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Introduction
Realgar, an arsenic disulfide compound, has long been
used as a therapeutic agent to treat some diseases in ancient
China and Europe, and its medicinal effects have attracted
increasing attention in recent years. It was found that
realgar was usually in the treatment of leukemia in China.
Previous studies showed that realgar could inhibit proliferation
and induce differentiation in the human promyelocytic
leukemia cell line HL-60 through activating some serine/threonine
protein phosphatases[1]. Although recent evidence has
suggested that in vitro realgar exhibits significant antiproliferation
effects on many kinds of leukemia, such as HL-60 and
K562[2,3], its mechanism of action in human histocytic lymphoma U937
is unclear. Our previous study has shown that in nanoparticle
realgar powder (NRP)-induced U937 cell death, the ratio of
the Bax/Bcl-2 protein expression was increased. Additionally,
the simultaneous activation of c-Jun NH2-terminal kinase
(JNK), but not p38 or the
extracellular regulated kinase (ERK),
was involved in the NRP-induced U937 apoptosis. In the
present study, we further investigated the mechanism
involved in the NRP-induced U937 cell death.
Apoptosis, or programmed cell death, is a genetically
regulated self-destructive cellular death process that is
important in development, tissue remodeling, immune
regulation, and many diseases[4_7]. Aberrant apoptosis is the
major cause for tumor development and progression. The
development of cancer is associated with several key events,
including deregulated cell growth in response to oncogene
activation that resists cells to
apoptosis[8,9]. It is well known that among the proteins involved in the regulation of cell
death, p53 and p53-regulated proteins, the mitochondrial
pathway-related protein a nicotinamide adenine dinucleotide
(NAD+)-dependent histone deacetylase (SIRT1) and the
phosphoinositide 3-kinase (PI3-K)/Akt pathways are the key
factors in promoting or inhibiting apoptosis.
As a tumor suppressor, p53 enforces normal growth
control and genomic stability. A direct inactivation of the gene
that encodes it is reflected in the fact that acquired
mutations in it or its upstream activators are found in all major
human cancers[10]. The pathway, which p53 controls,
normally helps the cell to respond to DNA damage, and then
leads to either growth arrest or
apoptosis[11_14]. Although in response to genotoxic stress, multiple serine residues on
p53 are phosphorylated, p53 activation is not always
accompanied by the specific phosphorylation events proposed to
be important for its function[15_17].
PI3-K, an upstream effector of serine/threonine kinase Akt
(also known as protein kinase B [PKB]), is the major mediator
of survival signals that protect cells from
apoptosis[18,19]. Large studies have shown that PI3-K provides an anti-apoptotic
signal in many types of cells. PKB/Akt, a downstream target
of PI3-K, is a key serine/threonine kinase required for the
inhibition of apoptosis[20,21]. Moreover, the activation of the
PI3-K pathway was capable of protecting from p53-mediated
apoptosis[22]. Therefore, PI3-K acting in cellular signaling
transduction pathways plays a central role in cancer cell
survival.
SIRT1, a nicotinamide adenine dinucleotide
(NAD+)-dependent histone deacetylase, is known to inhibit
stress-induced apoptosis. It not only regulates p53 function through
deacetylation p53, but also inhibits apoptosis by
deacetylating the DNA repair factor Ku70, which is bound with Bax in the
cytosol, causing it to sequester Bax away from mitochondria
[23]. Either the activation of SIRT1 or inactivation of p53
protects against tumor cell apoptotic stimuli induced by
anticancer drugs[24].
The aim of this study was to investigate the effector
mechanism of NRP-induced U937 cell death. It was found
that NRP-induced U937 cell death is through distinct
mechanisms and pathways, including apoptosis and necrosis.
Moreover, the inhibition of the PI3-K/Akt signaling
pathway enhances NRP-induced apoptosis and contributes to
the upregulation of p53 protein expression, indicating that
NRP-induced U937 cell death is closely associated with the
inactivation of PI3-K/Akt and the activation of p53.
