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Important roles of P2Y receptors in the inflammation and cancer of digestive system

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Purinergic signaling is important for many biological processes in humans. Purinoceptors P2Y are widely distributed in human digestive system and different subtypes of P2Y receptors mediate different physiological functions from metabolism, proliferation, differentiation to apoptosis etc. The P2Y receptors are essential in many gastrointestinal functions and also involve in the occurrence of some digestive diseases. Since different subtypes of P2Y receptors are present on the same cell of digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic functions on the same cell. Recently, growing lines of evidence strongly suggest the involvement of P2Y receptors in the pathogenesis of several digestive diseases. In this review, we will focus on their important roles in the development of digestive inflammation and cancer. We anticipate that as the special subtypes of P2Y receptors are studied in depth, specific modulators for them will have good potentials to become promising new drugs to treat human digestive diseases in the near future.
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Oncotarget28736
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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 19
Important roles of P2Y receptors in the inammation and cancer
of digestive system
Han-Xing Wan1, Jian-Hong Hu1, Rei Xie1, Shi-Ming Yang1 and Hui Dong1,2
1 Department of Gastroenterology, Xinqiao Hospital, Third Military Medical University, Chongqing, P.R. China
2 Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California,
USA
Correspondence to: Hui Dong, email: h2dong@ucsd.edu
Keywords: P2Y receptors, digestive inammation, digestive cancer
Received: November 04, 2015 Accepted: February 05, 2016 Published: February 19, 2016
ABSTRACT
Purinergic signaling is important for many biological processes in humans.
Purinoceptors P2Y are widely distributed in human digestive system and dierent
subtypes of P2Y receptors mediate dierent physiological functions from metabolism,
proliferation, dierentiation to apoptosis etc. The P2Y receptors are essential in
many gastrointestinal functions and also involve in the occurrence of some digestive
diseases. Since dierent subtypes of P2Y receptors are present on the same cell of
digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic
functions on the same cell. Recently, growing lines of evidence strongly suggest the
involvement of P2Y receptors in the pathogenesis of several digestive diseases. In
this review, we will focus on their important roles in the development of digestive
inammation and cancer. We anticipate that as the special subtypes of P2Y receptors
are studied in depth, specic modulators for them will have good potentials to become
promising new drugs to treat human digestive diseases in the near future.
INTRODUCTION
Purinoceptors are generally divided into P1
receptors which main ligand is adenosine, and P2 receptors
which main ligands are nucleotides. Based on their
signaling properties, P2 receptors are further subdivided
into ionotropic P2X receptors that are nucleotide-gated
ion channels and metabotropic P2Y receptors that are G
protein-coupled receptors (GPCRs). P2Y receptors are
consist of eight subtypes: ve Gq/G11-coupled subtypes
(P2Y1, P2Y2 , P2Y4, P2Y6 and P2Y11), usually
activating phospholipase C-IP3 pathway that modulates
endoplasmic reticulum calcium release, and three Gi/o-
coupled subtypes (P2Y12, P2Y13 and P2Y14), mainly
inhibiting adenylyl cyclase to regulate cyclic AMP/
protein kinase A (PKA) [1-2]. At present, all eight P2Y
receptor subtypes have been cloned in mammalian [3].
Different P2Y receptor subtypes are activated by different
nucleotides. ADP has been claimed to be selective agonist
of P2Y1, P2Y12 and P2Y13 receptors; however, UTP
predominantly binds to P2Y2 and P2Y4 receptors, and
to a lesser extent to P2Y6 receptors which preferential
agonist is UDP. P2Y14 receptors are mainly activated
by UDP-glucose and other UDP-sugars, or by UDP [4].
In recent decades, a growing line of evidence suggests
the involvements of P2Y receptors in the pathogenesis
of human diseases, and different subtypes of P2Y
receptors mediate various pathophysiological processes,
ranging from metabolism, proliferation, differentiation
to apoptosis. Recent studies also demonstrate that
P2Y receptors play important roles in the regulation of
physiological functions and pathological processes in
the digestive system. In this review, we will focus on the
pathophysiological roles of P2Y receptors in digestive
inammation and cancer.
PHYSIOLOGICAL FUNCTIONS OF
P2Y RECEPTORS IN THE DIGESTIVE
ORGANS
P2Y receptors are widely expressed in digestive
organs and their functions vary from neurotransmission,
gland secretion, contraction and relaxation of smooth
muscle to carbohydrate and lipid metabolism in the
digestive system. In accordance with evidence, we rst
highlight physiological roles of P2Y receptors in the
esophagus, stomach, liver, pancreas, and colon (Figure 1).
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Esophagus
The P2Y receptors are functionally expressed in the
esophagus to play an important role in the regulation of
esophageal motility. In human and porcine esophagus,
P2Y1 receptors mediate lower esophageal sphincter
(LES) relaxation by regulating neurotransmission [5-6].
P2Y1 receptors also mediate contraction of the circular
smooth muscle layer in porcine esophagus [7]. Electrical
eld stimulation (EFS)-induced contractions is mediated
through P2Y receptors in cat esophageal smooth muscle
[8]. Feline esophageal contraction is preferentially
mediated by P2Y receptors coupled to Gαi3 and Gαq
proteins, which activate PLCβ, subsequently increase
intracellular Ca2+ and activate PKC [9].
Stomach
Although gastric acid secretion is mainly regulated
by P1 adenosine receptors [10], P2Y receptors may
also regulate gastric acid secretion, gastric contraction,
relaxation and neurotransmission. However, the specic
receptor subtypes of P2Y receptors involved and their
underlying mechanisms still need further investigation.
ATP selectively inhibits histamine-stimulated gastric
acid secretion from rabbit parietal cells by acting on P2Y
receptors [11]. UTP and UDP can induce contraction of
gastric smooth muscle through activation of P2Y receptors
[12]. ATP also induces contraction of gastric smooth
muscle in guinea pig via activation of P2Y receptors [13].
At least two subtypes of P2Y purinoceptors are involved
in gastric contraction in guinea-pig and are related to the
elevation of intracellular Ca2+ [14]. Finally, ATP may
regulate NANC inhibitory neurotransmission in rat pyloric
sphincter through acting on P2Y receptors [15].
Liver
Several P2Y receptor subtypes regulate hepatic
physiological functions, such as carbohydrate metabolism,
lipid metabolism and proliferation [16-17]. P2Y1 receptors
induce glycogen phosphorylase of rat hepatocyte by
raising intracellular calcium concentrations but inhibiting
cyclic AMP accumulations [18]. P2Y2 receptors in
human hepatocytes regulate both glycogen metabolism
and proliferation-associated responses through Ca2+ and
MAPK pathways [19], and induce ERK phosphorylation,
Egr-1 expression, and cyclins and cell cycle progression,
which are essential for efcient hepatocyte proliferation
[20]. P2Y2 receptors also mediate extracellular ATP-
induced c-jun N-terminal kinase signaling and cell cycle
progression to promote hepatocellular proliferation [21].
P2Y13 receptors modulate reverse cholesterol transport
by increasing hepatic HDL cholesterol uptake, overall
hepatocyte cholesterol content, and biliary output [22-24].
Pancreas
P2Y1, 2, 4, 6, 11, 12 and 13 receptor subtypes have
been identied in INS-1βcells, mouse, rat and human
pancreaticβcells [25-26]. Activation of P2Y receptors
inβcells is conrmed to participate in the regulation of
insulin secretion, glucose metabolism and an increase in
intracellular Ca
2+
concentration [27]. Glucose stimulation
triggers exocytosis of insulin and ATP through activation
of P2Y1 receptors to result in PLC activation and DAG
production in MIN6 mouse pancreaticβcells [28]. P2Y1
and P2Y6 receptors in MIN6 cells induce intracellular
calcium release and insulin secretion, and prevent TNF-α
induced βcells apoptosis [29]. Stimulation of P2Y13
receptors in pancreatic βcells inhibits insulin secretion via
calcium inux and inhibition of cyclic AMP production
[30]. High glucose and free fatty acids can also induce
β cells apoptosis through stimulating P2Y13 to activate
apoptotic pathways [31].
