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Introduction
Cancers of the gastrointestinal tract account for 22% of
all cancers and represent a major health threat
worldwide[1]. Gastric cancer is the fourth most frequently diagnosed
malignancy worldwide, accounting for 12% of all cancer-related
deaths[2,3]. In Asia and parts of South America in particular,
gastric cancer is the most common epithelial malignancy and
a leading cause of cancer-related death.
The phosphatidylinositol 3-kinase (PI3K) pathway plays
a central role in the regulation of cell proliferation, growth,
differentiation, and survival[4,5]. Dysregulation of this
pathway is frequently observed in a variety of tumors, including
brain tumors and breast, ovarian and other
carcinomas[6_8]. Therefore, inhibition of PI3K signaling is under
investigation as a potentially useful approach for cancer treatment.
The PI3K family of enzymes is well characterized with
respect to the promotion of cellular growth, survival, and
suppression of apoptosis in cancer
cells[9_11]. These kinases can be activated by cell surface growth factor receptors (such
as epidermal growth factor receptor) and are known to play a
critical role in regulating the balance between cell survival
and apoptosis. PI3K enzymes are cytosolic and consist of
both regulatory and catalytic subunits, which regulate a vast
array of fundamental cellular
responses[12]. Members of the PI3K family can be divided into three classes: class I, II and
III. Class I PI3K members are heterodimers, each consisting
of a p110 catalytic subunit and a smaller regulatory subunit
with Src-homology 2 (SH2) domains. Mammals have three
catalytic subunits (p110α, p110β, p110γ) and five regulatory
subunits (p85α, p85β, p55γ, p55α,
p50α)[13,14]. One of the functions of class I PI3K is to inhibit apoptosis. Much
evidence has implicated PI3K in cancer. Mutations in the
3-phosphoinositide phosphatase PTEN[15] as well as
over-expression of the p110 catalytic subunits of the p85/p110 (class
IA) PI3K[16] are frequently found in human tumors. The
overexpressed p110 subunits are presumably active by
virtue of the excess free p85 that exists in many cell
types[17].
The AKT protein kinase transduces signals from growth
factors and oncogenes to downstream targets that control
crucial elements in tumor development. The AKT pathway
is one of the most frequently hyperactivated signaling
pathways in human cancers[18]. AKT is activated by PI3K, which
transmits signals from cytokines, growth factors, and
oncoproteins (eg RAS) to multiple targets, including AKT.
Activation of PI3K localizes AKT to the plasma membrane
via the pleckstrin homology domain of AKT, where AKT is
activated by phosphorylation at
Ser473[19]. p53 acts as a tumor suppressor primarily by inducing either cell cycle arrest
or apoptosis in response to cellular stress, leading to
oncogenic alteration[20,21]. These cellular responses are mediated
largely through the function of p53 as a transcriptional
activator or repressor, targeting a diverse range of genes.
LY294002 is a specific inhibitor of class I
PI3K[22]. In the present study we examined whether inhibition of class I PI3K
by LY294002 has cytotoxic effects in SGC7901 gastric cancer
cells, and how this affects the p53-mediated apoptotic
signaling pathway.
Materials and methods
Reagents RPMI-1640 medium was purchased from Gibco
(Rockville, MD, USA). Fetal bovine serum was purchased
from Hangzhou Sijiqing Biological Engineering Material Co
(Hangzhou, China), L-glutamine and 3-(4,5-dimethyl-2
thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were
purchased from Sigma (St Louis, MO, USA). Antibodies
against p53 (1:500, 1C12), p85 (1:500, 19H8), p-AKT (1:500,
Ser473) and PUMA (1:500, P-13) were purchased from Cell
Signaling Technology (Beverly, MA, USA). Primers, the
RNAiso reagent kit, the Primescript RT reagent kit and the
SYBR Premix EX Taq kit were purchased from TaKaRa (Dalian,
China).