Materials and methods
Materials The NRP (the chemical quality is
As2S3) were obtained by the high-energy ball-milling machine and
techniques. The PI3-K inhibitor wortmannin and Akt
inhibitor KP372-1 were purchased from Sigma (St Louis, MO, USA).
The SIRT1 inhibitor sirtinol and Ras inhibitor manumycin A
were purchased from Calbiochem (La Jolla, CA, USA).
Polyclonal antibodies against caspase-3, Akt,
phosphorylated Akt, SIRT-1, p53, phosphorylated p53, and horseradish
peroxidase (HRP)-conjuated secondary antibodies (goat
antirabbit and goat antimouse) were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture The U937 cells were purchased from
American Type Culture Collection (ATCC, Manassas, VA, USA).
The cells were cultured in RPMI-1640 medium (GIBCO, NY,
USA) supplemented with 10% fetal bovine serum (Shengma
Yuanheng, Beijing, China), 100 mg/L streptomycin, 100
IU/mL penicillin, and 0.03% L-glutamine, and maintained at 37
ºC with 5% CO2 in a humidified atmosphere.
Cytotoxity assay The U937 cells were incubated at
2×104 cells/well in 96-well plates (NUNC, Roskide, Denmark). The
cells were incubated with different concentrations (20~100
µg/mL) of NRP for different time periods, or wortmannin and
KP372-1 (Sigma, USA) at given concentrations for 1 h before
the introduction of 75 µg/mL NRP for 24 h. Then cell growth
was measured with 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) assay. The percentage
of cell growth inhibition was calculated as follows:
Cell death
(%)=(A570[control]_A570
[NRP])×100/A570(control).
Lactate dehydrogenase activity-based cytotoxicity assays
Lactate dehydrogenase (LDH) activity was assessed using
a standardized kinetic determination kit (Zhongsheng,
Beijing, China). LDH activity was measured in both apoptotic
and necrotic cells. The viable cells were collected from the
culture medium by centrifugation at
1000×g at 4 ºC for 5 min, and
3000×g for apoptosis. The LDH content from the pellets
by centrifugation at 3000×g was used as an index of apoptotic
cell death (LDHp)[19]. The LDH released in the culture
surpernatant (extracellular LDH [LDHe]) was used as an
index of necrotic death. The LDH present in the pellets by
centrifugation at 1000×g cells was used as an index of
intracellular LDH (LDHi). The percentage of apoptotic and
necrotic cell death was calculated as follows:
Apoptosis (%)=LDHp/(LDHp+LDHi+LDHe)×100
Necrosis (%)=LDHe/(LDHp+LDHi+LDHe)×100.
Fluorescence morphological examination Apoptotic
morphology was studied by staining the cells with the
fluorescent DNA-binding dye acridine orange (AO). The cells
were harvested and washed 3 times with
phosphate-buffered saline (PBS) after being incubated with 75 or 120 µg/mL
NRP, and were then stained with 20 µg/mL AO (Sigma, USA)
for 15 min. Then the color and structure of the different cell
types were observed under a fluorescence microscope
(Olympus, Tokyo, Japan)[25].
Flow cytometric analysis The U937 cells
(1×106) were harvested and washed once in cold PBS. The cell pellets
were fixed in 70% ethanol and washed in cold PBS. Then the
pellets were suspended in 1 mL of propidium iodide (PI)
solution containing 50 µg/mL PI, 1 g/L RNase A, and 0.1%
(w/v) Triton X-100 in 3.8 mmol/L sodium citrate, followed by
incubation on ice under dark conditions for 30 min. The samples
were analyzed by a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Western blot analysis The U937 cells were treated with
NRP for different time periods. Both adherent and floating
cells were collected, and then the Western blot analysis was
performed as previously described with some modifications.
Briefly, the cell pellets were resuspended in lysis buffer
consisting of 50 mmol/L HEPES (pH 7.4), 1% Triton-X 100, 2
mmol/L sodium orthovanadate, 100 mmol/L sodium fluoride,
1 mmol/L edetic acid, 1 mmol/L phenylmethylsulphonyl
fluoride (PMSF), 10 mg/L aprotinin (Sigma, USA), and 10 mg/L
leupeptin (Sigma, USA) and lysed at 4 ºC for 60 min. After
centrifugation at 13000×g centrifugation for 15 min, the
protein content of the supernatant was determined by a protein
assay reagent (Bio-Rad Laboratories, Hercules, CA, USA).
