Key words NSAIDs, angiogenesis, prostaglandin E2, EP3 receptor, tumor ... via EP3 receptors which exists on the stroma but on tumor cells. An EP3 receptor ...
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Review Article Prostanoid receptor in tumor-associated angiogenesis and tumor growth 炎症・再生 Vol.23 No.1 2003
Review Article Prostanoid receptor in tumor-associated angiogenesis and tumor growth Hideki Amano1), Izumi Hayashi2), Hirokuni Yoshimura1) and Masataka Majima2) Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase (COX) enzyme and suppress PG synthesis which are used commonly as anti-inflammatory, anti-pyretic and analgestic agents. Though NSAIDs is known to suppress incidence and progression of cancer especially colorectal cancer, the precise mechanism of their protective effect remain unknown. A wide range of mechanism about anti-tumor effect of NSAIDs have been reported. Some of them are unrelated to the inhibition of COX activity and subsequent PG formation. However, recent result from by using knockout mice and selective antagonists indicated that prostanoid receptor, especially PGE2 enhances angiogenesis and tumor growth. Here, we summarize significant of PGE2 signaling via EP3 receptors which exists on the stroma but on tumor cells. An EP3 receptor antagonist may be a candidate of chemopreventive agents for malignant tumor. Rec.12/14/2004, pp26-31 1)
Department of Thoracic and Cardiovascular Surgery, 2) Department of Pharmacology Kitasato University School of Medicine
Key w ords NSAIDs, angiogenesis, prostaglandin E2, EP3 receptor, tumor wo
Introduction
inhibitor were expected to be super aspirin that would not
Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the
exhibit the adverse effects typical of classical NSAIDs. It has
enzyme cyclooxygenase (COX-2) and suppress PG synthesis
been reported that some organs, such as the kidney, express
including PGE2 and PGI2 have been used as anti-inflammatory,
constitutively and that COX-2 is necessary for the kidney to
antipyretic and analgesic agents. Recent population-based
mature after birth. Thus, selective inhibition of prostanoid
studies reveal a 40-50% reduction in mortality from colorectal
receptor signaling might be a more effective form of treatment
cancer in individual taking NSAIDs, and evidence suggests
than COX-2 inhibition. Despite the efficacy of NSAIDs as
that they also affect the incidence and progression of other type
anticancer agents, the precise mechanism of their protective
of cancer. These observation are pointing to a possible role of
effective is unknown. In article, we discuss anti-cancer effect
COX in tumor formation.
of prostanoid receptor by using different prostanoid receptor
Two COX isoforms have been identified. COX-1 is consti-
knockout mice.
tutively expressed in various tissues, whereas COX-2 is induced by mitogen, cytokines, and tumor promoters. Selective COX-2
Inflammation and Regeneration Vol.25 No.1 JANUARY 2005
A
C
27
B
D
E Fig.1 Effect of COX-2 inhibitors on tumor growth A: Vehicle(Saline), B: SC560 (COX-1 inhibitor), C: NS-398 (COX-1 inhibitor), D: JTE-522 (COX-2 inhibitor), E: Aspirin (From Reference 23. with permission)
The Effect of NSAIDs on angiogenesis
the generation of colon tumors was verified genetically in mice
Angiogenesis contributes to various physiological processes,
with compound mutations in the adenomatous polyposis
including embryonic development, female reproductive cycling,
(APC) and COX-2 genes10). However, the mechanism by which
and wound repair, as well as to pathological conditions such as
inhibition of COX results in a reduction in the initiation and
tumor growth, ischemic heart desease, diabetic retinopathy, and
progression of cancer, or, more precisely, whether and if so how
1-5)
rheumatoid arthritis . Angiogenesis itself is a complex multi-
PGs contribute to these processes, remains unclear. With the use
step process that is regulated by a variety of bioactive agents.
of an in vitro system, Dubois and colleagues showed that PGE2
Prostaglandins (PGs) have been suggested also to participate
inhibits apoptosis of, and increases the tumorigenic potential
in this process. PGs comprise a group of oxygenated metabo-
of, colonic epithelial cells, and they suggested that inhibition
lites of arachidonic acid that are produced by the sequential
of apoptosis may contribute to tumor promotion by PGs11).
actions of cyclooxygenase (COX) and specific synthases, and
Angiogenesis is an important factor in tumor development.
they include PGD2, PGE2, PGF2α, PGI2, and thromboxane A2.
