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Introduction
Extracellular adenosine triphosphate (ATP) and adenosine (ADO) are important signaling molecules in both the
intracellular and extracellular microenvironments of cells. ATP exerts transient physiologic and pharmacologic effects on the
organism because it can be rapidly metabolized to ADO
by ectotriphosphatase, ectodiphosphatase, and 5¡¯-nucleotidase, and
maintained its low physiologic concentrations. However,
extracellular ATP may reach high concentrations when
released exocytotically from various cell types such as neurons, platelets, basophils, and mast
cells, or when released nonexocytotically from damaged
cells[1]. It is well known that extracellular ATP and ADO have widespread effects on physiologic activities and
play important physiologic and pathophysiologic roles in the cardiovascular system. Recently,
they have been implicated in the induction of apoptosis in several cancer cell types such as human histiocytic leukemia cell line
U-937[2], the mouse neuroblastoma cell line
N1E-115[3_4], pancreatic cancer
cells[5], colorectal carcinoma
cells[6], prostate carcinoma
cells[7], rat pheochromocytoma
cells[8_9], and so on.
The process of programmed cell death has been suggested to play an important role in cancer diseases. Cell death by
apoptosis progresses through a series of well-regulated morphological and biochemical phases, including chromatin
condensation and DNA fragmentation. Recent studies have shown that ATP and ADO induces apoptosis in various cell types
through receptor-mediated and non-receptor-mediated pathway, namely, the extrinsic pathway and the intrinsic
pathway[10_11]. For the extrinsic pathway,
A1, A2A, A2B and
A3 ADO receptors[12-16], and
P2X1, P2X2, P2X7,
P2Y1, P2Y2
receptors[1,4,6,17-20] appear to bear apoptosis
in normal cells and carcinoma cells. For the intrinsic pathway, extracellular ATP and ADO seem to
induce apoptosis in epithelial cancer cells originated from the breast, the colon, and the ovary or neuroblastoma cells by the
conversion of ATP to ADO and intracellular uptake of
ADO[3,21].
We have investigated the antiproliferation effects of
extracellular ATP and ADO on human gastric cell line,
HGC-27[22]. In an attempt to gain more insight into the mechanism of ATP- and ADO-induced apoptosis in tumor tissue, we report that ATP
and ADO can induce apoptosis in HGC-27 cells, in which cellular uptake of ADO plays an important role. In present study we
observed the growth inhibitory and apoptotic effects of ATP and its final metabolite, ADO, on the human undifferentiated
gastric cancer (HGC)-27 cells.
Materials and methods
Drugs and reagents ATP, ADO, ADO diphosphate (ADP), adenosine monophosphate (AMP), acridine orange (AO),
ethidium bromide (EB), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), aminophylline and dipyridamole
were purchased from Sigma. RNase, SDS, proteinase K, trypsin and agarose were purchased from Sino-American Biotec.
RPMI 1640 medium was purchased from GIBCO. Fetal
bovine serum (FBS) was purchased from
Hangzhou Sijiqing Biotec. Pyridoxal-phosphate-6-azophenyl-2, 4-disulphonic acid tetrasodium salt (PPADS) was purchased from Tocris Cookson.
ATP, ADP, AMP and ADO were dissolved in sterile phosphate-buffered saline (PBS) and stored at -20 ºC.
Cell culture HGC-27 cells, obtained
from Shanghai Cell Database of Chinese Academy of Sciences, were cultured in
RPMI 1640 medium supplemented with 10% (v/v) FBS, 100
U·mL-1 of penicillin, and 100
µg·mL-1 streptomycin at 37 ºC
in a humidified, CO2-controlled (5%) incubator.
MTT assays The cell viability was determined by MTT
assay[23]. The HGC-27 cells in exponential phase of growth were
harvested and seeded in 96-well plates (Costar, USA) at a density of 10 000 cells per well, and cultured for 24 h. ATP, ADP,
AMP or ADO (0.3 and 1 mmol·L-1) and control (PBS) was then added into the wells, and incubated continuously for 48 h at 37
ºC with 5% CO2. A 20 µL sample of MTT solution (5
g·L-1 dissolved in PBS) was added to each well and the plates were
incubated at 37 ºC for 4 h. The supernatant was discarded and 150 µL dimethylsulfoxide was added to dissolve the blue
insoluble MTT formazan produced by mitochondrial succinate dehydrogenase. The absorbance was measured at 490 nm in
a spectrophotometer (Zhengzhou Bosai Biotech, ht2010), the negative control well contained medium only. Other inhibitors
such as aminophylline, PPADS and dipyridamole were added to the medium 30 min before incubating cells with ATP or ADO.
