Download Antiangiogenic and Antitumor Activities of

[CANCER RESEARCH 60, 1306 –1311, March 1, 2000]
Antiangiogenic and Antitumor Activities of Cyclooxygenase-2 Inhibitors
Jaime L. Masferrer,1 Kathleen M. Leahy, Alane T. Koki, Ben S. Zweifel, Steven L. Settle, B. Mark Woerner,
Dorothy A. Edwards, Amy G. Flickinger, Rosalyn J. Moore, and Karen Seibert
G. D. Searle/Monsanto Company, St. Louis, Missouri 63167
ABSTRACT
We provide evidence that cyclooxygenase (COX)-2-derived prostaglandins contribute to tumor growth by inducing newly formed blood vessels
(neoangiogenesis) that sustain tumor cell viability and growth. COX-2 is
expressed within human tumor neovasculature as well as in neoplastic
cells present in human colon, breast, prostate, and lung cancer biopsy
tissue. COX-1 is broadly distributed in normal, as well as in neoplastic,
tissues. The contribution of COX-2 to human tumor growth was indicated
by the ability of celecoxib, an agent that inhibits the COX-2 enzyme, to
suppress growth of lung and colon tumors implanted into recipient mice.
Mechanistically, celecoxib demonstrated a potent antiangiogenic activity.
In a rat model of angiogenesis, we observe that corneal blood vessel
formation is suppressed by celecoxib, but not by a COX-1 inhibitor. These
and other data indicate that COX-2 and COX-2-derived prostaglandins
may play a major role in development of cancer through numerous
biochemical mechanisms, including stimulation of tumor cell growth and
neovascularization. The ability of celecoxib to block angiogenesis and
suppress tumor growth suggests a novel application of this anti-inflammatory drug in the treatment of human cancer.
enzyme in cancer cells suggests a predominant role for COX-2 in
tumor regulation and angiogenesis. In this study, we evaluated the
expression of COX-2 in different types of human cancers and, in
particular, the expression of COX-2 in tumor angiogenesis. Using
inhibitors of COX-2 and COX-1, we demonstrated that COX-2derived PGs mediate tumor growth and metastasis in two independent
animal models and in FGF-2-induced angiogenesis. Celecoxib demonstrated potent antiangiogenic and antitumor activity in vivo, suggesting the potential use of this anti-inflammatory drug in the treatment of human cancer.
MATERIALS AND METHODS
Immunohistochemistry Analysis. Cancer tissues were obtained from archival samples from Weill Medical College of Cornell University (New York,
NY) and from Washington University (St. Louis, MO). The study was approved by the Committees on Human Rights in Research at these institutions.
Paraffin-embedded sections were cut into 4-micron sections, mounted onto
polylysine-coated slides, dewaxed in xylene, rehydrated in alcohol, and
blocked for endogenous peroxidase (3% H2O2 in methanol) and avidin/biotin
(Vector Blocking Kit). Sections were treated with TNB-BB [0.1 M Tris (pH
INTRODUCTION
7.5), 0.15 M NaCL, 0.5% blocking agent, 0.3% Triton-X, 0.2% saponin], and
Inflammatory mediators such as cytokines, eicosanoids, and growth incubated with 1:500 dilution of COX-2-specific antibody (PG-27; Oxford
factors are thought to play a critical role in the initiation and main- Biomedical Research Inc.). Specificity of the antibody was determined by
tenance of cancer cell survival and growth (1). One of these media- using control sections that were incubated with antiserum in the presence of a
tors, PGE22, is produced in large amounts by tumors. PGE2 is pro- 100-fold excess of human recombinant COX-2 protein, or with isotypeduced from arachidonic acid by either of two enzymes: COX-1 or matched IgG normal rabbit serum. Immunoreactive complexes were detected
using tyramide signal amplification (TSA-indirect) and visualized with the
COX-2. COX-1 is constitutively expressed in most tissues, whereas
peroxidase substrate, AEC. Slides were counter stained with hematoxylin.
COX-2 is induced in inflammatory cells and in human tumors by
All slides were evaluated by a minimum of two board certified pathologists,
cytokines and tumor promoters (2–5). Both COX isozymes can be one of whom specializes in the specific cancer type.
inhibited by traditional NSAIDs, such as aspirin and indomethacin.