Angiogenesis associated with cancer is mediated by the migra-
Two COX isoforms have been identified: COX-1 is constitu-
tion and proliferation of host endothelial cells. With the use
tively expressed in various tissues, whereas COX-2 is induced
of a coculture system comprising endothelial cells and colon
6-8)
by mitogens, cytokines, and tumor promoters . Nonsteroidal
carinoma cells that overexpress COX-2, Tsujii et al showed that
antiinflammatory drugs (NSAIDs) that inhibit COX also in-
the increased production of PGs by the carcinoma cells was
hibit angiogenesis both in vivo in experimental animals and in
associated with endothelial cell migration and tube formation,
vitro in endothelial cell cultures, and exogenously applied PGs
suggesting a role for COX-2 in tumor-induced angiogenesis12).
induce proliferation of vascular endothelial cells both in vitro
However, the relevance of these in vitro observations to tumori-
and in vivo. These observations suggest that PGs may play an
genesis in vivo remains to be established. Furthermore, whether
important role in the regulation of neovascularization. However,
prostanoids actually contribute to the observed tumor-induced
the identities of the PGs and the PG receptors that mediate such
angiogenesis, and, if so, the identity of the responsible PGs and
regulation remain unknown, and the significance of PG action
PG receptors, remains unknown.
in angiogenesis under various pathological conditions is also
For mechanistic analysis of angiogenesis in vivo, we have
unclear. NSAIDs reduce mortality associated with colorectal
developed a sponge implantation model, in which a polyurethane
cancer, and evidence suggests that they also affect the incidence
sponge disk implanted subcutaneously in rats induces extensive
9)
and progression of other types of cancer . A role for COX-2 in
angiogenesis in surrounding proliferative granuloma tissue.
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Review Article Prostanoid receptor in tumor-associated angiogenesis and tumor growth 炎症・再生 Vol.23 No.1 2003
Fig.2 Effect of angiogenesis after injection of EP agonists (From Reference 23. with permission)
With the use of this model, we have previously shown that
quantified by measurement of the hemoglobin content of the
angiogenesis both occurs concomitantly with the induction of
sponge induced granulation tissue together with histological
COX-2 mRNA and is inhibited by administration either of a
examination and that the effect of exogenous substances can
nonselective NSAID (indomethacin) or of selective COX-2
be investigated by their direct injection into the sponge22).
inhibitors13). We further showed that PGE2 or the PGI2 analog
Compared to other EP agonists (EP1,EP2,EP4), EP3 agonist
beraprost topically injected into the sponge promoted angio-
ONO-AE-248 specifically enhanced angiogenesis in this model
genesis. Angiogenesis induced by either endogenous COX-2 or
in a dose-dependent manner, and further the angiogenic response
exogenous PGs was accompanied by increased expression of
was certainly reduced in EP3-/- with the reduction of VEGF
vascular endothelial growth factor (VEGF), and angiogenesis
expression, but not in IP-receptor. These result suggested that
was abolished by administration of an antisense oligonucleotide
endogenous PGE2 facilitates angiogenesis through the EP3
specific for VEGF mRNA. These results suggest that either
signaling and the induction VEGF in this model23). In this model,
PGE2 or PGI2 may mediate the angiogenic action of COX-2 in
we had already reported that PGE2 induced activation of ade-
situ.
nylate cyclase / protein kinase A signaling pathway22). One of the EP3 splicing variants receptor, which elevates intracellular
The prostanoid receptor subtype in angiogenesis
cAMP levels, may be involved in enhance angiogenesis.
PGs exert their biological actions by binding to specific different PG receptors have been defined pharmacologically
Host stromal prostanoid receptor signaling contributes to tumor-growth and angiogenesis
and cloned, including the PGD receptor (DP), four subtypes of
We have shown that Sarcoma-180 implanted mice were
PGE receptor (EP1, EP2, EP3, EP4), the PGF receptor (FP),
inhibited tumor growth and associated angiogenesis by using
receptors that contain seven transmembrane domains. Eight
14)
the PGI receptor (IP), and the thromboxane receptor (TP) .