Colon
Large numbers of studies in animals and humans
demonstrate that P2Y1 receptors mediate NANC
inhibitory transmission of intestinal smooth muscle in
mice through release of ATP and NO [10]. Activation
of P2Y1 receptors can induce nerve-mediated relaxation
via inhibitory neuromuscular transmission in human
intestines, guinea pig small intestine and rat colon [32-35].
Endogenous nucleotides acting on P2Y1, 2, 4 receptors
evoke intestinal Cl secretion [36]. The activation of P2Y2
and P2Y4 receptors stimulate Cl secretion in small and
large intestines. Basolateral UTP-induced Cl secretion in
jejunum was partially reduced in P2Y2 knockout (40%)
and P2Y4 knockout (60%) null mice [37]. Activation of
P2Y2 receptors induces duodenal mucosal bicarbonate
secretion via both intracellular Ca2+ release and
extracellular Ca2+ entry through store-operated channels
[38]. Stimulation of luminal P2Y2 and P2Y4 receptors
lead to K+ secretion in mouse distal colonic mucosa
[39]; however, activation of basolateral P2Y6 receptors
on rat colonic enterocytes induces NaCl secretion via a
synergistic increase of [Ca2+]i and cyclic AMP [40].
P2Y RECEPTORS IN DIGESTIVE
INFLAMMATION
Over the past decades, many studies have
highlighted fundamental roles of P2Y receptors in
inammatory diseases, particularly P2Y2, 6, 12 receptors
have been well studied, such as P2Y2 receptor agonists
can treat cystic borosis, and promote wound healing
and leukocyte functions [4, 41-45]. Whereas, P2Y2
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receptors have ambivalent functions, such as promoting
chronic inammatory states and brotic remodeling [46-
48]. However, in inammation of the digestive system,
the studies of P2Y receptors are mainly concentrated on
the liver and colon. The involvements of P2Y receptors
in the inammation of other digestive organs, such
as esophagitis, gastritis and pancreatitis, are poorly
understood and rarely presented in the literatures.
Liver
During inammatory responses, endogenous release
of ATP in the liver can activate purinergic P2 receptors.
It was found that large amounts of ATP released from
the liver increased the expression of P2Y2 receptors
in concanavalin A-induced model of acute hepatitis in
C57BL/6 mice. Liver damage and necrosis are largely
decreased in C57BL/6 wild-type mice injected with
suramin, an inhibitor of P2Y receptors, or in P2Y2
receptors knockout mice, in which acetaminophen-
induced liver damage is also alleviated. P2Y2 receptors
can promote neutrophil inltration, regulate cell survival,
and promote tumor necrosis factor-mediated cell death,
supporting the view that activation of P2Y2 receptors
stimulates the recruitment of neutrophils into the liver to
cause hepatocyte death [49].
Hepatic stellate cells play an important role in
formation of liver brosis and liver cirrhosis. ATP
increases intracellular Ca2+ in hepatic stellate cells, which
is inhibited by suramin. Interestingly, P2Y2 and P2Y4
receptors are expressed in quiescent hepatic stellate
cells, whereas P2Y6 receptors are expressed in activated
hepatic stellate cells. The activated hepatic stellate cells
express the ectonucleotidase nucleoside triphosphate
diphosphohydrolase-2 (NTPDase-2) that colocalizes with
activated HSC in CCl4-induced cirrhosis. UDP regulates
transcription of procollagen-1 in activated HSC via P2Y
receptors activation, which is partially inhibited by the
P2Y receptors inhibitor suramin, suggesting P2Y receptors
may be attractive targets to prevent/treat liver brosis
[50]. When macrophages in kupffer cells of the liver are
activated by various factors, such as lipopolysaccharide
(LPS), they produce various cytokines and chemokines
Figure 1: The physiological functions of P2Y receptors in digestive system. Different subtypes of P2Y receptors are expressed
in human esophagus, stomach, liver, pancreas and colon. They play different roles in the regulation of physiological processes, such as
neurotransmission, ion transports, metabolism, proliferation and apoptosis, muscle contraction and relaxation in the digestive organs.
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to play important roles in hepatitis and liver brosis.
P2Y2, 5, 6, 12, 13 receptors are strongly expressed in
the liver kupffer cells of C57BL/6 mice (KUP5 cells).
After stimulation with LPS, KUP5 cells produce IL-6 and
TNF-α. Non-selective P2 receptor antagonist, suramin,
and P2Y13 receptors selective antagonist, MRS2211,
markedly inhibit LPS-induced IL-6 increase in KUP5
cells, whereas both suramin and MRS2211 do not inhibit
LPS-induced TNF-α production, suggesting that P2Y13
and other P2Y receptors may be involved in LPS-induced
IL-6 production in Kuffer cells and liver inammation
[51].
Figure 2: P2Y receptors-mediated Ca2+ signaling in proliferation or apoptosis of digestive cancer cells. Stimulation of
Gq/G11-coupled P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11) activates PLC/IP3 pathway to induce intracellular calcium
release from the endoplasmic reticulum (ER). An increase in intracellular calcium concentrations would increase the proliferation or
apoptosis of different digestive cancer cells.
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Colon
Intestinal inammation can upregulate mRNA
expression of P2Y2 and P2Y6 receptors in the colonic
mucosa of colitic mice. The mRNA of P2Y2 and P2Y6
receptors are increased in both Crohn’s and ulcerative
colitis of intestinal human samples compared with
noninamed tissues. The mRNA expression of P2Y2
receptors is also increased in Caco-2 and IEC-6 cells
during intestinal inammation, but it is unknown how
inammation up-regulates expression of P2Y2 receptors.
ATP or UTP stimulation of P2Y2 receptors in intestinal
epithelial cells increases ICAM-1 expression and promotes
transepithelial migration and adhesion of neutrophils and
macrophage to the apical surface of IEC. In addition, ATP
or UTP facilitates the migration of neutrophil-like PLB-
985 cells and macrophage across the Caco-2 monolayer
and promotes macrophage-like U-937 cells adhere to
IEC monolayers. CD68+ macrophage inltrates from
the colonic epithelium and presents at the apical surface
of colonocytes during intestinal inammation. P2Y2
receptors mediate neutrophil adhesion to the surface
of IEC need presence of adherent macrophage. This
investigation is helpful to identify potential therapeutic
targets to treat inammatory bowel diseases [52]. Recent
studies found that NF-κB p65 could regulate P2Y2
receptors transcription, and activation of P2Y2 receptors
by UTP increased both cyclooxygenase-2 (COX-2)
expression and PGE2 release in IEC [53]. Further studies
showed the effects of CCAAT/enhancer-binding proteinβ
(C/EBPβ) and NF-κB p65 on P2Y2 receptor transcription
are synergistic during inammation in IEC [54]. In the
enteric nervous system, ATP has long been established
as an inhibitory neurotransmitter, 75% of Hirschsprung’s
disease patients, aganglionosis is conned to the
intestine. The expression of P2Y1 and P2Y2 receptors
are absent from the submucosal and myenteric plexuses
of aganglionic tissue compared to ganglionic tissue and
normal controls. The deciency of P2Y receptors in
ganglionic intestine in Hirschsprung’s disease suggests
the absence of the inhibitory neurotransmitter, ATP. This
explains the contracted state of the aganglionic gut in
Hirschsprung’s disease [55].