The protein lysates were separated by electrophoresis in
12% SDS-PAGE and blotted onto a nitrocellulose membrane.
The proteins were detected using a polyclonal antibody and
visualized using antirabbit immunoglobulin G conjugated with
peroxidase (HRP) and 3,3'-diaminobenzidine tetrachloride
(DAB) as the HRP substrate.
Statistical analysis The data are expressed as mean±SD.
Statistical comparisons were made by Student's
t-test. P<0.05 was considered
significant.
Results
Preparation of NRP The NRP were prepared with
ZJM-20/25 Ball Mill (210th Hospital of the People's Liberation Army,
Dalian, China) in PBS. The particles of the NRP were
homogeneously decentralized (Figure 1A, 1B). Diameters above
85% NRP were smaller than 200 nm. The suspension was
filtrated with a 0.22 μm membrane and diluted with
RPMI-1640.
Effect of NRP on the growth of U937 cells
The NRP induced U937 cell death in a time- and dose-dependent
manner. The incubation of U937 cells with different doses of
NRP (20, 40, 60, 80, or 100 µg/mL) for different time periods
(12, 24, 36, or 48 h) resulted in a significant increase in the
death rate (Figure 2). 24 h after treatment with 75 µg/mL NRP,
the cell death rate reached nearly 50% and the
IC50 (half number of the cells are dead) at 24 h was 71.7 µg/mL. Therefore,
24 h of incubation with 75 µg/mL NRP is sufficient for the
half induction of cell death.
NRP-induced apoptotic cell death in U937
cells To determine the characteristics of NRP-induced U937 cell death,
morphological changes were observed using AO staining
by fluorescence microscopy. The control cells exhibited
uniformly green fluorescence (Figure 3A), while exposure of the
U937 cells to 75 µg/mL NRP for 24 h resulted in
morphological alterations characteristic of apoptosis, including
membrane blebbing, nuclear condensation, and granular apoptotic
bodies (Figure 3A).
It was reported that the ratio of LDH release from viable
cells, apoptosis, and the culture medium might be used to
distinguish the number of apoptotic and necrotic
cells[26]. In this study, in the presence of NRP (80 µg/mL), the number of
apoptotic cells was 30.1% at 24 h; the necrotic cells were still
below 10.8%. As the concentration of NRP increased (120
µg/mL), the apoptotic ratio began to decrease with an
enhanced necrotic ratio (Figure 3B). Therefore, NRP induced
U937 cell death by affecting the balance between apoptosis
and necrosis.
However, the activation of caspase-3, another hallmark
of apoptosis, was further demonstrated. As the results
showed, procaspase-3 was gradually decreased, whereas
activated caspase-3 was increased, indicating that NRP
time-dependently induced U937 apoptosis (Figure 3C).
Inactivation of PI3-K contributes to NRP-induced U937
cell death Previous studies showed that the activation of
the PI3-K pathway plays an important role in tumor cell
proliferation and invasion[18]. Therefore, the U937 cells were
pretreated with 200 nmol/L PI3-K inhibitor wortmannin for 1 h,
followed by 75 µg/mL NRP treatment. The inhibitory ratio at
24 h was significantly increased compared with the NRP
alone-treated group (Figure 4), while Ras inhibitor manumycin
A, p38 inhibitor SB203580, and ERK inhibitor PD98059 failed to
enhance NRP-induced growth-inhibitory effects in U937 cells.
These results indicated that the inhibition of PI3-K
contributed to NRP-induced cell death.
To further confirm the above results, a flow cytometric
analysis was performed (Figure 5). It was found that in the
presence of 75 µg/mL NRP, the apoptotic ratio (the
percentage of cells in
sub-G0/G1 phase, a hallmark of apoptosis)
reached 26.6% (Figure 5D) from the baseline of the control
(2.62%; Figure 5A), whereas pretreatment with wortmannin
significantly increased the apoptotic ratio (35.9%; Figure 5E)
compared with the group treated by NRP alone.