Drug preparation LY294002 and Pft-α (Cell Signaling
Technology) were diluted in DMSO to create a stock
solution that was stored according to the manufacturer's
instructions. Adriamycin (ADM) was diluted in RPMI-1640
to create a stock solution that was stored according to the
manufacturer's instructions. The final concentration of the
LY294002 solution used was 50 µmol/L, ADM was 0.04
µg/mL and that of the Pft-α solution was 30 µmol/L. This
concentration of LY294002 was selected on the basis of
our experiments on SGC7901 cells, the concentration of
ADM and the concentration of Pft-α was selected on the
basis of the manufacturer's recommendations.
Cell culture SGC7901 cells and SMMC7721 cells were
maintained in RPMI-1640 medium (Gibco) containing 10%
heat-inactivated fetal bovine serum (Hangzhou Sijiqing
Biological Engineering Material Co), 0.03% L-glutamine
(Sigma) and incubated in a 5% CO2 atmosphere at 37 °C.
Cells in the mid-log phase were used in the experiments.
Cell viability assay Cell viability was assessed using an
MTT assay. To determine the time-course of the effects of
LY294002, SGC7901 cells and SMMC7721 cells were plated
onto 96-well microplates (7×104 cells/well). LY294002 (12. 5,
25, 50 µmol/L) was added to the culture medium and cell
viability was assessed using an MTT assay 24, 48 and 72 h
after drug treatment. To determine the synergistic effect of
LY294002 and ADM on growth in combination with ADM,
SGC7901 cells were plated onto 96-well microplates
(7×104 cells/well). LY294002 (50 µmol/L) and ADM (0.04 µg/mL)
were added to the culture medium and cell viability was
assessed using an MTT assay 24, 48 and 72 h after drug
treatment. To determine if p53 plays a critical role in
LY294002-induced cytotoxicity, SGC7901 cells were pretreated with the
p53-specific inhibitor Pft-α for 6 h before the addition of
LY294002 and cell viability was assessed using an MTT
assay 24, 48 and 72 h after drug treatment. The MTT solution
was added to the culture medium (500 µg/mL final
concentration) 4 h before the end of treatment and the
reaction was stopped by addition of 100 µL 10% acidic SDS. The
absorbance value (A) at 570 nm was read using an
automatic multi-well spectrophotometer (Bio-Rad, Richmond,
CA, USA). The percentage cell death was calculated as
follows: cell death (%) = (1_A of experimental
well/A of positive control well )×100%.
Detection of cell cycle To analyze the effect of LY294002
on cell cycle progression, we incubated SGC7901 cells with
LY294002 (50 µmol/L) for 12 or 24 h. The cells were
harvested using 0.25% trypsin, washed with
phosphate-buffered saline (PBS), counted and adjusted to
1×106 cells/mL. The cells were fixed in 70% ethanol, treated with 100 mg/L
RNase at 37 °C for 30 min and stained with 50 mg/L propidium
iodide (Sigma) for 30 min. The cells were analyzed using
flow cytometry (Epics XL; Beckman Coulter, Fullerton CA,
USA). Percentage apoptotic cells was taken as the
percentage of cells with a DNA content lower than that of cells
in G0_G1 in the propidium iodide intensity-area histogram
plot[23]. These hypodiploid cells were considered to
represent apoptotic cells, and the rate of apoptosis was analyzed
using Multicycle software (Beckman Coulter).
Detection of mitochondrial potential (Δψ)
Mitochondrial Δψ was determined using the KeyGEN Mitochondrial
Membrane Sensor Kit (KeyGEN, Nanjing, China). The
Mitosensor dye aggregates in the mitochondria of healthy
cells and emits red fluorescence against a green monomeric
cytoplasmic background staining. However, in cells with a
collapsed mitochondrial Δψ, the dye cannot accumulate in
the mitochondria and remains in monomeric form
throughout the cells with green
fluorescence[24]. Briefly, SGC7901 cells were incubated with LY294002 in
24-well plates for the indicated times and then pelleted, washed with PBS, and
resuspended in 0.5 mL of diluted Mitosensor reagent
(1 µmol/mL in incubation buffer). After the cells were
incubated with the Mitosensor reagent for 20 min, 0.2 mL of
incubation buffer was added and cells were centrifuged then
resuspended in 40 µL of incubation buffer. Finally, the cells
were washed and resuspended in 1 mL PBS for flow cytometry
analysis.