The expression of P2Y6 receptors is enhanced
by inammation with TNF-α and IFN-γ both in IEC-
6 and Caco-2/15 cells. In Caco-2/15 cells, stimulation
P2Y6 receptors by UDP results in an increased
expression and release of CXCL8, partially depending
on ERK1/2 phosphorylation. UDP also increases
ERK1/2 phosphorylation of IEC-6 cells, suggesting
the involvement of P2Y6 receptors [56]. Indeed, UDP
stimulation of P2Y6 receptors promotes CXCL8
transcription through ERK1/2 activation and the AP-1
complex of transcription factors, aggravating colitis-like
disease in mice by stimulating neutrophil recruitment
at the site of inammation. CXLC8 gene expression
is regulated at the transcriptional level by mediating
ERK1/2-dependent phosphorylation of c-fos in IEC
through P2Y6 receptors activation. P2Y6 regulation of
CXCL8 expression requires PKCδactivation upstream of
the signaling pathway composed of MEK1/2-ERK1/2 and
c-fos [57]. Previous reports revealed that T cells play an
important role in the pathogenesis of IBD and extracellular
nucleotides can regulate colonic epithelial cell damage
during inammation. Interestingly, UDP, P2Y6 receptors
selective agonist, activates peripheral T cells and increases
mRNA levels of P2Y6 receptors, and raises intracellular
calcium concentration. Although P2Y6 receptors are
expressed in human T cell inltrating IBD,the roles of
P2Y6 receptors in the pathogenesis of IBD need further
studies [58].
P2Y RECEPTORS IN DIGESTIVE
CANCER
Different subtypes of P2Y receptors are expressed
in many cancer cells and tissues to be likely involved in
cancer development, such as P2Y receptors in melanoma
[59], skin squamous cell carcinoma [60], lung cancer
[61-62], prostate cancer [63-66], glioma [67-68], breast
cancer [69-75], ovarian cancer [76], and haematological
malignancies [77], etc. Recent growing lines of evidence
suggest an important role of P2Y receptors in digestive
tumorigenesis. Different subtypes of P2Y receptors are
present in cancer cells and primary cancer tissues of
digestive system; however, the mechanisms by which
these receptors play in the devolvement and progression
of cancers are still poorly understood. The involvement of
P2Y receptors in digestive cancer is mainly investigated
in esophageal cancer, hepatocellular carcinoma, biliary
cancer, pancreatic cancer and colorectal cancer, which are
summarized in Table 1 and Figure 2. Although expression
and function of P2Y receptors are well documented in
normal human and animal stomach, their involvements in
the pathogenesis of gastric cancer have not been explored
so far.
Esophageal cancer
The squamous esophageal cancer cell line, Kyse-140
cells express mRNA of P2X4, P2X5, and P2Y2 receptors,
but not P2X1 and P2X7 receptors that are mainly
associated with apoptosis. The mRNA of P2Y2 and P2X4
receptors are also found in biopsies of human squamous
esophageal cancer; however, in accordance with the Kyse-
140 cells, the mRNA of the P2X1 and P2X7 receptors are
not expressed in human squamous esophageal cancer
biopsy. Although mRNA of P2Y2 and P2X4 receptors was
found in both Kyse-140 and esophageal primary culture
cells, only P2Y2 receptors protein specic uorescence
was detected in the membrane of the Kyse-140 and
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esophageal primary culture cells. ATP, UTP and ATPγS,
responding to P2Y2 receptors activation, result in an
increase of intracellular Ca2+ levels. Incubation of Kyse-
140 cells with the phospholipase C inhibitor U73122
dose-dependently inhibits ATP-induced intracellular Ca
2+
level, suggesting that P2Y2 receptors mediate intracellular
Ca2+ via phospholipase C (PLC) activation in Kyse-
140 cells. Extracellular ATP as well as ATP analogue
dose-dependently increase the proportion of cells in the
S-phase of the cell cycle in return inhibiting proliferation
of primary cell cultures of human esophageal cancer
as well as Kyse-140; however, only ATP but not ATP
analogue dose-dependently induces caspase-3 activity and
increases in apoptosis of Kyse-140 cells through activation
of P2Y2 receptors [78]. Together, these ndings suggest
that purinergic nucleotides may be tumor preventers
through P2Y2 receptors/PLC/Ca
2+
signaling in squamous
esophageal cancer.
Hepatocellular carcinoma
P2Y1 purinergic receptors were found to play a
role in the response of hepatocellular carcinoma (HCC)
cells to osmotic swelling and involved in the volume-
regulatory response [79]. However, in HCC cells, copper
inhibits thapsigargin-sensitive Ca2+ stores by acting
P2Y2 receptors to lead to inhibition of regulatory volume
decrease (RVD) [80]. The mRNA and protein levels of
P2Y13 receptors are conrmed in Huh-7 hepatoma
cells that can release ATP when exposed to hypotonicity
medium. On the other hand, ADP can activate P2Y13
receptors to potentiate volume regulatory decrease (RVD)
and then mediate cell metabolism [81].
A recent study showed that both mRNA and protein
expression levels of P2Y2 receptors were dramatically
higher in native human HCC and human HCC cell lines
compared with human normal hepatocytes. Extracellular
nucleotides-induced intracellular Ca
2+
increase is markedly
higher in human HCC cells than normal hepatocytes.
Activation of P2Y2 receptors signicantly promotes
proliferation and migration of HCC cells and volume
growth of HCC in nude mice through store-operated
calcium channels (SOCs)-mediated Ca2+ signaling [82].
Insulin and ATP induce a dose-dependent increase in
p44/42 MAPK phosphorylation in rat HCC cells and
chelation of extracellular Ca2+ with EGTA diminishes ATP-
and insulin-induced p44/42 MAPK phosphorylation. Patch
clamp electrophysiology and uorescence microscopy
showed that insulin and ATP induced monophasic
Table 1: Involvement of P2Y receptors in various types of digestive cancer
Cancer types Tissue
cell line
P2Y receptor
subtypes
Signaling
pathway
Pathological
mechanisms
Reference
Esophageal
cancer
Kyse-140 P2Y2 PLC/Ca2+ anti-proliferative
apoptosis-inducing [78]
Hepatic
carcinoma
Huh-7
Rat hapatoma
cell line
P2Y1,2,13 Ca2+
volume-regulatory
cell metabolism [79-81]
HePG2
BEL-7404 P2Y2 Ca2+
promoting proliferation
migration
growth
[82]
Rat hepatoma
HTC cells
P2Y2 Ca2+
MAPK glucose metabolism [83]
HePG2
huh-7 P2Y1,2,4,6 Ca2+ unknown [84]
Biliary cancer Mz-Cha-1 P2Y1,2,4,6 Ca2+ unknown [85]
Pancreatic
cancer
PANC-1 P2Y1,2, 6 PLC
IP3/PKC pro-proliferative [86-87]
Colon cancer
HT-29 P2Y2 ECAR tumor cell metabolism [90]
HT-29
calo320MD
P2Y2 Ca2+
cyclic AMP
anti-proliferative
apoptosis-inducing [91]
HT-29
Primary cancer
P2Y2,4 Ca2+ anti-proliferative
apoptosis-inducing [92-93]
HCT8
Caco2 P2Y1,2,4,6,11,12 Ca2+ pro-proliferative
apoptosis-inducing [94]
HT-29 P2Y2 ERK
P38 MAPK
anti-proliferative
apoptosis-inducing [95-96]
Caco2 P2Y2,4 Ca2+
MAPK
proliferative [97-99]
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and multiphasic changes in membrane potential and
intracellular Ca2+ in HCC cells. Therefore, insulin and
ATP effects are synergistic to regulate glucose metabolism
of HCC cells [83]. Although the mRNA of P2Y1, P2Y2,
P2Y4 and P2Y6 receptors were detected in HepG2 and
HuH-7 cells, P2Y1 and P2Y6 receptor agonists, ATP and
UDP, did not alter intracellular Ca2+, suggesting that these
receptors are not expressed at functional levels. However,
UTP through activation of P2Y2 and P2Y4 receptors can
mobilize internal Ca2+ via inositol 1,4,5-trisphosphate (IP3)
[84]. Therefore, these P2Y receptors may play major roles
in the pathogenesis of HCC.
