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Introduction
Recently, the antineoplastic properties of arsenic trioxide
(As2O3) have aroused wide interest.
In vitro studies have shown that
As2O3 induced the apoptosis of acute promyelocytic leukemia (APL) cell lines at micromolar
concentrations[1]. These preclinical studies were confirmed by controlled clinical trials showing that treatment with
As2O3 led to complete remission in APL
patients[2]. More recently, studies have revealed that the apoptosis-inducing properties of
As2O3 were not restricted to APL, and the viability of different types of tumor cells, including hepatocellular carcinoma cell lines, were shown
to be altered in vitro by exposure to
As2O3[3,4]. Key mediators of sensitivity to
As2O3-induced apoptosis included intracellular
glutathione (GSH) and reactive oxygen species (ROS). The loss of inner mitochondrial membrane potential
is also an important step in
As2O3-mediated cell
apoptosis[5].
Ascorbic acid (AA), regarded as an anti-oxidant, has been found to have pro-oxidative properties which could enhance
the sensitivity of myeloid and lymphoid malignant cells to
As2O3 in
vitro[6_8]. In a previous study, we found that AA could
promote As2O3-induced apoptosis of human hepatocellular carcinoma cell line, HepG2,
in vitro[9]. As ROS played a key role
in AA combined with As2O3-induced apoptosis in other cell
lines[6,7], we investigated ROS and
downstream activation by ROS in the mechanisms of apoptosis induced by
AA/As2O3 combination in HepG2 cells.
Materials and methods
Reagents Ascorbic acid,
As2O3 and rhodamine 123 (RH123) were purchased from Sigma Chemical (USA) (chemical
purification >99%). The 6-carboxy-2¡¯,7¡¯
dichloro-dihydrofluorescein diacetate (DCFH-DA) and
N-acetyl-L-cysteine (NAC) were purchased
from Beyotime Institute of Biotechnology; Annexin V-FITC Apoptosis Detection Kit was purchased from
Alexis (USA); GSH, glutathione peroxidase
(GPx) and superoxide dismutase (SOD) assay kits were
purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); antibodies to Bcl-2, Bax, and caspase-3 (p17) were obtained from
BeiJing Zhongshan Golden Bridge Biotechnology Corporation (Beijing, China);
and anti-b-actin antibody was purchased from Santa Cruz (USA).
Cell culture and drug treatment Human hepatocellular carcinoma cell line HepG2 was obtained from the China Center for
Type Culture Collection. Cells were cultured in Dulbecco¡¯s Modified Eagle¡¯s Medium (DMEM) (HyClone, USA)
supplemented with 10% newborn calf serum (HyClone,
USA) in a humidified atmosphere of 95% air and
5% CO2 at 37 °C in a carbon
dioxide incubator (Shellab,USA). The cells in exponential growth state were divided into 4 groups:
As2O3 treatment group, cells were incubated with 2.0 µmol/L
As2O3 for 24 h; AA treatment group, cells were treated with 100 µmol/L AA for 24 h;
combined treatment group, cells were cultured in 2.0 µmol/L
As2O3 combined with 100 µmol/L AA for 24 h; control group, cells
were incubated for 24 h with medium alone. In the experiments on apoptosis rate and ROS level, the above 4 groups were
treated with or without 10 mmol/L NAC for 24 h.
Detection of intracellular ROSIntracellular ROS was detected by means of an
oxidation-sensitive fluorescent probe (DCFH-DA). After treatment with
As2O3 2.0 µmol/L and/or AA 100 µmol/L, with or without NAC 10 mmol/L for 24
h, cells were washed twice in phosphate-buffered saline (PBS). They were then incubated with 10
µmol/L DCFH-DA at 37 ºC for 20 min
according to the manufacturer¡¯s instructions. DCFH-DA was deacetylated intracellularly by nonspecific esterase, which was
further oxidized by ROS to the fluorescent compound 2,7-dichlorofluorescein (DCF). DCF fluorescence was detected by
FACScan flow cytometer (Becton Dickinson). For each sample 10 000 events were collected.