Lewis Lung Carcinoma. Lewis Lung Carcinoma cells (1 ⫻ 106) were
Several studies show that regularly taking aspirin or other conven- implanted into the paws of C57/Bl6 male mice and divided into five treatment
tional NSAIDs provides a 40 –50% reduction in relative risk of death groups of 20 animals each. Group 1 received normal chow, whereas the other
by colon cancer, indicating that inhibition of COX in humans has a groups received celecoxib in the diet, from date of implant until end of study,
chemopreventive effect (6). In rodent models of FAP, a genetic at doses between 160-3200 ppm. All of the animals in the control group
disease leading to colon carcinoma, blockade of COX-2, either by developed tumors reaching the size of 1.5–2.0 ml in approximately 35 days.
gene deletion or by pharmacological inhibition of enzyme activity, Tumor volume was determined biweekly using a plethysmometer. At the end
of the study, the animals were sacrificed and the tumor and lungs were excised.
suppresses intestinal polyp formation (7). COX-2 inhibition also demAnalysis of lung metastasis was done in all of the animals by counting surface
onstrates chemopreventive activity against colon carcinogenesis (8). metastatic lesions under stereomicroscope and verified by histochemical analTaken together, these data provide strong evidence for the importance ysis of consecutive lung sections.
of COX-2 enzyme activity in oncogenesis.
HT-29 Human Colon Tumor in Nude Mice. HT-29 human colon carciOne of the mechanisms by which PGE2 supports tumor growth is noma cells were also implanted in the hind paws (1 ⫻ 106 cells) of nude mice
by inducing the angiogenesis necessary to supply oxygen and nutri- (n ⫽ 15/treatment group). Celecoxib therapy was initiated in the diet at doses
ents to tumors ⬎2 mm in diameter (9, 10). We have observed COX-2 between 160-1600 ppm when tumors reached a mean volume of 100 mm3 and
expression in newly formed blood vessels within tumors grown in maintained for the duration of the experiment. At the end of the experiment,
animals, whereas under normal physiological conditions the quiescent the lungs were excised, fixed in Strecks (Strecks Laboratory, Omaha, NE), and
vasculature expresses only the constitutive COX-1 enzyme (11). This the total number of neoplastic nodules present in the lung surface were counted
as described previously.
novel observation together with studies showing the expression of the
Corneal Angiogenesis Model. An intrastromal pocket was surgically created in the cornea of an anesthetized rat. A slow release hydron/sucralfate
Received 7/13/99; accepted 1/7/00.
pellet containing either 100 ng of FGF-2 or saline (placebo) was inserted into
The costs of publication of this article were defrayed in part by the payment of page
the pocket approximately 1.4 mm from the temporal corneal limbus. The
charges. This article must therefore be hereby marked advertisement in accordance with
pocket was self-sealing, and antibiotic ointment was placed on the eye to
18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom requests for reprints should be addressed, at G. D. Searle/Monsanto
prevent infection. COX inhibitors were administered by gavage in a 0.5-ml
Company, T3G, 800 North Lindbergh Boulevard, St. Louis, MO 63167. Phone: (314)
suspension of 0.5% methylcellulose (Sigma Chemical Co., St. Louis, MO),
694-8950; Fax: (314) 694-3415; E-mail: [email protected].
0.025% Tween 20 (Sigma Chemical Co.) twice daily at 12-h intervals, begin2
The abbreviations used are: PGE2, prostaglandin E2; NSAID, nonsteroidal antining the day before surgery and continuing the length of the study. Four days
inflammatory drug; COX, cyclooxygenase; FGF-2, basic fibroblast growth factor; FAP,
after surgery, the corneas were examined under a slit lamp microscope and the
familial adenomatous polyposis.
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COX-2 INHIBITION: ANTITUMOR AND ANTIANGIOGENIC EFFECTS
neovascular response was quantified by measuring the average new vessel
length (VL), the corneal radius (r ⫽ 2.6 mm), and the contiguous circumferential zone (CH ⫽ clock hours where 1 CH ⫽ 30 degrees), and applied to the
formula: area (mm2)⫽ (CH/12 ⫻ 3.14(r2-(r-VL) (2). All animal treatment
protocols were reviewed by and were in compliance with Monsanto’s Institutional Animal Care and Use Committee.