Real-time quantitative RT-PCR analysis of p53 and
PUMA Total RNA was extracted using the RNAiso kit
(TaKaRa). For extracting total mRNA, SGC7901 cells were
treated with LY294002 (50 µmol/L) for 6 h before being
harvested. To detect whether the inhibitor of p53 inhibits
the expression of p53 and PUMA, SGC7901 cells were
pretreated with Pft-α 6 h before incubation with LY294002.
First-strand cDNA was generated via reverse transcription of 2 µg
of total RNA using random primers and the Primescript RT
Reagent Kit (TaKaRa) in a total reaction volume of 20 µL
according to the manufacturer's instructions. The sequences
of the forward and reverse oligonucleotide primers, specific
to the chosen candidate and housekeeping genes, were
designed using Primer5 software (available from frodo. wi. mit.
edu/cgi-bin/primer5/primer5_www. cgi). For p53 the
primers were: forward, 5'-ACTAAGCGAGCACTGCCCAAC-3';
reverse, 5'-CCTCATTCAGCTCTCGGAACATC-3' (GenBank NM_000546; nucleotides 1161_1290. For PUMA the primers
were: forward, 5'- CGACCTCAACGCACAGTACGA-3'; reverse, 5'-GGCACCTAATTGGGCTCCATC-3' (GenBank
NM_014417; nucleotides 719_868). For β-actin the primers
were: forward, 5'-ATTGCCGACAGGATGCAGA-3'; reverse,
5'-GAGTACTTGCGCTCAGGAGGA-3' (GenBank NM_001101; nucleotides 998_1086). Real-time quantitative
RT-PCR was performed using the iCycler 5 thermal cycler
(BioRad, Hercules, CA , USA). An 80-fold dilution of each
cDNA was amplified in a 20 µL volume, using the SYBR Premix
EX Taq kit (TaKaRa), with a 500 nmol/L final
concentration of each primer. The amplification specificity was
checked using melting curve analysis. Threshold cycle Ct,
which correlates inversely with the target mRNA level, was
calculated using the second derivative maximum algorithm
provided by the Light-Cycler software. For each cDNA,
all target gene mRNA levels were normalized to β-actin
mRNA levels. Results are expressed as the ratio of
normalized target gene mRNA levels in treated cells relative
to those in untreated cells.
Western blotting analysis Cells were harvested
and rinsed twice with ice-cold PBS. Five volumes of Western
blot lysing buffer [containing 10 mmol/L Tris-HCl (pH
7.4), 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate,
0.1% sodium dodecyl sulfate, 5 mmol/L EDTA, 1 mmol/L
phenylme-thylsulphonyl fluoride (PMSF), 0.28 U/mL aprotinin,
50 µg/mL leupeptin, 1 mmol/L benzamidine, and 7 µg/mL
pepstatin A] was added to each volume of cell pellets and
the mixture was sonicated on ice (1 s/mL per sonication, with
30 s intervals, 5 times). The mixture was microcentrifuged at
10 600×g at 4 °C for 10 min and the supernatant was
preserved at _70 °C for later use. The protein concentration
was determined using the BCA kit (Pierce, Rockford, IL, USA).
Proteins were separated on a 12% SDS-PAGE gel, transferred
to a nitrocellulose membrane and immunoblotted with a
1:500 dilution of primary antibody (Cell Signaling Technology) at
4 °C overnight. Immunoreactivity was detected using
horseradish peroxidase (HRP)-conjugated anti- rabbit antibody
(Sigma) at a dilution of 1:5000 in blocking solution for 1 h at
room temperature. Immunoreactivity was detected using
enhanced chemiluminescence (ECL kit; Amersham Pharmacia
Biotech, Rockford, IL, USA) and visualized by
autoradiography. β-actin protein (1:5000; Sigma) was used as a loading control.