All determinations were performed in quadruplicate and each experiment was repeated at least three times.
Morphological assessment of apoptotic cells induced by ATP or ADO
Morphological assessments of apoptotic cells were
carried out using the AO/EB double-staining
method[24]. HGC-27 cells in exponential phase of growth were harvested and
seeded in a 25 mL cultured flask. The cells were
incubated for 24 h at 37 ºC with 5%
CO2, and then treated with 0.3
mmol·L-1 ATP or ADO for 48 h. Freshly isolated HGC-27 cells
(1×106) were harvested in an Eppendorf centrifuge tube, centrifuged for
5 min at 1000 rpm and suspended in PBS containing fluorescence dye AO/EB (AO and EB were both at the concentration of
100 mg·L-1 in PBS). The cells were prepared and dropped on slides. The morphology of the cells was observed under
fluorescence light microscope (UFX-¢ò; Nikon, Japan) and photographed.
Agarose gel electrophoresis of
DNA[25] After treatment with ATP or ADO (0.03, 0.1, 0.3, and 1
mmol·L-1) for 72 h, HGC-27 cells
(1×106) were harvested in an Eppendorf centrifuge tube and washed twice with PBS. The cells were resuspended in a cell
lysis buffer (50 mmol/L Tris-HCl buffer, 20 mmol/L EDTA pH 8.0, 1% SDS) and then mixed by vortexing.
After the cells were left to stand for 30 min on ice, proteinase K was added at a final concentration of 0.25
g·L-1. The cell lysates were incubated
at 37 ºC overnight in a water bath, and RNase was added at a final concentration of 0.5
g·L-1 and incubated at 37 ºC for 1 h. The
lysates were mixed with an equal volume of Tris-saturated phenol (1:1,
v/v) and mildly shaken for 30 min. The mixture was
centrifuged at 3000 rpm for 10 min at room temperature to separate the aqueous phase from the organic phase. Extraction of
each aqueous phase was repeated, using the Tris-saturated phenol/chloroform/isopropanol (25:24:1,
v/v) mixture. The aqueous phase was further extracted with an equal volume of chloroform. Two volumes of ice-cold ethanol and 0.1 volume
of 3 mol·L-1 NaAc precipitated DNA were mixed in the final aqueous phase. At this point, the mixture could be stored
overnight. DNA was recovered by centrifugation at 13 000 rpm for 20 min in an Eppendorf centrifuge tube. The supernatant
was discarded, the DNA pellet was washed once with 70% ethanol, air-dried, and then redissolved in an appropriate volume
of deionized distilled-water and electrophoresed for 3 h at a constant voltage of 60 mV on a 1.8% agarose gel containing 0.5
mg·L-1 EB, using an electrophoresis buffer (40
mmol·L-1 Tris/acetate buffer, 1
mmol·L-1 EDTA, pH 8.0). Each DNA sample
contained bromophenol blue as a front-running dye. Ladder formation of oligonucleosomal DNA was made visible by
ultraviolet transillumination and photographed using a Gel Imaging System (PE Company, USA).
Determination of apoptosis by flow cytometric analysis
After the cells were incubated with the different concentration of
ATP or ADO for 48 h, they were harvested by centrifugation, washed with ice-cold PBS once and fixed in 70% ethanol at 4 ºC
overnight. The cells were then washed once with ice-cold PBS and resuspended in PBS (pH 7.4) containing 0.5% pepsin, 5
mg·L-1 EB and RNase at room temperature for 30 min. Finally cells were analyzed by flow cytometry on a FACS420 (Becton
Dickinson, USA) equipped with an argon ion laser (488 nm), using the HP-300 Consort 30 software to determine percentage
of the apoptotic cells and the proportion of cells in
G0/G1, S, G2/M phases of the cell cycle. The proliferation index (PI) of cells
was calculated by the following formula:
Statistical analysis The data shown were mean values of at least three independent experiments and expressed
as mean±SD. Statistical analysis was performed by one-way ANOVA and the Student¡¯s
t-test, using statistical software SPSS 10.0. Statistical significance was set at a level
of P<0.05.