Biliary cancer
Although the mRNAs for P2Y1, P2Y2, P2Y4
and P2Y6 purinergic receptors subtypes are found in
biliary epithelial cancer cells (Mz-Cha-1), but only
P2Y2 receptors are present at the protein level. Not
only extracellular ATP dose-dependently results in
an intracellular Ca2+ increase, but also UTP produces
a similar Ca2+ response and cross-desensitation. ATP
induces cytosolic and nuclear Ca
2+
transients [85]. To date,
only expression of P2Y receptors is observed in biliary
epithelial cancer cells, however, the roles of P2Y receptors
in biliary cancer need further investigation.
Pancreatic cancer
P2Y receptors, especially P2Y1, P2Y2 and P2Y6
receptors are highly expressed in PANC-1, a duct epithelial
cell derived from human primary pancreatic cancer cells.
P2Y1 and P2Y6 proteins were also found in PANC-1 cells.
ADP activation of P2Y1 receptors and UDP acting P2Y6
receptors increase proliferation of PANC-1 cell through
PLC/IP3/PKC pathway. This proliferative action of P2Y
receptors may potentially apply to recover pancreatic
duct epithelial damage by physiological or pathological
processes [86]. UTP or P2Y2 receptor selective agonist
MRS2768 can increase proliferation of PANC-1, which
is signicantly decreased by P2Y receptor antagonist
suramin and siRNA against P2Y2 receptors. UTP/P2Y2
receptor regulation of pancreatic cell proliferation depends
on PLC/IP
3
/PKC and phosphorylation of Akt [87]. In INS-
1 cells and rat pancreatic islets, ATP at low concentrations
increases insulin release via P2Y receptors and PLC;
however, ATP at high concentrations inhibits insulin
release after metabolizing to adenosine [88]. So far, the
roles of P2Y receptors in pancreatic cancer are poorly
understood and need lucubrating.
Colorectal cancer
P2Y2 and P2Y4 receptors are overexpressed
in human colon cancer compared with normal colon
tissues although their functional signicance need
further studies [89]. Immunocytochemistry and western
blot analysis also demonstrate the protein expression of
P2Y2 receptors in HT-29 human colon carcinoma cells.
ATP or UTP elicits a biphasic effect of extracellular
acidication rate by activating P2Y2 receptors in HT-
29 cells, but effects of UTP or ATP are resistant to
suramin, suggesting that agonists of purinoceptors may
affect tumor cell metabolism [90]. The mRNA of P2Y2
receptors is expressed in two colorectal carcinoma cell
lines (HT29, Colo320DM) and short-term stimulation of
P2Y2 receptors cause both intracellular Ca2+ release and
transmembrane Ca2+ inux, and a subsequent increase
in cyclic AMP. This effect is inhibited by BAPTA-AM.
Prolonged stimulation of P2Y2 receptors induces a time-
dependent increase in apoptosis in both cell lines and
causes a dose-dependent inhibition of cell proliferation
up to 85% (Colo320 DM) or 64% (HT29). Chelating
[Ca2+]i with BAPTA-AM almost completely abolishes
this effect. Moreover, forskolin or cyclic AMP derivatives
cause a rise in intracellular cyclic AMP and lead to
synergistic anti-proliferative effect in both cell lines. This
nding demonstrates P2Y2 receptors play major roles in
anti-proliferative and apoptosis-inducing in colorectal
carcinoma cell lines [91]. The primary cell cultures of
human colorectal carcinomas and HT29 cell line express
functional P2U-receptors (P2Y2 and P2Y4). ATP or UTP
at micromolar concentrations leads to a rapid biphasic
increase of [Ca
2+
]
i
and cross-desensitization between two
nucleotides. P2U-receptor agonist ATP derivative ATP-γ-S
inhibits proliferation and induces apoptosis of HT 29 cells
[92-93].
Two human colorectal carcinoma cell lines (HCT8
and Caco-2) express mRNA of P2Y1, 2, 4, 6, 11, 12
receptors and proteins of P2Y1 and P2Y2 receptors. ATP,
at high concentrations, induces apoptosis through P2Y1
receptors; conversely, ATP, at lower concentrations,
and UTP stimulates proliferation of human colorectal
carcinoma cells, probably acting on P2Y2 receptors.
UTP can trigger calcium inuxes through either P2Y2 or
P2Y4 receptors, which is inhibited by suramin. Therefore,
stimulation of purinergic receptors may contribute to the
modulation of epithelial carcinoma cell proliferation and
apoptosis [94]. Ursolic acid could inhibit proliferation of
HT29 and induce apoptosis via P2Y2 receptors-mediated
inhibition of ERK phosphorylation and activation of p38
MAPK pathway [95-96]. The mRNA of P2Y2 and P2Y4
receptors is found in Caco-2 cells. ATP, UTP and UDP
increase phosphorylation of MAPK by stimulating P2Y
receptors, probably through subtypes of P2Y2, P2Y4,
P2Y6 and P2Y11 receptors. ATP increases proliferation
of Caco-2 cells via activation of P2Y purinergic receptors
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[97-98]. On the contrary, higher concentrations (1-10 mM)
of extracellular ATP or the unhydrolyzed ATP analogue
5’-adenylyimido-diphosphate (AMP-PNP), suppress
Caco-2 cell proliferation arresting cells cycling at the S
phase by inhibiting PKC, ERK and MAP kinase [99]. The
sustained activation of P2 receptors by ATP may lead to
IL-8 secretion from human colorectal epithelial cells and
may play an important role in tumor progression as well
as in the pathology of IBD [100].
CONCLUSIONS
Growing lines of evidence suggest that P2Y
receptors are involved in inammation-associated
diseases of liver and colon. P2Y receptors can also
regulate metabolism, proliferation, differentiation and
apoptosis of digestive cancer cells and tissues. It has
been demonstrated that different P2Y receptor subtypes
are present on the same cell and that various subtypes of
receptors may produce opposite functions. Such as HCT8
and Caco-2 cells express P2Y1, P2Y2, P2Y4, P2Y6,
P2Y11, and P2Y12 receptors. Lower concentrations of
ATP and UTP stimulate proliferation of these cells via
P2Y2 receptors activation, but high concentrations of
ATP induce apoptosis and anti-proliferation through
P2Y1 and P2X7 receptors. This suggests that the control
of cell proliferation by extracellular nucleotides might
be regulated by a crucial balance of the activities of the
receptor subtypes. The subtypes of P2Y receptors as new
therapeutic targets for drug discovery to treat digestive
diseases may have extensive clinical signicance. We
therefore anticipate that as these subtypes of P2Y receptors
in digestive organs are further studied, their specic
modulators may become promising new drugs to treat
digestive diseases, such as inammation and cancer in the
near future.
Abbreviations
GPCRs:G protein-coupled receptors; PKA: protein
kinase A; LES: lower esophageal sphincter; NTPDase-2:
nucleoside triphosphate diphosphohydrolase-2; LPS:
lipopolysaccharide; COX-2: cyclooxygenase-2; IBD:
inammatory bowel diseases; RVD: volume regulatory
decrease; SOCs: store-operated calcium channels; Mz-
Cha-1: biliary epithelial cancer cells; AMP-PNPATP:
analogue 5’-adenylyimido-diphosphate; HCC:
hepatocellular carcinoma; PLC: phospholipase C; IP3:
inositol 1,4,5-trisphosphate.