Intracellular GSH, and GPx, SOD activity
assay The GSH activity was measured by reacting on
DTNB(5,5¡¯-DITHIO-BIS(2-NITROBENZOIC ACID) to form a yellow substance, which can be determined by colorimetry. The GPx activity was
detected by the oxidizing speed of the GSH, which can be expressed by the GSH reduction in a certain time. One unit of GPx
activity was defined as 1 µmol/L GSH oxidized to GSSG (glutathione disulphide) per milligram of protein per minute. The SOD
activity was determined by hydroxylamine assay-developed from xanthine oxidase assay, which was presented as units per
milligram of protein. The 4 groups of treated cells mentioned in "cell culture and drug
treatment" were washed twice and then resuspended in PBS, sonicated for 30 s on ice, and centrifuged at
1000×g for 15 min according to the manufacturer¡¯s instructions.
The supernatants were subjected to intracellular GSH, and GPx, SOD activity assays, which were determined with commercial
kits from Jiancheng Bioengineering Institute.
Flow cytometry analysis of mitochondrial membrane potential
RH123, the anion dye, could enter the mitochondrial
matrix and cause photoluminescent quenching dependent on mitochondrial transmembrane potential. If mitochodrial
membrane potential depleted, the RH123 could be released from the mitochondria and produce fluorescence. The mean
fluorescence intensity of RH123 was measured to determine the loss of
mitochondrial membrane potential. Treated cells were
collected and resuspended at a concentration of
1×105/mL in PBS, which contained 1 µmol/L RH123 and incubated at 37 ºC for
30 min. Samples were analyzed by FACScan flow cytometer (Becton Dickinson, USA) with an excitation wavelength of 488
nm and an emission wavelength of 525 nm. Fluorescent signal intensity was recorded and analyzed by CellQuest software
(Becton Dickinson). For each sample 10 000 events were
collected[10].
Western blotting analysis of Bcl-2, Bax and p17 subunit of caspase-3
expression Approximately
5×106 treated cells were washed with cold PBS (containing PMSF, phenyl-methylsulfonyl fluoride) three times, and collected by scraping. Collected
cells were solubilized in 150 µL ice-cold lyse buffer, and then sonicated. The cell-free supernatants were recovered by
centrifugation at 12 000×g for 25 min at
4 °C. Loading buffer was added to each supernatant, which was subsequently boiled for 10 min. Then 20 µL lysate was
electrophoresed on a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel. Proteins were then transferred onto
nitrocellulose membrane. After blocking of the membrane with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20
for 1 h at 25 °C, the blots were incubated for 12 h at 4°C with the appropriate dilution of the studied antibody (anti-Bcl-2,
anti-Bax and anti-caspase-3 p17 antibody was used at 1:500 dilution;
anti-b-actin antibody was used at
1:3000 dilution). After washing, the blots were incubated with peroxidase-conjugated secondary antibody (1:2500) in the
second reaction. Binding of antibodies was detected by chemiluminescence staining using the ECL detection kit (Amersham)
and quantified by densitometry. Comparable loading of proteins on the gel was verified by re-probing the blots with an
antibody specific for the housekeeping gene product
b-actin. The optical density scores of bands were calculated to analyze
the relative content of Bcl-2, Bax, and caspase-3 after normalization with
b-actin values in the same lane.
Apoptosis assessment by Annexin V-FITC and propidium iodide staining
Cell apoptosis was measured by Annexin V-FITC and propidium iodide (PI) staining. Cells were
treated with As2O3 2.0 µmol/L and/or AA 100 µmol/L, with or without NAC
10 mmol/L for 24 h, and then washed twice in 4 °C
PBS and resuspended in binding buffer at a cell density of
1×106/mL. Cells were then stained with 5 µL Annexin V-FITC and
10 µL PI (20 µg/mL) according to the manufacturer¡¯s instructions. They were then incubated in the dark at 25 °C for 15 min.
Samples were acquired on a FACScan flow cytometer (Becton Dickinson) and analyzed with Cellquest software (Becton
Dickinson); 10 000 cells were analyzed.
Statistical analyses All values were presented as the mean±SD. Statistical analyses were performed by using SPSS
11.0 software. Representative data were analyzed for statistical significance by one-way ANOVA. A
P-value of less than 0.05 was considered as statistically significant.