COX-2 inhibitor, but not COX-1. COX-2 inhibitor blocked
Genes for each of these receptors have been disrupted and the
tumor growth by about 80% similar to the extent of the effect
15-19)
. Fur-
of such inhibitors on the growth of COX-2 overexprssing tumor
thermore, with the use of the cloned receptors, agonists and
cells transplanted into nude mice. These results indicated that
antagonists highly selective for each of the four EP subtypes
PGs is essential for tumor growth and angiogenesis. What type
corresponding knockout mice have been produced
20-21)
have been or are in the process of being developed
.
of PG is most important for tumor-associated growth and
To mimic stromal angiogenic response, we developed the
angiogenesis?
sponge implantation model. Two advantages of this model for
We tested tumor-associated angiogenesis by using knock-
studies of angiogenesis are that angiogenesis can be readily
out mice of EP subtypes and IP receptor. Tumor-associated
Inflammation and Regeneration Vol.25 No.1 JANUARY 2005
29
angiogenesis in EP3-receptor -/- mice is suppressed by ∼80% and partial reduction of angiogenesis is observed in EP2receptor -/- mice. Histological examination of tumor in EP3receptor -/- mice reveals a low level of vascularization and distinct boundaries with the surrounding normal tissues. We identified that the cells which produced VEGF to facilitate angiogenesis and tumor growth were CD3 and Mac-1 double negative fibrobrast. In immunohistochemical study, host stromal cells expressed VEGF in wild type but not in EP3-receptor -/mice. These result suggested that these host stromal cell express VEGF in response to activation of EP3 receptors by endogenous PGE2. Gel shift assay revealed that activator protein (AP-1) might be important in the expression of VEGF and angiogenesis, although other transcript factor such as HIF-1, whose activation is related to EP1, EP2 and EP4 receptor mediated signaling, have been ruled out. It is widely accepted that the host microenviromental induces tumor progression. In addition, PGs might be one of the important regulator of tumor-host communication. The study in EP3-/- knockout mice reveale d that either stromal cells around tumors express COX-2 or tumor cells themselves (or both) synthesized release PGE2 into the tumor microenviroment, which acts on the stromal cell that contain EP3 receptors to induce the production of pro-angiogenetic factors and consequent angiogenesis.
Prostanoid receptor antagonist has possibility to preventive approach for cancer and the control of inflammatory response and tumor angiogenesis Highly selective EP3 and EP2 receptor antagonists exhibit
Fig.3 Tumor growth and angiogenesis in prostanoid knockout mice A: Microvessel density, B: weight of tumor, C: Hemoglobin contents *p < 0.05 (From Reference 23. with permission)
beneficial effect on stromal cells might be a good choice as novel therapeutic tools against cancer. Daily topical injection of EP3 antagonist to the subcutaneous tissues around the tumor,
including fibroblast, which related to angiogenesis and
where functionally active EP3 receptor localized significantly
lymphogenesis. The stromal fibroblast might be derived from
suppressed tumor-associated angiogenesis and tumor growth
the bone marrow and from tumor associated macrophages, such
in wild type mice, whereas those of an EP1 and EP4 antagonist
as polarized M2 mononuclear phagocytes. Recetnly, tumor
did not. These results certainly confirm the result from EP3-
angiogenesis has been targeted by transplanting genetically
receptor -/- mice. Therefore, a highly selective EP3 receptor
modified hematopoietic stem cell. Controlling inflammatory,
antagonists has a preventive action, on the stromal cells and is
responses in the tumor microenvironment in area in which cells
expected to become a novel therapeutic against cancer.
that express EP receptors accumulate is also likely to be a novel
Recent paper shows that inflammation is an important factor
therapeutic approach to cancer, which now caeses ∼ 55000
in facilitating tumor growth, and many cancer arise from the
deaths worldwide each years.
sites of infection, chronic inflammation. The tumor stromal reaction can be characterized by the proliferation of cells,
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Review Article Prostanoid receptor in tumor-associated angiogenesis and tumor growth 炎症・再生 Vol.23 No.1 2003
EP3 WT EP3 KO
COX-2
Fig.4 Expression of COX-2 and VEGF in tumors and surrounding stroma tissues in WT mice and EP3 knockout mice
VEGF
(From Reference 23. with permission)
Fig.6 Schematic drawing of the major signaling pathway of PGE2 generated through the action of COX-2 (From Reference 24. with permission)
Fig.5 Effect of the EP3 antagonists on tumor growth and tumor angiogenesis A: Microvessel density, B: weight of tumor, C: Hemoglobin contents *p < 0.05 (From Reference 23. with permission)
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