Results
Effects of ATP or ADO on the cell cycle and PI of HGC-27 cells
The cell cycle phase and PI value of HGC-27 cells
changed, when exposed to ATP or ADO at concentrations
of 0.03, 0.1, 0.3, 1
mmol·L-1 for 48 h. The proportion of cells in the
G0/G1- phase of cell cycle was significantly increased, S-phase of cells and PI value were significantly decreased after
sustained incubation of HGC-27 cells with ATP or ADO (0.1, 0.3, 1
mmol·L-1). The proportion of cells in
G2/M-phase of cell cycle was significantly decreased, when exposed to ATP (0.1, 0.3, 1
mmol·L-1) or ADO (0.1, 0.3
mmol·L-1). These data suggest that ATP and ADO inhibited the cell proliferation via
G0/G1- phase delay (Table 1 and Figure 1A, 1B).
Morphological changes of HGC-27 cells induced by ATP or ADO
Under fluorescence light microscope, the tumor cells
exposed to 0.3 mmol·L-1 ATP or ADO displayed morphological changes of apoptosis by AO/EB double-staining, such as cell
shrinkage, chromatin condensation, cell nuclear fragmentation, cell nucleous disappearance, increased nuclei fluorescence
or labeled orange or red-orange color (Figure 2A_2C).
Agarose gel electrophoresis results of HGC-27 cells induced by ATP or ADO
By the agarose gel electrophoresis, a ladder-like pattern of DNA fragmentation obtained from HGC-27 cells treated with 0.1_1
mmol·L-1 ATP or ADO appeared in agarose gel electrophoresis, indicating that ATP and ADO induced apoptosis of HGC-27 tumor cells (Figure 3A, 3B).
Apoptotic rate of HGC-27 cells induced by ATP or
ADO The method used for this part was the analysis of the
sub-G1 peak in the cell cycle. ATP or ADO induced the apoptosis of HGC-27 cells in a dose-dependent manner at concentrations between
0.03_1 mmol·L-1 for 48 h. The apoptotic rate of
HGC-27 cells treated with ATP or ADO were markedly higher than the
control. The maximum apoptotic rate of HGC-27 cells exposed to ATP or ADO (1
mmol·L-1) for 48 h was
(13.53±1.52)% or (15.90±1.15)%, respectively (Table 2 and Figure 4A_4E).
Effects of ATP metabolites, ADO-uptake inhibitors and antagonists of P receptors on cell
viability The effects of ADP and AMP on HGC-27 cells were examined to find out whether the metabolites of ATP also inhibit the growth of HGC-27 cells.
Both ADP and AMP reduced cell viability (Figure 5). We previously mentioned that ADO also caused apoptosis in these port
cells. Therefore, the inhibitors that were able to block ADO-induced apoptosis were tested to investigate the involvement
of ADO in ATP-induced apoptosis. Dipyridamole is an inhibitor of the nucleoside
transporter and blocks the trans
of ADO into the cell. We showed that this inhibitor (10
µmol·L-1) was able to block ATP- and ADO-induced apoptosis in
HGC-27 cells. The antagonists of P1 and P2 receptors, aminophylline and PPADS, were used. Both were non-selective antagonists
of P receptors. Neither aminophylline (0.1
mmol·L-1) nor PPADS (30
mmol·L-1) were able to block the apoptosis induced by ATP
and ADO (Figure 6).
Discussion
ATP and its related compounds are widespread transmitters for extracellular communication in many cell types. By
coupling to specific purinergic receptors, ATP is involved in a large variety of cellular functions. Receptors for purines and
pyrimidines (P receptors) are divided into two major classes termed as ADO or P1 receptors, for which ADO is the principal
natural ligand, and P2 receptors, for which ATP, ADP, UTP and UDP is the principal natural ligand. To date four P1 receptor
subtypes have been identified, A1,
A2A, A2B and A3, all coupled to G proteins with distinct tissue distribution and
pharmacological properties. The P2 receptors are divided into two families: the ligand-gated ion channels (P2X) and the G
protein-coupled receptors (P2Y)[20, 26]. ATP and ADO are known to inhibit cell growth and to induce apoptosis in various tumor
models[2-8, 5,16,18,19,21,22,25,27,28]. Both growth inhibition and programmed cell death were previously considered to be mediated
mainly by P1-receptors and
P2-receptors[1,4,6,10,17-20]. Here we provide evidence that extracellular ATP induces apoptosis and
causes cell-cycle arrest in human undifferentiated gastric cancer cells, and that ADO plays an important role in this.