Effect of Akt on NRP-induced U937 apoptosis
Activated PI3-K converts phosphatidylinositol 4,5 phosphate into
phosphatidylinositol 3,4,5 phosphate, which results in the
recruitment of Akt and the phophorylation of PI3-K at
residues T308 and S473[19]. Here, the U937 cells were cultured
with NRP for 24 h after being pretreated with 10 µmol/L Akt
inhibitor KP372-1 for 60 min, and the ratio of cell death was
detected (Figure 4). Consistent with the result of the
pre-incubation of wortmannin, KP372-1 promoted NRP-induced
U937 cell death, and the apoptotic ratio increased from
26.6% to 33.6% (Figure 5F).
Then the Western blot analysis was performed to detect
the protein expressions of Akt and p-Akt. As shown in
Figure 6, Akt and Akt phosphorylation were reduced by NRP
treatment and this suppressive effect became more
significant with the extension of the incubation time with NRP.
Inactivation of SIRT1 contributes to p53 activation in
NRP-induced U937 cell apoptosis SIRT1 has been shown to
be associated with apoptosis, and the expression of the
inactive SIRT1 protein can potentiate p53-mediated
apoptosis[27,28]. In order to demonstrate whether such a mechanism is
involved in NRP-induced cell apoptosis, SIRT1 inhibitor sirtinol
was pretreated for 1 h before NRP was introduced. After 24
h, the growth inhibitory ratio was detected by MTT. It was
found that the inactivation of SIRT1 facilitates NRP-induced
U937 cell death (Figure 7A). In order to further confirm this
mechanism, a Western blot analysis was performed to detect
the expressions of p53 and p-p53. As shown in Figure 7B,
the expression of p53 and its phosphorylation were
upregulated by NRP treatment, and the inhibition of SIRT1
activity with sirtinol contributed to the activation of p53.
Decreased expression of SIRT1 and p53 activation were
dependent on PI3-K/Akt inactivation The cells were
pretreated with 200 nmol/L wortmannin for 1 h, followed by the
addition of 75 µg/mL NRP. After 24 h, the cell lysates were
separated by 10% SDS-PAGE, and SIRT1, p53, and the
phosphorylated p53 protein bands were detected by Western blot
analysis. The results showed that wortmannin pretreatment
executed a more significant inhibitory effect on the
expression of SIRT1, as well as more marked augmented effect on
p53 activation, compared with the NRP-alone treatment
group (Figure 8).
Discussion
In this study, we demonstrated that NRP inhibits U937
cell growth and induces apoptosis. These biochemical events
might be associated with the inhibition of the PI3-K/Akt
signaling pathway, the inactivation of SIRT1, and the
activation of the p53 tumor suppressor gene. Importantly, the
inhibition of PI3-K activity significantly augmented NRP-induced
U937 cell apoptosis, leading to the downregulated
expression of the SIRT1 protein and the activation of p53.
It is well known that the majority of death signals pass
through 2 distinct pathways: apoptosis and
necrosis[29]. Some signaling pathways, such as death receptors,
mitogen-activate protein kinase, and caspase cascades, participate in
both processes, and it is possible to switch apoptosis to
necrosis by regulating the signaling
pathways[30]. In this study, we demonstrated that NRP inhibited the proliferation
of U937 cell in vitro in a dose- and time-dependent manner.
There were significant growth inhibitory effects at 20_100
µg/mL NRP. Based on the changes of cellular morphology,
the LDH activity-based assay, and the activation of caspase-3,
we concluded that NRP induced U937 cell death by
mediating the balance between apoptosis and necrosis.
The enzyme PI3-K is composed of a 110 kDa catalytic
subunit and an 85 kDa adaptor subunit. When growth factors,
such as insulin, insulin-like growth factor-1, fibroblast growth
factor-2, and others bind to their cognate cell-membrane
receptor, the p85 subunit is recruited to the C-terminal
(intracellular) part of the growth factor receptor. Subsequent
dimerization with the P110 unit then leads to the full
enzymatic activity of PI3K[31]. Accumulating evidence suggests
that PI3-K is an important regulator of cell survival.