Statistics. The data are expressed as the mean ⫹/⫺ SEM. Student’s and
Mann-Whitney tests were used to assess differences between means using the
InStat software package. Significance was accepted at P ⬍ 0.05.
RESULTS
Immunohistological Localization Of COX-2 in Human Tumor
Angiogenesis. We used a highly sensitive immunohistochemical
method to carefully examine incidence and distribution of COX-2 in
the major human cancers, including its expression in tumor angiogenesis. Immunohistological analysis revealed COX-2 is consistently
expressed in the neoplastic lesions in colon (17 COX-2-positive
neoplastic colons/20 neoplastic colons examined), prostate (20 of 20),
lung (19 of 20), and breast (16 of 18) cancers (Fig. 1). Moderate-to-
strong COX-2 expression was detected in approximately 40 – 80% of
the total neoplastic cells in most tumors, and moderately and welldifferentiated carcinomas showed significantly higher COX-2 immunoreactivity than poorly differentiated cases. In addition to expression
of COX-2 within neoplastic cells per se, COX-2 was also detected in
the angiogenic vasculature present within the tumors and preexisting
vasculature adjacent to cancer lesions (Fig. 1). In contrast, COX-2 was
not detected in normal colonic epithelium or stroma, with the exception of a small number of colonic crypt epithelial cells in which the
enzyme was weakly detected (Fig. 2A). Interestingly, during colon
oncogenesis COX-2 is expressed not only in the neoplastic adenomas
in patients with FAP (Fig. 2B), in colon cancer (Fig. 1), and robustly
in metastatic lesions in liver (Fig. 2C), but also in the neovasculature
associated with the adenomas (Fig. 2B), colonic lesions (Fig. 1), and
metastatic liver lesions (Fig. 2D). The expression of COX-2 in the
neovasculature of human tumors seems to be a general characteristic
of epithelial tumors. In addition to the cancers mentioned above,
COX-2 was observed in the angiogenic vessels in most of the human
cancers analyzed thus far, including head and neck and pancreas (12,
Fig. 1. Immunohistological localization of COX-2 in human
cancer. Representative pictures of tissues obtained from 20
patients with cancer of the colon, lung, breast, and prostate were
analyzed for the expression of COX-2 using specific antibodies.
COX-2-positive stain was observed in 85% of colon, 100% of
prostate, 95% of lung, and 88% of breast samples analyzed. It is
important to indicate that not all cancer cells express COX-2. In
fact, for colon and lung cancers between 40% and 70% of the
cancer cells express COX-2. In the cases of prostate and breast,
between 5% and 70% of the cancer cells are COX-2 positive.
COX-2-positive stain was found in at least 50% of the samples
containing angiogenic blood vessels. COX-2 expression is
clearly seen (red) in neoplastic epithelium and in some inflammatory cells present within the stroma. COX-2 was also detected in the endothelial cells (red) forming the angiogenic
vasculature present within the tumors (arrows).
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COX-2 INHIBITION: ANTITUMOR AND ANTIANGIOGENIC EFFECTS
Fig. 2. COX-2 expression in colon oncogenesis.
A, COX-2 is generally not found in normal tissues,
with the exception of weak expression in some colonic crypt epithelial cells (red, small arrows). B,
COX-2 is clearly expressed in the adenomas of FAP
patients (red, FAP arrows) and in the blood vessels
associated with the adenomas (red, BV arrows). C,
striking homogeneity and robust levels of COX-2
immunoreactivity were detected in metastatic lesions in liver in eight of eight colon cancer patients
with metastatic disease (red). D, COX-2 was also
clearly detected in the vasculature within metastatic
tissues and adjacent to neoplastic clusters (red).
Similar results were observed in patients with pancreatic cancer, cholangiocarcinoma, and Kaposi’s
sarcoma.
13). These results imply COX-2 may play a functional role in tumorinduced angiogenesis and subsequent progression to metastatic disease and may partially explain the potent antimetastatic activity of
COX-2 inhibitors observed in rodent models.