In the present study, the HGC-27 cell line was chosen to study the possible role of ATP and ADO in human gastric cancer
diseases. Our results show that ATP and ADO reduced cell viability, caused cell arrest and induced apoptosis in this cell line.
Therefore, we investigated the mechanisms involved, in particular the role of ATP and ADO receptors and ADO transporters.
We assessed which of the P receptors were present in HGC-27 cells and also whether nucleoside transporters were present.
We used a series of inhibitors, including antagonists of P1 receptors and P2 receptors (ie, aminophylline and PPADS,
respectively) and a blocker of ADO transporters,
dipyridamole[3,4,26]. The effects of ATP and ADO on HGC-27 cell death were
not affected by aminophylline and PPADS, indicating that P1 and P2 receptors might not mediate them. In contrast, they were
significantly inhibited by dipyridamole, suggesting that ADO transporter might play an important role on the apoptosis
induced by ATP and ADO.
Apoptosis induced by ADO in GT3-TKB human gastric cancer cells has already been suggested by Saitoh
et al, who determined the effects of the ADO
(0.1-20 mmol·L-1) on mitochondrial membrane
potentials[27]. They demonstrated that
ADO-induced GT3-TKB cell death was significantly inhibited by dipyridamole. Their results also rule out the possibility for the
implication of P1 receptors. But the effects of ATP on GT3-TKB cells were not involved in their studies. Other studies,
however, showed that some subtypes of P2 receptor had been implicated in the induction of apoptosis in several cell lines,
such as P2X7 receptor in macrophages, microglial and dendritic cells;
P2X2 receptor in rat pheochromocytoma cells, PC12;
P2Y2 receptor in colorectal carcinoma cell lines (HT29, Colo320 DM) and oesophageal cancer cells,
Kyse-140[4,6,17-19]. Abbracchio et al
[29] and Kohno et al[16] also suggested the involvement of the
A3 receptor in ADO-induced apoptosis using
2-Cl-IB-MECA (in rat astrocytes and the human promyelocytic leukemia cell line HL-60 cells, respectively). Both studies used
high-agonist concentrations (10
mmol·L-1), rendering a selective effect question.
Normally, ATP is very rapidly broken down to ADP and AMP, and ADO is the final metabolite. Schrier
et al[4] reported, ATP breakdown in a medium without cells was not as fast as that in medium with cells
(t1/2: 4 h vs 30 min). ATP itself was
completely broken down by N1E-115 cells in 60-120 min. We have shown that ADO could cause apoptosis in this cell line.
ATP- and ADO-induced apoptosis were decreased by the inhibition of ADO-uptake using the nucleoside transport blocker
dipyridamole. These results suggest that ATP induces apoptosis in HGC-27 cells via extracellular breakdown to ADO, and
extracellular ATP and ADO induces apoptosis by their uptake into cells through ADO trans-porters. Thus, it was argued that
the effects of ATP and ADO might be subsequent to the uptake of ADO by the cells. However, we could not exclude the
possibility that the mechanism of apoptosis induced by ATP and ADO might also be due to the possible involvement of other
routes as most antagonists of P receptors to date are non-selective and have other pharmacological effects. Therefore,
further pathways leading to ATP- and ADO-induced apoptosis in HGC-27 cells need to be identified. Whether these same
effects occur in vivo is yet to be established.
Additionally, Lu et al[28] reported growth inhibition of ATP (0.23 mg/mL) on MGC-803 human gastric cancer cells. It was
found that ATP inhibited the proliferation and arrested cell cycle in the S-phase. Our results showed that ATP arrested cell
cycle in the G0/G1 phase. The reason why our results were not in agreement with those of Lu
et al[28] may be that we chose different kinds of gastric-cancer cell
lines.
In conclusion, our data shows that extracellular ATP and ADO reduce cell viability, cause cell-cycle arrest and induce
apoptosis. P receptors may not play a major role in ATP- and ADO-induced apoptosis in HGC-27 cells, but the uptake of ADO
is required for apoptosis in this cell line. These data show that extracellular ATP and ADO are potent inducers of apoptosis.
This makes ATP and ADO important compounds to consider when examining apoptosis in cancer diseases. We think that the
investigation of further effects of ATP and ADO on tumor cells may provide innovative treatment strategies for gastric
cancer.
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