFH-DA (Fig. 4). The mean fluorescence intensity in C6
cells was 7.58, 200.37, 344.80 and 501.74 at 0, 24, 48 and 72 h, respectively. The mean
fluorescence intensity in 9 L cells was 3.01, 180.27, 248.32 and 485.90 at 0, 24, 48 and 72 h,
respectively. Thus, the level of ROS level positively correlates with DCF intensity. In both C6
(Fig. 4a and b) and 9 L (Fig. 4b and c) cells, intracellular ROS increased significantly with
increasing incubation time with 5 μmol/l As2O3 (p < 0.01). Cells pretreated with NAC
significantly inhibited the increase in ROS in response to 24 h exposure to As2O3 (Fig. 4a and b).
Effects of As2O3 on the expression of apoptotic proteins Bcl-2, Bax and Fas
To validate the apoptosis process, the expression levels of apoptosis markers, including
Bcl-2, Bax and Fas, were examined in C6 and 9 L glioma cells using western blotting.
As2O3 significantly inhibited expression of the anti-apoptotic gene Bcl-2 and upregulated
the pro-apoptotic gene Bax in both C6 and 9 L glioma cells in a time-dependent manner
(Fig. 5a and b). The expression of Fas did not significantly change after exposure to As2O3
(Fig. 5a and b).
Because of its ability to induce apoptosis in various malignant tumor cells, As2O3 has
potential as a treatment agent for malignant tumors [
]. Gliomas are highly
aggressive tumors that respond poorly to existing clinical therapeutic agents. In
previous studies, it was shown that As2O3 treatment could inhibit cell growth of glioma
cells, but the studies did not yield guidance on the effective doses [
Here, we investigated the effective doses of As2O3 using rat glioma cells and
comparing them with non-tumor glial cells. Our results showed that As2O3 inhibited the
growth of glioma cells in time- and concentration-dependent manners, and that
5.0 μmol/l As2O3 is the optimum concentration for inhibiting cell viability in both C6
and 9 L glioma cells. The inhibitory rate for non-tumor cells was less than 10% of that
for the glioma cells, indicating that As2O3 is a promising drug. Due to the exist of the
blood–brain barrier, it remains unclear how the 5 μmol/l concentration can be obtained
in human blood such that it would be useful for treating glioma cells. Further studies
using in vivo animal models are needed.
Both the HOE/PI and annexin-V/PI assays showed that 5.0 μM As2O3 induced
apoptosis. However, the mechanism of apoptosis in solid tumor cells is far from clear. In
glioma cells treated with As2O3, one of the most likely mechanisms for triggering an
antitumor effect is the induction of ROS [
]. Like other heavy metals, including
iron, copper, chromium, cadmium, lead and mercury, arsenic affects cells by causing
Fig. 5 Western blot of Bcl-2, Bax and Fas in C6 glioma and 9 L sarcoma cells with or without treatment with
As2O3 (5 μmol/l) for 24, 48 and 72 h. Bcl-2 expression reduced in a time dependent manner, while Bax
expression increased in a time dependent manner. a Blots and quantification data for C6 cells. b Blots and
quantification data for 9 L cells. *p < 0.05 vs. control
oxidative damage, primarily through disruption of the endogenous cellular
antioxidant–redox balance [
]. Cysteine thiol is the functional site for most redox
proteins. Arsenic can directly bind to this site and destroy protein function, thereby
affecting ROS production and clearance [
]. Cell viability, ROS levels, apoptosis
and autophagy in human glioblastoma cell line have been shown to be regulated by
] and/or As2O3 in combination with other agents [
]. As2O3 induces
ROS production and apoptosis in glioma cells through the upregulation of the
mitoferrin-2 gene [
]. Consistently with the results of those studies, we also found that
intracellular ROS levels increased significantly after As2O3 treatment.
The brain appears to be especially sensitive to ROS stress when compared to other
organs. Although comprising only 2% of human body weight, the human brain
consumes up to 20% of the oxygen supply. Such a high level of oxygen consumption
indicates that large quantities of ROS are generated during oxidative phosphorylation in
brain tissue. In addition, iron content has been shown to increase brain sites in which
ROS production may be greater [
]. Tumor cells are vulnerable to ROS stress. Thus,
therapeutic approaches directed at ROS intervention may have an antitumor effect, and
As2O3 is a promising antitumor reagent for gliomas.
As2O3 downregulated the expression of Bcl-2, an anti-apoptotic protein, and
upregulated the expression of Bax, a pro-apoptotic protein, thus shifting the Bax/Bcl-2 ratio in
favor of apoptosis. Fas protein expression remained unchanged. These findings indicate
that Bcl-2 and Bax play an important role in As2O3-induced apoptosis in C6 and 9 L
Our results hinted at the possible involvement of mitochondrial dysfunction in
As2O3-induced apoptosis. The Bcl-2 family of proteins appear to control cell death by
regulating mitochondrial physiology [
]. A change in the mitochondrial
electrochemical potential results in the release of apoptotic proteins, such as cytochrome c, Smac/
DIABLO, pro-caspases 2, 3 and 9, and apoptosis-inducing factor.
Under physiological and pathophysiological conditions, ROS contributes to trigger
and mediate apoptosis [
]. The mitochondria are highly susceptible to oxidative
damage, and Bcl-2 exerts its anti-apoptotic function by reducing intracellular ROS. As2O3
downregulated Bcl-2 and rendered C6 and 9 L glioma cells vulnerable to apoptotic cell
death. In cells pretreated with NAC, As2O3-induced apoptosis was inhibited, suggesting
that a mitochondrial death pathway plays an important role in As2O3-induced
As2O3 strongly inhibits cell viability and induces apoptosis of rat C6 and 9 L glioma
cells in vitro when used at an optimal concentration of 5 μmol/l. This action is related
to the induction of ROS generation. Moreover, As2O3 showed lower cytotoxicity to
normal glial cells than glioma cells, indicating that As2O3 may be a potentially potent
chemotherapeutic agent for treating brain tumors.
APL: Promyelocytic leukemia; As2O3: Arsenic trioxide; DMEM: Dulbecco’s modified Eagle’s medium;
ECL: Enhanced chemiluminescence; FCS: Fetal calf serum; FITC: Fluorescein isothiocyanate; MTT:
3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS: Phosphate buffered saline; PI: Propidium iodide;
ROS: Reaction oxygen species
This study was supported by the National Natural Science Foundation of China (Grant No. 30600641) and the
Administration of Education, Heilongjiang Province (Grant No. 11511209).
Availability of data and materials
Please contact the author with data requests.
YS, CW, LW and ZD performed the experiments. All the authors contributed to the data analysis and manuscript
preparation. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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