ACKNOWLEDGMENTS
supported by the National Natural Science
Foundation of China ( No.31371167 and No.81570477 to
HD).
CONFLICTS OF INTEREST
The authors declare no conicts of interest.
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... Nesse processo, estão presentes diversos receptores relacionados ao sistema purinérgico, que atuam com uma ampla gama de efeitos, conforme exposto na Figura 2. Marcadamente, no cólon humano, existe a presença de diferentes linhagens de receptores P2Y, dentre eles, P2Y1, P2Y2, P2Y4 e P2Y6. Esses receptores são acoplados à proteína G e ativados por nucleotídeos, como ADP, UTP e UDP (Wan et al., 2016). Fica evidente a importância da sinalização purinérgica no contexto do CCR quando analisados parâmetros inflamatórios no ambiente tumoral. ...
... Para que ocorra a carcinogênese, são necessárias algumas condições que favoreçam o aparecimento de células neoplásicas, dentre elas a inflamação, levando à perda da homeostase e prejudicando o potencial regenerativo dos tecidos. Pacientes com doenças inflamatórias como retocolite ulcerativa ou doença de Crohn demonstram aumento na expressão de RNAm correspondente aos receptores P2Y2 e P2Y6, enquanto nos tumores colônicos em geral ocorre superexpressão dos receptores P2Y2 e P2Y4, em comparação a tecidos intestinais normais (Wan et al., 2016). ...
... Ou seja, o estudo do sistema purinérgico aplicado aos tumores de modo geral possui grande importância para o desenvolvimento de terapias mais eficazes e direcionadas às células neoplásicas. Para que isso seja possível, é fundamental que sejam compreendidos os processos de sinalização celular e os atores envolvidos nesse processo (Wan et al., 2016;Di Virgilio, Adinolfi, 2017). ...
Chapter
Embora existam diversos grupos brasileiros de pesquisa da sinalização purinérgica, o conhecimento sobre o tema tem sido amplamente difundido em língua inglesa em periódicos internacionais. Em língua portuguesa, os estudos, de um modo geral, constituem dissertações e teses. Todos os colaboradores do livro são pesquisadores brasileiros que estudam alguma situação fisiológica ou patológica envolvendo o sistema purinérgico. O livro trata do funcionamento da sinalização purinérgica em condições fisiológicas gerais e sobre a história das enzimas e dos receptores purinérgicos. Aborda situações fisiológicas, como a modulação do sistema purinérgico pelo exercício físico e por moléculas nutracêuticas. E dedica alguns capítulos para a relação da sinalização purinérgica em condições patológicas: diversos tipos de câncer; doenças endócrinas, como diabetes e disfunções da glândula tireoide; doença renal; hipertensão arterial sistêmica; doenças degenerativas, como doença de Alzheimer e doença macular relacionada à idade; doenças parasitárias e virais, dentre outras.
... P2X7R is involved in the pathogenesis of IBD by mediating the NLRP3/ Caspase-1 inflammasome and NF-κB pathways, and regulating the balance of Th17 and Treg cells [13]. In intestinal samples of patients with IBD, mRNA levels of both P2Y2 and P2Y6 receptors were found to be increased during flare-ups [14]. Studies increasingly Ivyspring International Publisher support the role of P2 purinergic receptors in the pathogenesis of IBD. ...
... The P2Y receptor is a G protein-coupled receptor (GPCRs) that can recognize ATP, ADP, UTP, UDP and UDP-glucose. Different subtypes of P2Y receptors mediate many pathophysiological processes, including apoptosis, immune regulation, cell proliferation and differentiation [14]. Increasing evidence show that P2Y receptor is involved in the pathogenesis of inflammatory diseases and tumors [14]. ...
... Different subtypes of P2Y receptors mediate many pathophysiological processes, including apoptosis, immune regulation, cell proliferation and differentiation [14]. Increasing evidence show that P2Y receptor is involved in the pathogenesis of inflammatory diseases and tumors [14]. While the P2Y1, P2Y2 and P2Y6 of P2Y receptor involved in the development of UC has been described [12,[26][27], the role of P2RY13 in UC has never been investigated. ...
Article
The pathogenesis of ulcerative colitis (UC) is unclear, while genetic factors have been confirmed to play an important role in its development. P2RY13 is a G protein-coupled receptor (GPCRs), which are involved in the pathogenesis of inflammation and immune disorders. According to GEO database analysis, we first observed that the expression of P2Y13 was increased in UC patients. Therefore, we sought to determine the role of P2Y13 in the development of colitis. Our data showed that P2RY13 was highly expressed in the inflamed intestinal tissues of UC patients. In mice, pharmacological antagonism of P2Y13 can significantly attenuate the intestinal mucosal barrier disruption. In LPS-induced NCM460 cell, knockdown or pharmacological inhibition of P2RY13 increased the expression of intestinal tight junction protein and reduced apoptosis. In addition, we found that the effect of P2Y13 on colitis is related to the activation of the IL-6/STAT3 pathway. Activation of P2Y13 increases IL-6 expression and promotes STAT3 phosphorylation and nuclear transport. Deletion of the STAT3 gene in the intestinal epithelial cells of mice significantly mitigated the exacerbation of colitis due to P2Y13 activation. Thus, P2Y13 can aggravate intestinal mucosal barrier destruction by activating the IL-6/STAT3 pathway. P2Y13 might be a potential drug target for UC.
... The IC50 results evidenced the higher anticancer activity of ATP-SeNPs in the caco-2 cell line compared to HEK293 cells. Although the purinoceptor is found in all cells, it is found in higher expression in cancer cells such as hepatocellular carcinoma cells (HepG2 cell line) and human colon cancer cells (caco-2 cells) [3,20,21]. Moreover, the cellular uptake of nanoparticles was found to be ...
... The IC 50 results evidenced the higher anticancer activity of ATP-SeNPs in the caco-2 cell line compared to HEK293 cells. Although the purinoceptor is found in all cells, it is found in higher expression in cancer cells such as hepatocellular carcinoma cells (HepG2 cell line) and human colon cancer cells (caco-2 cells) [3,20,21]. Moreover, the cellular uptake of nanoparticles was found to be higher with ATP-SeNPs (0.92 ± 0.02 µg/10 4 cells) compared to SeNPs (0.24 ± 0.1 µg/10 4 cells) due to the purinoceptor targeted penetration of the ATP-SeNPs. ...
... These results indicated that the SeNPs and ATP-SeNPs did not show significant changes in total cell death. The necrosis was found to be higher in the ATP-SeNPs (46.20%) compared to SeNPs (14.76%) (Figure 8c), which indicated the successful penetration of the ATP-SeNPs in caco-2 cells and resulting purinoceptor-targeted cell death in the human colon cancer cells (caco-2 cells) [3,20,21]. SeNPs exhibited significant necrosis and cell washout due to toxicity-induced floating of the dead cells. FACS was used to determine the apoptosis stages in SeNPs or ATP-SeNPs treated caco-2 cells using the Annexin FITC/PI staining kit (Figure 8c) (Figure 8c). ...