Downstream of PI3-K lies PKB/Akt and PLCγ, which stimulate the
process of cell proliferation[32]. Our results show that PI3-K
inhibitor wortmannin, and Akt inhibitor KP372-1, significantly
augmented NRP-induced apoptosis, while other signaling
molecule inhibitors, such as the Ras inhibitor manumycin A,
ERK inhibitor PD98059, and P38 inhibitor SB203580 did not
show these apoptosis-augmented effects. NRP
downregulated the expressions of Akt and p-Akt, indicating that the
PI3-K/Akt signaling cascade might be partially involved in the
apoptosis-inducing effects of NRP in U937 cells.
The p53 protein is a regulator of cell cycle progression
and a mediator of apoptosis in many cell lines. A large body
of data documented that the p53 gene is often mutated in
many tumor cells and the mutations contribute to clonal
cellular expansion and genomic instability because of decreased
DNA repair and apoptosis[33,34]. MDM2 (transformed 3T3 cell
double minute 2) is an E3 ligase, which can ubiquitylate the
lysine residues at the C terminus of
p53[35_37]. It inhibits p53 using at least 2 mechanisms, namely, by ubiquitination of
p53 on the lysine residues at its C terminus, leading to
proteosomal degradation, and by blocking a transactivation
domain of p53[38,39]. Here in our experiments, both the total
p53 and p-p53 protein levels increased. It is possible that
there may be a portion of total p53 becoming acetylated
under the condition of NRP treatment, leading to the upregulation
of p-p53 protein expression. At the same time, a part of
ubiquitylated p53 was downregulated. Therefore, the result
of the balance between acetylation and ubiquitylation in this
system showed that total p53 increased.
Moreover, MDM2 also serves as a good substrate for
Akt. A recent study showed that the PI3-K/Akt signaling
pathway inhibited p53-mediated transcription and apoptosis
through inducing the phosphorylation of MDM2 and
enhancing the degradation of p53[40]. In the present study, Akt
and p-Akt were downregulated, p53 and p-p53 were
correspondingly upregulated. The inhibition of PI3-K activity
contributed to the upregulated expressions of the p53 and p-p53
proteins and increased the ratio of cell death. These
phenomena suggest that a tight association between the
PI3-K/Akt signaling pathway and p53 in NRP-induced U937 cell
apoptosis exists. Covering this pathway will be essential in
understanding how p53 acetylation is regulated, and how
Akt regulates its function is a very important subject of our
study, so our further study will concentrate on this aspect.
SIRT1 is a key regulator involved in cell aging and
response to DNA damage and is a member of the sirtuin family
of NAD+-dependent deacetylase known to inhibit
stress-induced apoptosis[26,41]. It also regulates p53 function via
deacetylation with a specificity for the p53 C-terminal Lys382
residue, a modification of which has been implicated in the
activation of p53 as a transcription
factor[27]. Our present results show that 75 µg/mL NRP abrogates SIRT1
expression while facilitating p53 activation. Moreover, the
inhibition of PI3-K activity further attenuates SIRT1 expression by
the administration of wortmannin. These results indicate that
the inactivation of SIRT1 might be in part associated with
PI3-K inactivation-induced cell death in NRP-treated U937
cells.
Noticeably, the inhibition of SIRT1 activity by using
SIRT1 inhibitor sirtinol augmented p53 activation. These
results indicate that the activation of p53 was due to the
downregulated deacetyl-function of SIRT1 induced by the
inhibition of the PI3-K/Akt signaling pathway in NRP-treated
U937 cells. However, further study will be required to
elucidate the relationship of SIRT1 and p53 in the apoptotic
action of NRP in U937 cells.
NRP induced U937 cell apoptosis and inhibited cell
growth in a time- and dose-dependent manner. The
mechanism is in part due to the inhibition of the PI3-K/Akt
signaling pathway. The potential effects of wortmannin on
NRP-induced cell death reflect that the inhibition of the PI3-K
survival pathway is necessary for the SIRT1 inactivation
and p53 activation in NRP-induced U937 cell apoptosis.
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