In contrast to the COX-2 expression in human cancer tissues,
COX-1 is expressed in both normal and neoplastic regions in all
tissues, and seemed to be particularly expressed in the tumor stroma
that included fibroblasts, smooth muscle cells, and the vasculature.
The broad distribution of this isoform in both normal and neoplastic
tissues makes it difficult to determine whether changes in COX-1
expression occurred during tumorigenesis (14, 15).
Inhibition of Tumor Growth and Lung Metastasis by Celecoxib
in Mice. The COX-2 inhibitor celecoxib, a 1,5 diarylpyrazole with
⬎300-fold selectivity for COX-2 versus COX-1 (16, 17) in vitro, was
tested in two animal models of lung and colon cancer to evaluate its
effect on tumor growth and lung metastases. Lewis Lung carcinoma
cells implanted into the right paws of a series of 20 C57/B16 male
mice develop tumors reaching the size of 1.5–2.0 cm3 in approximately 35 days. Celecoxib supplied in the diet continuously from date
of implant at doses between 160-3200 ppm significantly retarded the
growth of these primary tumors (Fig. 3A). The inhibitory effect of
celecoxib was dose dependent and ranged from 48 – 85% when compared with untreated tumors (P ⬍ 0.001, Student’s t test). In the same
model, cyclophosphamide (50 mg/kg, i.p., days 5, 7, and 9) produced
a 34% reduction in tumor volume. A pharmacological effect of
celecoxib on lung metastasis was evaluated in all of the animals by
counting the number of metastases in a stereomicroscope and by
histochemical analysis of consecutive lung sections. Celecoxib did not
affect the number and size of lung metastases at the lower dose of 160
ppm, however, the number of metastatic nodules was reduced by
⬎50% in animals treated with doses between 480 and 3200 ppm.
Moreover, histopathological analysis revealed that celecoxib dosedependently reduced the size of the metastatic lesions in the lung (data
not shown).
Celecoxib was also tested in nude mice that had been implanted
with the human colon cancer cell line HT-29. These tumors reached
1.5–2.0 cm3 in size by 40 –55 days after implantation. Celecoxib was
introduced to the diet at doses ranging from 160-1600 ppm when the
tumors had reached 0.1 cm3 in volume. Maximal inhibition of 67% of
tumor growth was achieved with the lowest dose of celecoxib
(Fig. 3B). In addition, celecoxib dose-dependently inhibited tumor
lung metastases (Table 1, Fig. 3C). The inhibitory effect was remarkable with a 65% inhibition at 160 ppm and a maximal effect of 91%
at 1600 ppm. Celecoxib peak plasma levels were approximately
between 0.2 and 9.0 ␮g/ml for the 160 and 3200 ppm, respectively.
The peak plasma concentrations achieved in the studies were similar
to those reported by Reddy et al. (8) in rats, in which a potent
chemopreventive properties of celecoxib in colon carcinogenesis was
observed. Similar to the study by Reddy et al. (8), no toxicity was
observed in any of the animals as measured by weight gain/loss, as
well as gross pathological examination of the gastrointestinal tract of
the animals at necropsy.
Effect of COX Inhibitors on FGF-induced Corneal Angiogenesis. The contribution of COX-2 to angiogenesis was evaluated
using an in vivo rat corneal model (18). The pharmacological
effects of the COX-2 inhibitor celecoxib, which at therapeutic
doses in humans does not inhibit COX-1, the COX-1 inhibitor
SC-560 and the conventional NSAID (COX-1/COX-2) indomethacin (16, 17) were measured. Celecoxib caused a substantial reduction in the number and length of sprouting capillaries (Fig. 4A).
Celecoxib dose-dependently inhibited the angiogenic response
with an ED50 of 0.3 mg/kg/day, and with maximal inhibitory
activity of 80% at a dose of 30 mg/kg/day (Fig. 4B). Plasma levels
were determined 4 h after the last dose and found to be approximately 0.3 and 2.0 ␮g/ml for the 3 and 30 mg/kg/day, respectively.