Statistical analysis All data are presented as mean±SD.
Statistical analysis was carried out using ANOVA followed
by Dunnett's t-test, with P<0.05 taken to indicate significance.
Results
LY294002 reduced cell viability LY294002 reduced
SGC7901 viability in a dose- and time-dependent fashion.
MTT assays revealed that after 24 h of treatment, the rate of
inhibition reached 20.71% ± 4.13% at the highest dose used,
50 µmol/L. The rate of inhibition rose when the incubation
time was prolonged, reaching 41.54%± 2.06% at 48 h and
64.09%±1.5% at 72 h after treatment (Figure 1A). In order
to assess the clinical value of the PI3K inhibitor in tumor
treatment, and to test the synergistic inhibitory effect of
the PI3K inhibitor on growth in combination with a
chemotherapy drug, we used the chemotherapy drug ADM
(0.04 µg/mL). We found that LY294002 has a more marked
effect when used in combination with ADM than when used
alone (Figure 1B). As shown in Figure 1C, LY294002 also
inhibited the proliferation of SMMC7721 liver cancer
cells: MTT assays revealed that after 24, 48 and 72 h of
treatment, the rates of inhibition were 33.4%±2.23%,
39.82%±3.14% and 55.7%±2.41% at the highest dose used,
50 µmol/L. Thus, LY294002 inhibited the proliferation of
SGC7901 gastric cancer and SMMC7721 liver cancer cells.
LY294002 arrested the cell cycle and induced apoptosis
of SGC7901 cells The effect of LY294002 on the cell cycle
progression of SGC7901 cells was studied after 12 and 24 h
of LY294002 (50 µmol/L) exposure. Flow cytometry analysis
indicated that LY294002 halted the cell cycle of SGC7901
cells at the G1 and G2 phases after both 12 and 24 h treatment.
The proportion of cells in the G1 phase increased with the
duration of LY294002 (50 µmol/L) exposure (0, 6, 12, or 24 h),
being 48.9%, 58.1%, 68.9% and 75.1%. The proportion of
cells in the G2 phase also increased with the duration of
exposure, being 7.08%, 8.51%, 13.1% and 31.6%, respectively.
In contrast, the proportions of cells in the S phase decreased
with exposure, being 34.8%, 28.2%, 17.3% and 0.63%,
respectively; but the apoptosis rate increased with exposure,
being 0.33%, 5.58%, 29.5% and 43. 4%, respectively (Figure
2 and Table 1). There was a significant difference between the
50 µmol/L LY294002 group and the control group at every
time point. The results indicate that LY294002 induces cell
cycle arrest and apoptosis of SGC7901 cells.
LY294002 induced mitochondrial dysfunction
In the present study, mitochondrial membrane potential was
examined using the fluorescent dye JC-1. We detected a
collapse in mitochondrial membrane potential (Δψ) as early
as 6 h after LY294002 treatment, as indicated by increased
emission of green fluorescence. This change reached a
maximum 24 h after LY294002 treatment (Figure 3). A
collapse in mitochondrial membrane potential always
indicates cell apoptosis or necrosis, LY294002 induced
mitochondrial dysfunction and activated cell apoptosis in
SGC7901 cells, and the results described here prove that
LY294002 induced apoptosis in SGC7901 cells.
LY294002 decreased expression of p85 and
p-AKT To distinguish the specific class I PI3K/AKT-mediated cell
proliferation blockage from nonspecific LY294002 toxicity, we
measured the phosphorylation of one of the downstream
targets of PI3K, p-AKT(Ser473), and the expression of p85
after treatment with LY294002 (50 µmol/L) for durations
ranging from 6 to 24 h. As shown in Figure 4A expression of p85
in the SGC7901 cell line was inhibited by LY294002 treatment.