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The adenosine triphosphate (ATP)-conjugated biogenic selenium nanoparticles (SeNPs) for P2 (purinoceptors) receptor-targeted anti-colon cancer activity were developed in this study. First, the SeNPs were synthesized using Trichoderma extracts (TE) and then conjugated with ATP to enhance their anticancer activity. The developed SeNPs had an oval crystalline structure with an average diameter size of 26.45 ± 1.71 d. nm, while the ATP-SeNPs were 78.6 ± 2.91 d. nm. The SeNPs contain Se, and less persistence of P while the ATP-SeNPs have high level of P, and Se in the energy-dispersive spectroscopy (EDS). Further, both nanoparticles exhibited larger sizes in the dynamic light scattering (DLS) analysis than in the transmission electron microscopy (TEM) analysis. The DLS and Fourier transform infrared spectroscopy (FTIR) results provide evidence that the amine group (–NH2) of ATP might bind with the negatively charged SeNPs through covalent bonding. The IC50 concentration was 17.25 ± 1.16 µg/mL for ATP-SeNPs and 61.24 ± 2.08 µg/mL against the caco-2 cell line. The IC50 results evidenced the higher cytotoxicity of ATP-SeNPs in the caco-2 cell line than in HEK293 cells. ATP-SeNPs trigger the anticancer activity in the caco-2 cell line through the induction of mitochondrial membrane potential (MMP) loss and nucleus damage. The biocompatibility test of hemolysis and the egg CAM assay confirmed the non-toxicity of these nanoparticles. Overall, the results proved that the newly developed ATP-SeNPs exhibited higher cytotoxicity in the caco-2 cell line than SeNPs. However, further molecular and in vivo experiments are required to develop the ATP-SeNPs as a candidate drug for cancer-targeted therapeutics.
... ATP is actively released in the pericellular environment in response to several stimuli, including (1) inflammation-related biological processes, (2) cellular stress cells. We focused our work on CRC because of the somewhat contradictory data available so far [47,48]. Moreover, most of the work published to date is mainly focused on two purinergic receptors (e.g., P2RX7 and P2RY2) and only on a few cell lines [23,[48][49][50][51][52][53][54]. ...
... We focused our work on CRC because of the somewhat contradictory data available so far [47,48]. Moreover, most of the work published to date is mainly focused on two purinergic receptors (e.g., P2RX7 and P2RY2) and only on a few cell lines [23,[48][49][50][51][52][53][54]. In order not to limit our study to selected genes, we first explored gene expression profiles through "RNA-Sequencing" (RNA Seq) technology. ...
... In order not to limit our study to selected genes, we first explored gene expression profiles through "RNA-Sequencing" (RNA Seq) technology. Experiments were performed on cellular extracts prepared from four different CRC cell lines, including the HT29 cells which are likely the most widely characterized colorectal cancer cells in terms of purinergic signaling [48][49][50][51][52]. The four chosen cell lines show distinct genetic and phenotypic profiles and were grown in either 2D or 3D cell culture to determine whether 3D cell organization might impact the expression profiles of the purinergic signaling components. ...
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Article
The purine nucleotide adenosine triphosphate (ATP) is known for its fundamental role in cellular bioenergetics. However, in the last decades, different works have described emerging functions for ATP, such as that of a danger signaling molecule acting in the extracellular space on both tumor and stromal compartments. Beside its role in immune cell signaling, several studies have shown that high concentrations of extracellular ATP can directly or indirectly act on cancer cells. Accordingly, it has been reported that purinergic receptors are widely expressed in tumor cells. However, their expression pattern is often associated with contradictory cellular outcomes. In this work, we first investigated gene expression profiles through "RNA-Sequencing" (RNA Seq) technology in four colorectal cancer (CRC) cell lines (HT29, LS513, LS174T, HCT116). Our results demonstrate that CRC cells mostly express the A2B, P2X4, P2Y1, P2Y2 and P2Y11 purinergic receptors. Among these, the P2Y1 and P2Y2 coding genes are markedly overexpressed in all CRC cells compared to the HCEC-1CT normal-like colonic cells. We then explored the cellular outcomes induced by extracellular ATP and adenosine. Our results show that in terms of cell death induction extracellular ATP is consistently more active than adenosine against CRC, while neither compound affected normal-like colonic cell survival. Intriguingly, while for the P2Y2 receptor pharmacological inhibition completely abolished the rise in cytoplasmic Ca 2+ observed after ATP exposure in all CRC cell lines, Ca 2+ mobilization only impacted the cellular outcome for HT29. In contrast, non-selective phosphodiesterase inhibition completely abolished the effects of extracellular ATP on CRC cells, suggesting that cAMP and/or cGMP levels might determine cellular outcome. Altogether , our study provides novel insights into the characterization of purinergic signaling in CRC.
... To evaluate ER activity 15 in primary hepatocytes under APAP treatment, we examined Ca 2+ release capacity in the ER by using the Grynkiewicz 43 method and a specific FURA-2 labeling. A lack of Cnnm4 led to a more functional hepatic ER with a higher release of Ca 2+ under the different stimuli, including thapsigargin (a specific inhibitor of the ER SERCA pump) 44,45 and ATP (which triggers Ca 2+ release through P2Y receptor) 46 (Fig. 4a) (Supplementary Fig. 5A). ...
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Acetaminophen overdose is one of the leading causes of acute liver failure and liver transplantation in the Western world. Magnesium is essential in several cellular processess. The Cyclin M family is involved in magnesium transport across cell membranes. Herein, we identify that among all magnesium transporters, only Cyclin M4 expression is upregulated in the liver of patients with acetaminophen overdose, with disturbances in magnesium serum levels. In the liver, acetaminophen interferes with the mitochondrial magnesium reservoir via Cyclin M4, affecting ATP production and reactive oxygen species generation, further boosting endoplasmic reticulum stress. Importantly, Cyclin M4 mutant T495I, which impairs magnesium flux, shows no effect. Finally, an accumulation of Cyclin M4 in endoplasmic reticulum is shown under hepatoxicity. Based on our studies in mice, silencing hepatic Cyclin M4 within the window of 6 to 24 h following acetaminophen overdose ingestion may represent a therapeutic target for acetaminophen overdose induced liver injury. Drug induced liver injury (DILI) is an important cause acute liver failure. Here the authors report that serum Mg2+ serum levels decrease in patients with DILI as well as in preclinical animal models treated with acetaminophen overdose, and that early intervention targeting the Mg2+ transporter Cyclin M4 may be beneficial for acetaminophen overdose in preclinical models.
... In turn, ATP and other nucleotides have already been related to increased drug resistance by exerting inflammatory and pro-invasive functions acting on P2 purinergic receptors [14][15][16]. Several types of P2 receptors were shown to be involved in regulating the proliferation of numerous tumor cells, including P2X3, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4 P2Y11, and P2Y12 [17][18][19][20][21][22][23][24]. Specifically, the P2Y12 is a G-protein-coupled receptor that is activated by ADP and is expressed in gliomas by platelets and microglia, besides the tumor [25][26][27]. ...
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Gliomas are the most common malignant brain tumors in adults, characterized by a high proliferation and invasion. The tumor microenvironment is rich in growth-promoting signals and immunomodulatory pathways, which increase the tumor's aggressiveness. In response to hypoxia and glioma therapy, the amounts of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) strongly increase in the extracellular space, and the purinergic signaling is triggered by nu-cleotides' interaction in P2 receptors. Several cell types are present in the tumor microenvironment and can facilitate tumor growth. In fact, tumor cells can activate platelets by the ADP-P2Y12 engagement , which plays an essential role in the cancer context, protecting tumors from the immune attack and providing molecules that contribute to the growth and maintenance of a rich environment to sustain the protumor cycle. Besides platelets, the P2Y12 receptor is expressed by some tumors, such as renal carcinoma, colon carcinoma, and gliomas, being related to tumor progression. In this context , this review aims to depict the glioma microenvironment, focusing on the relationship between platelets and tumor malignancy.