Indomethacin, an inhibitor of both COX-1 and COX-2, also inhibited angiogenesis with an inhibition of 60% at the near maximally
tolerated dose of 1 mg/kg/day (Fig. 4B). Inhibition of angiogenesis
in rats dosed with indomethacin at 3 mg/kg/day was not determined due to the peritonitis, and subsequent death of these rats
before the 4th day of the study. In contrast to the effects of
celecoxib and indomethacin, the COX-1-specific inhibitor SC-560
did not affect angiogenesis when given at doses of 3 and 10
mg/kg/day. To determine the specificity of the inhibitors in vivo,
sera from the same rats were immunoassayed for TxB2, a product
of platelet COX-1 activity. While potently inhibiting angiogenesis,
celecoxib did not diminish platelet COX-1 activity in blood taken
from the same animals (Fig. 4, B and C). Because indomethacin
inhibits both isozymes at doses that suppressed angiogenesis, it
inhibited platelet COX-1 activity, as well (Fig. 4, B and C). The
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Fig. 3. Inhibition of tumor growth and lung metastasis by celecoxib in mice. A, Lewis Lung carcinoma (106 cells) was implanted into the hind paws of C57/B16 male mice to develop
tumors. Groups of 20 animals were treated with doses of celecoxib (160,480,1600, and 3200 ppm) in their food from the day of implant until the end of the study, and tumor volume
was determined biweekly using a plethysmometer. The data are expressed as the mean ⫹/⫺ SEM. Student’s and Mann-Whitney tests were used to assess differences between means
using the InStat software. B, HT-29 human colon carcinoma cells were also implanted in the hind paws (106 cells) of nude mice. Celecoxib therapy was initiated when tumors reached
a mean volume of 100 mm3 and maintained for the duration of the experiment. C, lung with several metastatic nodules (arrows) from a HT-29-implanted mouse. D, a lung without
metastases from a mouse bearing HT-29 tumor treated with 1600 ppm celecoxib (Table 1).
COX-1 inhibitor SC-560 inhibited platelet COX-1-derived TXB2
by 89% and 97% at doses of 3 and 10 mg/kg/day, respectively,
without any effect on the angiogenic response in the cornea (Fig.
4, B and C). To determine whether the antiangiogenic activity of
celecoxib was due to the inhibition of PG synthesis, we tested an
inactive isomer (1,3- versus 1,5-diarylpyrazole) of celecoxib. This
compound was completely devoid of antiangiogenic activity at a
maximal dose of 30 mg/kg/day (data not shown).
Histological examination of the angiogenic cornea revealed the
presence of new angiogenic blood vessels and several types of COX2-positive cells (Fig. 4, E and F). No angiogenesis was observed in
corneas implanted with placebo (Fig. 4D). In contrast, corneas implanted with the growth factor FGF-2 induced a strong neovascularization that is accompanied by corneal thickening with an expanded
stroma filled with an influx of COX-2-positive cells, including fibroblasts, macrophages, and endothelial cells (Fig. 4, E and F). Whereas
endothelial cells present in the established limbic vessels expressed
the COX-1 enzyme, newly vascularized endothelium expressed
COX-2 (Fig. 4, E and F).
In agreement with the studies of Kenyon et al. (18), the angiogenic
response we studied seemed to be mediated directly by FGF-2 and
was not due to an inflammatory reaction to the implants. Indeed, the
insertion of placebo pellets induced an initial inflammatory response
similar to the FGF-2-containing pellets; however, this inflammation
resolved within the first 24 h without a subsequent formation of new
blood vessels (Fig. 4D).
Table 1 Celecoxib dose-dependently inhibits metastasis to the lung
Mice injected in the paws with human colon cancer cells (HT-29) developed surface
lung metastasis that are readily detected by 50 – 60 days. Celecoxib was administered to
the animals (n ⫽ 15/treatment group) in the food. Therapy was initiated when primary
tumors reached 100 mm3 (around day 14), and lung metastasis was determined at the end
of treatment (Fig. 3C) by counting the total number of neoplastic nodules present in the
lung surface, using a stereomicroscope, and verified via histopathologic examination.
Celecoxib (ppm)
Lung metastasis
(average ⫾ SE)
0
160
480
1600
55 ⫾ 17
19 ⫾ 11
9⫾4
5⫾3
DISCUSSION
There is ample evidence to suggest an important role for COX-2 in
cancer. This newly discovered enzyme is not normally present under
physiological conditions in most tissues, however, it is rapidly induced by
a variety of cytokines and mitogens (2–5). Many reports indicate that
COX-2 is up-regulated in most human tumors (12–15, 19 –21). We
extended this observation to examine the expression of COX-2 in more
than 150 samples of different types of human cancers. We consistently
find COX-2 expression in most cancer tissues, including colon, lung,
breast, and prostate. Furthermore, we can clearly detect the presence of
COX-2 in the angiogenic vasculature in most of the tumors analyzed.