Results
Effects of AA and As2O3 on intracellular ROS level in HepG2 cells
Because the production of ROS is a critical
contributor to As2O3-induced
apoptosis[5], the level of ROS was measured to test whether oxidative stress played an important role in
AA potentiated As2O3-induced apoptosis.
Exposure of HepG2 cells to 2.0 µmol/L
As2O3 plus 100
µmol/L AA led to a remarkable increase in intracellular ROS compared with
As2O3 alone, with increased ROS level represented
by the DCF intensity from 127.61±5.12 to 152.60±5.88 (Figure
1, Table 1).
Effects of AA and As2O3
on intracellular GSH level in HepG2
cells A remarkable depletion in cellular GSH was found after
AA or As2O3 exposure. A higher decrease in GSH was observed in the combined treatment than
As2O3 treatment (Table 2). As
GSH was recognized as the predictor for response to
As2O3
treatment[5], the results suggested that AA potentiated
As2O3-induced apoptosis by depleting intracellular GSH.
Effects of AA and
As2O3 on intracellular GPx and SOD activity
In this research, significant reductions in GPx and
SOD activity were observed after
As2O3/AA exposure (Table 2). These results also proved that oxidative insults occurred in the
combined treatment, because GPx and SOD were recognized as the most important antioxidant members. However, SOD and
GPx depletion were found to be equal to
As2O3 treated alone
(P>0.05).
Effects of AA and
As2O3 on mitochondria membrane potential
ROS can result in free radical attack of membrane
phospholipids, leading to a loss of mitochondrial membrane potential. Mitochondria depolarization was considered as an
irreversible step in the apoptosis
process[11]. Therefore, the ability of HepG2 cell mitochondria to maintain membrane
potential in the four groups was measured by using flow cytometry techniques.
As2O3 treatment caused
depolarization of the inner mitochondrial membrane, while AA alone had
no significant effect on mitochondrial membrane potential (Figure 2). The
combination of
As2O3 and AA increased the
depolarization of the inner mitochondrial membrane compared to
As2O3 treatment
alone (Figure 2).
Effects of AA and
As2O3 on the ratio of Bcl-2/Bax
Proto-oncogene Bcl-2 appeared to function by inhibiting the
mitochondria depolarization. Conversely, Bax induced mitochondria depolarization. The ratio of Bcl-2 to Bax has been
reported to be correlated with apoptosis in cancer cells. The anti-apoptotic Bcl-2 protein bound a pro-apoptotic Bax protein,
and if Bcl-2 was in excess, all available Bax was bound and apoptosis was blocked. If Bax was in excess, all Bcl-2 was bound
and the cell proceeded to die[12]. Our results showed that there was an approximate 9-fold decrease in the ratio of Bcl-2/Bax
expression in the As2O3 treatment and combined treatment compared with the control, but there was no significant alteration
between As2O3 treatment and the combined treatment (Figures 3_5). Therefore, we could infer that AA potentiated
As2O3-induced apoptosis was in a Bcl-2 and Bax independent manner.
Effects of AA and
As2O3 on activation of
caspase-3 With the mitochondrial membrane¡¯s potential depletion by ROS, the
permeability transition pore (PTP) opened and intermembrane proteins were released out of the mitochondria,
which in turn activated a downstream executive
caspase-3[13]. Caspase-3 is expressed in cells as an inactive 32 kDa precursor from which
the 17 kDa (p17) and 11 kDa (p11) subunits of the mature caspase-3 are proteolytically generated during apoptosis. The
active caspase-3 enzyme is a heterodimer composed of two p17 and two p11 subunits. In the present
study, a marked increase of caspase-3 p17-activated
caspase-3 subunit in As2O3 or combined treatment was observed. Furthermore, a significant
increase in the combined treatment group compared with the
As2O3 treatment group was found (Figures 3 and 4).