As shown in Figure 4B, phosphorylation of AKT at
Ser473 in the SGC7901 cell line was inhibited in a time-dependent
manner. These results suggest that LY294002 may induce
tumor cell apoptosis by regulating p-AKT and p85. They
also suggest that AKT and p85 have a role in regulating
apoptosis and proliferation.
LY294002 upregulated p53 levels To determine
whether LY294002-induced apoptosis involves the p53
signaling pathway, real-time quantitative RT-PCR analysis was
used to measure p53 mRNA expression. The basal levels of
p53 mRNA in SGC7901 cells were relatively low. Six hours
after incubation with LY294002, p53 mRNA expression was
significantly increased (Figure 5A). The level of p53 mRNA
reached a peak at 6 h, but gradually decreased thereafter,
although remaining higher than the level of the control. To
determine whether LY294002 also increases the expression
of p53 protein, Western blot analysis was used. The level of
p53 protein increased between 6 and 24 h after LY294002
treatment (50 µmol/L). These results showed that p53
expression was induced by LY294002 treatment (Figure 5B).
P53 mRNA and protein began to increase at the same time
after treatment, but expression of p53 mRNA peaked at 6 h
and expression of p53 protein peaked at 24 h after treatment.
LY294002 upregulated PUMA levels To determine
whether LY294002 also increases the expression of the p53
target gene PUMA, real-time quantitative RT-PCR analysis
was used to measure PUMA mRNA expression and Western
blot analysis was used to measure PUMA protein expression.
The level of PUMA mRNA peaked at 6 h after treatment, but
gradually decreased thereafter, although remaining higher
than the level of the control. These results showed that
PUMA expression was induced by LY294002 treatment (Figure 6A). To determine whether LY294002 also
increases the expression of PUMA protein, Western blot analysis was
used. The PUMA protein level increased between 6 and 24 h
after LY294002 treatment (50 µmol/L). These results show
that PUMA protein expression was induced by LY294002
treatment (Figure 6B). PUMA mRNA and protein began to
increase at the same time after treatment, but expression of
PUMA mRNA peaked at 6 h and expression of PUMA protein peaked at 24 h after treatment.
Pft-α inhibited LY294002-induced apoptosis
Pft-α was originally thought to be a specific inhibitor of signaling by
the tumor suppressor protein p53[25]. To confirm the
contribution of p53 to the regulation of proliferation and apoptosis
involved in LY294002-induced SGC7901 cell death, cells were
pretreated with the p53-specific inhibitor Pft-α for
6 h before the addition of LY294002. As shown in Figure 7A,
Pft-α significantly attenuated the inhibitory effects of
LY294002 in a time-dependent manner.
Pft-α inhibited LY294002-induced expression of p53 and
PUMA mRNA and protein SGC7901 cells were pretreated
with Pft-α 6 h before incubation with LY294002. For
extracting total mRNA, SGC7901 cells were treated with LY294002
(50 µmol/L) for 6 h before being harvested. For extracting
total protein, SGC7901 cells were treated with LY294002 (50
µmol/L) for 24 h before being harvested. Expression of p53
mRNA and protein was inhibited and expression of PUMA
mRNA and protein was blunted by Pft-α treatment (Figure
7B, 7C, 7D, 7E).
Discussion
In the present study, we showed that the class I PI3K
inhibitor LY294002 reduced the viability of and induced
apoptosis in SGC7901 gastric cancer cells, thus
demonstrating the cytotoxic effects of LY294002. We also showed that
LY294002 increased the expression of p53 and PUMA, while
decreasing the expression of p-AKT and p85.
LY294002-induced apoptosis was blocked by the p53 inhibitor
Pft-α. These results show that LY294002 may induce cytotoxicity
in SGC7901 cells via activation of p53-mediated apoptotic
signaling. These findings suggest that inhibition of the class
I PI3K signaling pathway is a potential strategy for
managing gastric cancers.