Article
P2Y purinoceptor 6 (P2RY6) is highly expressed in skin keratinocytes, but its function in skin diseases is unclear. We use two-step chemical induction method to induce mouse skin tumor formation. Multiple in vitro and in vivo assays were used to explore the role of P2RY6 in skin tumor. We report that P2ry6-deficient mice exhibit marked resistance to DMBA/TPA-induced skin papilloma formation compared with wild-type mice. Consistent with these findings, epidermal hyperplasia in response to TPA was suppressed in the P2ry6 knockout or MRS2578 (P2RY6 antagonist)-treated mice. The dramatic decrease in hyperplasia and tumorigenesis due to P2ry6 disruption was associated with the suppression of TPA-induced keratinocyte proliferation and inflammatory reactions. Notably, P2ry6 deletion prevented the TPA-induced increase in YAP nuclear accumulation and its downstream gene expression in an MST/LATS1-dependent manner. Upon TPA stimulation, enhanced activation of MEK1 and β-catenin were also impaired in P2ry6 knockout primary keratinocytes, tumor tissues or MRS2578-treated HaCaT cells. Moreover, mutual promotion of the YAP and β-catenin signaling pathways was observed in normal skin cells treated with TPA, while P2ry6 deletion could inhibit their crosstalk by regulating MEK1. Thus, P2RY6 is a critical positive regulator of skin tumorigenesis via modulation of the Hippo/YAP and Wnt/β-catenin signaling pathways.
Article
The human ATP- and UTP-activated P2Y2 receptor (P2Y2R) is a Gq protein-coupled receptor involved in several pathophysiological conditions including acute and chronic inflammation, cancer, and pain. Despite its potential as a novel drug target, only few P2Y2R antagonists have been developed so far, all of which suffer from severe drawbacks. These include (i) high polarity due to one or several negative charges resulting in low oral bioavailability, (ii) metabolic instability and generally poor pharmacokinetic properties, and/or (iii) lack of selectivity, which limits their utility for in vitro and in vivo studies aimed at target validation. In search of new druglike scaffolds for P2Y2R antagonists, we employed a structure-based virtual high-throughput screening approach utilizing the complex of a P2Y2R homology model with one of the most potent and selective orthosteric antagonists described so far, AR-C118925 (10). After virtual screening of 3.2 million molecules, 58 compounds were purchased and pharmacologically evaluated. Several novel antagonist scaffolds were discovered, and their binding modes at the human P2Y2R were analyzed by molecular docking studies. The investigated antagonists likely share a similar binding mode with 10 which includes accommodation of bulky, lipophilic groups in the putative orthosteric binding site of the P2Y2R. The discovered scaffolds and the elucidated structure-activity relationships provide a basis for the development of future drug candidates for the P2Y2R which have great potential as novel drugs.
Article
G-protein-coupled receptors for extracellular nucleotides are known as P2Y receptors and are made up of eight members that are encoded by distinct genes and can be classified into two classes based on their affinity for specific G-proteins. P2Y receptor modulators have been studied extensively, but only a few small-molecule P2Y receptor antagonists have been discovered so far and approved by drug agencies. Derivatives of indole carboxamide have been identified as P2Y12 and P2X7 antagonist, as a result, we developed and tested a series of indole derivatives4a-lhaving thiourea moiety as P2Y receptor antagonist by using a fluorescence-based assay to measure the inhibition of intracellular calcium release in 1321N1 astrocytoma cells that had been stably transfected with the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Most of the compounds exhibited moderate to excellent inhibition activity against P2Y1 receptor subtype. The series most potent compound, 4h exhibited an IC50 value of 0.36 ± 0.01 µM selectivity against other subtypes of P2Y receptor. To investigate the ligand-receptor interactions, the molecular docking studies were carried out. Compound 4h is the most potent P2Y1 receptor antagonist due to interaction with an important amino acid residue Pro105, in addition to Ile108, Phe119, and Leu102.
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Prostate cancer is a heterogeneous, slow growing asymptomatic cancer that predominantly affects man. A purinergic G-protein coupled receptor, P2Y1R, is targeted for its therapeutic value since it plays a crucial role in many key molecular events of cancer progression and invasion. Our previous study demonstrated that indoline derivative, 1 (1-(2-Hydroxy-5-nitrophenyl) (4-hydroxyphenyl) methyl)indoline-4-carbonitrile; HIC), stimulates prostate cancer cell (PCa) growth inhibition via P2Y1R. However, the mode of interaction of P2Y1R with HIC involved in this process remains unclear. Here, we have reported the molecular interactions of HIC with P2Y1R. Molecular dynamics simulation was performed that revealed the stable specific binding of the protein-ligand complex. In vitro analysis has shown increased apoptosis of PCa-cells, PC3, and DU145, upon specific interaction of P2Y1R-HIC. This was further validated using siRNA analysis that showed a higher percentage of apoptotic cells in PCa-cells transfected with P2Y-siRNA-MRS2365 than P2Y-siRNA-HIC treatment. Decreased mitochondrial membrane potential (MMP) activity and reduced glutathione (GSH) level show their role in P2Y1R-HIC mediated apoptosis. These in silico and in vitro results confirmed that HIC could induce mitochondrial apoptotic signalling through the P2Y1R activation. Thus, HIC being a potential ligand upon interaction with P2Y1R might have therapeutic value for the treatment of prostate cancer.
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As one member of G protein-coupled P2Y receptors, P2Y2 receptor can be equally activated by extracellular ATP and UTP. Our previous studies have proved that activation of P2Y2 receptor by extracellular ATP could promote prostate cancer cell invasion and metastasis in vitro and in vivo via regulating the expressions of some epithelial-mesenchymal transition/invasion-related genes (including IL-8, E-cadherin, Snail and Claudin-1), and the most significant change in expression of IL-8 was observed after P2Y2 receptor activation. However, the signaling pathway downstream of P2Y2 receptor and the role of IL-8 in P2Y2-mediated prostate cancer cell invasion remain unclear. Here, we found that extracellular ATP/UTP induced activation of EGFR and ERK1/2. After knockdown of P2Y2 receptor, the ATP -stimulated phosphorylation of EGFR and ERK1/2 was significantly suppressed. Further experiments showed that inactivation of EGFR and ERK1/2 attenuated ATP-induced invasion and migration, and suppressed ATP-mediated IL-8 production. In addition, knockdown of IL-8 inhibited ATP-mediated invasion and migration of prostate cancer cells. These findings suggest that P2Y2 receptor and EGFR cooperate to upregulate IL-8 production via ERK1/2 pathway, thereby promoting prostate cancer cell invasion and migration. Thus blocking of the P2Y2-EGFR-ERK1/2 pathway may provide effective therapeutic interventions for prostate cancer.
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P2 receptors are membrane-bound receptors for extracellular nucleotides such as ATP and UTP. P2 receptors have been classified as ligand-gated ion channels or P2X receptors and G protein-coupled P2Y receptors. Recently, purinergic signaling has begun to attract attention as a potential therapeutic target for a variety of diseases especially associated with gastroenterology. This study determined the ATP and UTP-induced receptor signaling mechanism in feline esophageal contraction. Contraction of dispersed feline esophageal smooth muscle cells was measured by scanning micrometry. Phosphorylation of MLC20 was determined by western blot analysis. ATP and UTP elicited maximum esophageal contraction at 30 s and 10 μM concentration. Contraction of dispersed cells treated with 10 μM ATP was inhibited by nifedipine. However, contraction induced by 0.1 μM ATP, 0.1 μM UTP and 10 μM UTP was decreased by U73122, chelerythrine, ML-9, PTX and GDPβS. Contraction induced by 0.1 μM ATP and UTP was inhibited by Gαi3 or Gαq antibodies and by PLCβ1 or PLCβ3 antibodies. Phosphorylated MLC20 was increased by ATP and UTP treatment. In conclusion, esophageal contraction induced by ATP and UTP was preferentially mediated by P2Y receptors coupled to Gαi3 and Gαq proteins, which activate PLCβ1 and PLCβ3. Subsequently, increased intracellular Ca2+ and activated PKC triggered stimulation of MLC kinase and inhibition of MLC phosphatase. Finally, increased pMLC20 generated esophageal contraction.