NAC attenuates As2O3 and AA-mediated ROS increase and cell
apoptosis In our previous studies,
AA/As2O3 combined treatment could increase the apoptosis rate significantly compared to
As2O3 treatment
alone[9]. Similarly, in this test, untreated cultures contained 7.89%±0.98% apoptotic
cells. However, if the cells were treated
with 2.0 µmol/L
As2O3 and 100 µmol/L AA in combination, an almost 2-fold increase to 15.60%±1.14% apoptotic cells was
observed. In order to find out the relationship between ROS and the apoptosis effect of combined treatment, antioxidant NAC was used to scavenge
intracellular ROS. The results showed that NAC abolished the apoptosis effect of
As2O3 and AA-combined treatment, with the
apoptosis rate of
9.48%±0.67% (Figure 6), while attenuating combinative treatment mediated ROS elevation (Figure 1, Table 1). These results
suggested that ROS played a very important role in the enhancement effect of AA on the apoptosis of HepG2 cells induced
by As2O3.
Discussion
ROS played a very important role in apoptosis induction under both physiological and pathological conditions. Interestingly,
mitochondria were both the source and target of ROS. ROS, which was predominantly produced in the mitochondria, led to
the free radical attack of membrane phospholipids and
loss of mitochondrial membrane potential, which caused the
intermembrane proteins, such as cytochrome c, to be released out of the mitochondria and ultimately triggered caspase-3 activation.
Caspase-3 activation led to DNA breakage, nuclear chromatin condensation, and cell
apoptosis[5,11,13,17]. Anti-apoptotic
Bcl-2 and pro-apoptotic Bax, which resided upstream of mitochondria,
focused much of their efforts at the level of mitochondria.
Bcl-2 appeared to inhibit the mitochondria depolarization and ROS production, while Bax induced mitochondria depolarization
and ROS production[12].
Oxidative damage has been postulated to play the key role in the apoptosis process induced by
As2O3[14_16,23,24]
. The evidence was listed as follows: first,
As2O3 disturbed natural oxidation and reduction equilibria through various mechanisms
involved in complex redox reactions with endogenous oxidants and cellular antioxidant systems; second,
As2O3, by oxidizing thiols, acted directly on mitochondria to destroy the mitochondrial inner transmembrane potential, then promoted ROS
production; and third, As2O3, by downregulating Bcl-2 expression, resulted in mitochondria depolarization and ROS
production[1, 22]. A major mechanism for detoxification of radicals in mammalian cells is the breakdown of ROS by scavenger, such as
GPx, and SOD. The former could specifically catalyse the GSH to reduce the
hydroperoxide[14], and the latter could clear the
superoxide anion free radical.
AA, which was regarded as an anti-oxidant, was found to have pro-oxidative properties. In the plasma, AA was rapidly
oxidized to dehydroascorbic acid (DHA), which was then transported into the cell. AA was then regenerated from DHA via
glutaredoxin, which converted GSH to GSH disulfide. Recent evidence showed that AA enhanced the sensitivity of myeloid
and lymphoid malignant cells to
As2O3 in vitro through the oxidative
pathway[6_8] and the increased level of ROS was the
result of GSH depletion. GSH was a main nonprotein antioxidant in the cell, and it could clear away the superoxide anion free
radical and provide electrons for enzymes such as GPx, which reduce
H2O2 to H2O. Additionally, GSH could combine with
As2O3 directly, forming a transient
As(GS)3 molecule, which was more easily excreted through drug efflux
pumps[18_21].
In the present study, ROS was found to be a critical contributor to the potentiating effect of AA on
As2O3-induced apoptosis in HepG2 cells. A higher level of ROS production, much more depletion of antioxidant enzymes, and the activation
of ROS downstream, such as mitochondria depolarization and caspase-3 activation, were detected in the combined treatment
as compared to the As2O3 treatment alone. This suggests that AA potentiated
As2O3 -induced apoptosis in HepG2 cells
through the oxidative pathway. More importantly,
As2O3 and AA mediated
cell apoptosis was abrogated in the presence of
an antioxidant, NAC, which at the same time attenuated ROS elevation caused by the
As2O3 and AA combination. Additionally,
we found that AA enhanced
As2O3-induced ROS production was related to GSH depletion and was independent of Bcl-2 and
Bax, although As2O3 alone has been shown to cause downregulation of the ratio of Bcl-2 to Bax protein in HepG2 cells.
In conclusion, AA, by depleting intracellular GSH, potentiated
As2O3 mediated ROS production, which enhanced the loss
of mitochondrial membrane potential and activation of caspase-3, thus potentiating apoptosis in HepG2 cells induced by
As2O3.
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