The lipid kinase PI3K is a proto-oncogene that
generates 3'-phosphoinositides at the cell membrane. The
best-characterized inhibitors of PI3K are LY294002 and
wortmannin, which are commercially used compounds that
target the p85 regulatory subunit of PI3K. LY294002
effectively inhibits the growth of many types of tumor cells
in vitro and in vivo, which occurs via the inhibition of PI3K
and downstream components of the
pathway[26_38].
In the dose range used in the present study, LY294002
inhibited the expression of p85, a regulatory subunit of PI3K.
LY294002 also inhibited the phosphorylation of p-AKT (ie
activation of AKT). Thus LY294002 probably inhibits
proliferation and induces apoptosis in SGC7901 cells by
inhibiting class I PI3K. However, the downstream molecules
involved in the apoptotic death of tumor cells following
inhibition of PI3K/AKT by LY294002 remain to be identified.
The mitochondria play critical roles in integrating cell
death signals. Apoptosis is a cellular process involving the
selective degradation of membranous organelles such as the
mitochondria. The mitochondrial permeability transition
(MPT) represents an important event in initiating apoptosis.
Thus, it is not surprising that apoptosis and even necrosis
share a common mechanism through induction of the MPT.
Observations made in the present study suggest that the
mitochondrial Δψ collapsed after treatment of LY294002, thus
mitochondria may have initiated an apoptotic pathway.
The tumor suppressor p53 plays a central role in sensing
various genotoxic stresses. The basal levels of p53 were low
in SGC7901 gastric cancer cells, and LY294002 upregulated
the expression of p53. Upregulation of p53 after treatment
with LY294002 induced apoptotic cell death. Moll and Zaika
have proposed that the induction of apoptotic cell death by
p53 occurs via both target gene activation and
transactivation-independent mechanisms at
mitochondria[29]. In response to various forms of cellular stress, the levels of p53 increase and
a proportion of p53 rapidly localizes to the
mitochondria[30]. In the present study, the mitochondrial
Δψ collapse after LY294002 treatment may have been caused by upregulation
of p53. p53 accumulates in the nucleus, where it
trans-activates a number of proapoptotic target
genes[31], and induces apoptotic cell death.
p53 protein can engage apoptosis by inducing
expression of PUMA, which leads to release of cytochrome c from
the mitochondria and apoptosis of the cell. We found that
expression of p53 and its target gene PUMA was induced
after LY294002 treatment. Baseline PUMA expression was
very low, and LY294002 markedly increased the expression
of PUMA. PUMA is a downstream target of the p53 tumor
suppressor gene and a member of the BH3-only group of
Bcl-2 family proteins[32,33]. Activation of PUMA by DNA
damage is dependent on p53 and is mediated by the direct
binding of p53 to the PUMA promoter
region[33]. PUMA plays an essential role in the p53-dependent and
-independent apoptosis induced by a variety of
stimuli[34]. Here, we demonstrated that the class I PI3K inhibitor LY294002
activates p53 and upregulates PUMA, suggesting that the
LY29402-induced apoptosis of SGC7901 cells may be
mediated by the p53 apoptotic signaling pathway.
Although induction of p53 by LY294002 has been
previously reported, the role of p53 induction in
LY294002-induced tumor inhibition remains unclear. In the present study,
we evaluated the contribution of p53 to the
LY294002-induced apoptotic death of SGC7901 cells using a specific p53
inhibitor, Pft-α. We found that the LY294002-induced
induction of p53 and PUMA, as well as apoptosis, were partially
blocked by Pft-α. This is the first time that p53 and PUMA
induction have been shown to contribute to the antitumor
effects of LY294002.
All these observations suggest that p53 and apoptosis
activation may contribute significantly to the
LY294002-induced death of SGC7901 cells. Further investigation of the
downstream signals of PI3K/AKT involved in regulating cell
proliferation and apoptosis may reveal new strategies for
targeting therapies at the PI3K/AKT signaling pathway.
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