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Extracellular nucleotides, via activation of P2 purinergic receptors, influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G-protein coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. In order to evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild type (WT) and P2Y2 purinergic receptor knockout (P2Y2-/-) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1), and activator protein-1 (AP-1) DNA-binding activity (30 min) as well as subsequent hepatocyte proliferation (24-72 hr) in response to 70% PH were impaired in P2Y2-/- mice. Interestingly, early induction of cytokines (TNFα, IL-6) and cytokine-mediated signaling (NF-kB, STAT-3) were intact in P2Y2-/- remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from WT and P2Y2-/- livers were treated with ATP for 5-120 min and 12-24 hr. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH.
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Interleukin-8 (IL-8) plays key roles in both chronic inflammatory diseases and tumor modulation. We previously observed that IL-8 secretion and function can be modulated by nucleotide (P2) receptors. Here we investigated whether IL-8 release by intestinal epithelial HT-29 cells, a cancer cell line, is modulated by extracellular nucleotide metabolism. We first identified that HT-29 cells regulated adenosine and adenine nucleotide concentration at their surface by the expression of the ectoenzymes NTPDase2, ecto-5′-nucleotidase, and adenylate kinase. The expression of the ectoenzymes was evaluated by RT-PCR, qPCR, and immunoblotting, and their activity was analyzed by RP-HPLC of the products and by detection of P i produced from the hydrolysis of ATP, ADP, and AMP. In response to poly (I:C), with or without ATP and/or ADP, HT-29 cells released IL-8 and this secretion was modulated by the presence of NTPDase2 and adenylate kinase. Taken together, these results demonstrate the presence of 3 ectoenzymes at the surface of HT-29 cells that control nucleotide levels and adenosine production (NTPDase2, ecto-5′-nucleotidase and adenylate kinase) and that P2 receptor-mediated signaling controls IL-8 release in HT-29 cells which is modulated by the presence of NTPDase2 and adenylate kinase.
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Tumor microenvironmental hypoxia induces hypoxia inducible factor-1α (HIF-1α) overexpression, leading to the release of lysyl oxidase (LOX), which crosslinks collagen at distant sites to facilitate environmental changes that allow cancer cells to easily metastasize. Our previous study showed that activation of the P2Y2 receptor (P2Y2R) by ATP released from MDA-MB-231 cells increased MDA-MB-231 cell invasion through endothelial cells. Therefore, in this study, we investigated the role of P2Y2R in breast cancer cell metastasis to distant sites. ATP or UTP released from hypoxia-treated MDA-MB-231 cells induced HIF-1α expression and LOX secretion by the activation of P2Y2R, and this phenomenon was significantly reduced in P2Y2R-depleted MDA-MB-231 cells. Furthermore, P2Y2R-mediated LOX release induced collagen crosslinking in an in vitro model. Finally, nude mice injected with MDA-MB-231 cells showed high levels of LOX secretion, crosslinked collagen and CD11b+ BMDC recruitment in the lung; however, mice that were injected with P2Y2R-depleted MDA-MB-231 cells did not exhibit these changes. These results demonstrate that P2Y2R plays an important role in activation of the HIF-1α-LOX axis, the induction of collagen crosslinking and the recruitment of CD11b+ BMDCs. Furthermore, P2Y2R activation by nucleotides recruits THP-1 monocytes, resulting in primary tumor progression and pre-metastatic niche formation.
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Extracellular nucleotides are released and detectable in a high concentration within the tumor microenvironment. G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is activated equipotently by adenosine triphosphate (ATP) and uridine 5′-triphosphate (UTP), which mediate proinflammatory responses such as cell migration and proliferation. However, the role of P2Y2R in the process of cancer metastasis remains unclear. This study aimed to determine the role of P2Y2R in the proliferation, migration and invasion of highly metastatic MDA-MB-231 breast cancer cells through crosstalk with endothelial cells (ECs). ATP release and P2Y2R activity between high metastatic breast cancer cell MDA-MB-231 and low metastatic breast cancer cell MCF-7 were compared. Then, the role of P2Y2R on tumor growth and invasion via crosstalk with ECs was examined in vitro, using MDA-MB-231 cells and ECs transfected with control- or P2Y2R-siRNA, and in vivo, using an animal model injected with control-shRNA- or P2Y2R-shRNA-transfected MDA-MB-231 cells. We found that this highly metastatic breast cancer cell line released higher levels of ATP and showed a higher P2Y2R activity in comparison to a low metastatic breast cancer cell line, MCF-7. In MDA-MB-231 cells, P2Y2R activation by ATP or UTP increased proliferation at 24 or 72 hours, which was abolished by P2Y2R knock-down. In addition, the adhesion of MDA-MB-231 cells to ECs and cell migration were both significantly increased by ATP or UTP through the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in MDA-MB-231 or ECs but not in cells where P2Y2R was knocked down. Furthermore, ATP- or UTP-mediated activation of P2Y2R induced MDA-MB-231 invasion through ECs, increased matrix metalloproteinase-9 (MMP-9) activity and vascular endothelial growth factor (VEGF) production in MDA-MB-231 and induced the phosphorylation of vascular endothelial (VE)-cadherin in ECs. Tumor growth and metastasis to other tissues were dramatically reduced, and body weight was increased in mice injected with P2Y2R-shRNA-transfected MDA-MB-231 cells compared to mice injected with control shRNA-transfected MDA-MB-231 cells. This study suggests that P2Y2R may play an important role in cancer metastasis via modulation of the crosstalk between cancer cells and ECs.
Article
Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Pro-apoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the anti-apoptotic protein Bcl-2 demonstrated more sensitivity to ATP than pro-apoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression.
Article
Tumor metastasis is considered the main cause of mortality in cancer patients, thus it is important to investigate the differences between high- and low-metastatic cancer cells. Our previous study showed that the highly metastatic breast cancer cell line MDA-MB-231 released higher levels of ATP and exhibited higher P2Y2R activity compared with the low‑metastatic breast cancer cell line MCF-7. In addition, P2Y2R activation by ATP released from MDA-MB-231 cells induced hypoxia-inducible factor-1α expression, lysyl oxidase secretion and collagen crosslinking, generating a receptive microenvironment for pre-metastatic niche formation. Thus, in the present study, we investigated which P2Y2R-related signaling pathways are involved in the invasion of breast cancer cells. The highly metastatic breast cancer cells MDA-MB-231 and SK-BR-3 showed higher invasion than MCF-7 and T47D cells at a basal level, which was abolished through P2Y2R knockdown or in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides. MDA-MB‑231 cells also showed high levels of mesenchymal markers, such as Snail, Vimentin and N-cadherin, but not the epithelial marker E-cadherin and this expression was inhibited through ATP degradation or P2Y2R knockdown. Moreover, SK-BR-3 and MDA-MB231 cells exhibited higher ERK and PKC phosphorylation levels than T47D and MCF-7 cells and upregulated phospho-ERK and -PKC levels in MDA-MB-231 cells were significantly downregulated by apyrase or P2Y2R knockdown. Specific inhibitors of ERK, PKC and PLC markedly reduced the invasion and levels of mesenchymal marker expression in MDA-MB-231 cells. These results suggest that over-activated ERK and PKC pathways are involved in the P2Y2R-mediated invasion of breast cancer cells.