Another important finding of this study was the detection of COX-2 in
the angiogenic blood vessels present in livers from patients with metastatic colon carcinoma (Fig. 2). These novel findings suggest COX-2 may
play a role in tumor angiogenesis and suggest that inhibitors of this
pathway may affect tumor growth by inhibiting the formation of blood
vessels necessary for tumor survival (10).
Two animal models, the Lewis lung carcinoma and the human colon
carcinoma HT-29, were selected to evaluate the antitumorigenic effects
of celecoxib. Both models develop primary tumors as well as lung
metastases when the tumors reach volumes larger than 1.5 cm3 in size.
Celecoxib dose-dependently inhibited tumor growth and the number and
size of lung metastases. These data confirm the efficacy of the COX-2
inhibitor in blocking the growth of the primary tumor, as well as its
antimetastatic potential. The anticancer efficacy observed in both animal
models with celecoxib was devoid of any signs of gastrointestinal toxicity
that is typical of nonselective NSAIDs and preclude their use in cancer
therapy. Interestingly, the expression of COX-2 in the animal models of
cancer was mainly restricted to the angiogenic blood vessels, the preexisting vasculature adjacent to the primary tumor, the blood vessels invading the metastatic lesions in the lungs of these animals, and not in the
tumor cells themselves. On the basis of these findings, we suggest that
COX-2-derived PGs contribute to tumor growth in these rodent models
by inducing newly formed blood vessels that sustain tumor cell viability
and growth. Furthermore, the results suggest that a potent antiangiogenic
activity of celecoxib seems to be the primary mechanism of action in
these animal models of cancer and results in substantial inhibition of
tumor growth and metastasis.
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Fig. 4. Celecoxib is a potent inhibitor of angiogenesis. A, a representative photograph of neovascularization induced by basic FGF-2 in the rat cornea (left) and its inhibition by
celecoxib (right). B, pharmacological inhibition of angiogenesis by COX inhibitors. The COX-2 inhibitor celecoxib, the COX-1-specific inhibitor SC-560, and the nonspecific COX
inhibitor indomethacin were dosed p.o. b.i.d. (n ⫽ 6 per dose) for 5 days. Celecoxib and indomethacin inhibited angiogenesis (P ⬍ 0.001, Students t test) at all doses tested, whereas
SC-560 had no effect. C, representation of COX-1 activity in vivo. Sera from the same rats were immunoassayed (Cayman Chemical) for TxB2, a product of platelet COX-1 activity.
While potently inhibiting angiogenesis, celecoxib had no effect on platelet COX-1 activity. SC-560 did not affect angiogenesis at 3 and 10 mg/kg/day, doses that clearly inhibited COX-1
by 68% and 86%, respectively. Because indomethacin inhibits both isozymes, at doses that suppressed angiogenesis, it inhibited platelet COX-1, as well. These experiments were
repeated at least three times with six rats per group. D, COX-2 expression (red) and immunohistological examination of a transverse section of the temporal limbic region of the rat
cornea 4 days after implant with a placebo pellet. The eyes were resected, fixed in 10% formalin, and stained for COX-2 using specific antiserum. The photograph shows the presence
of COX-2-positive macrophages (M) near the limbic vessels (L). No evidence of inflammation or neovascularization was observed in placebo corneas. E, an identical region of the
FGF-2-implanted cornea showed an expanded stroma filled with an influx of COX-2-positive cells, including macrophages (M), fibroblasts, and endothelial cells. Mature endothelial
cells present in the established limbic vessels (L) did not express COX-2. F, high-power photomicrograph of a section of corneal stroma showed the presence of newly developed
angiogenic blood vessels (arrows) with some luminal RBCs. The endothelium of these vessels expressed the inducible COX-2 enzyme.
To test this hypothesis, we evaluated the antiangiogenic activity of
celecoxib in the rat corneal model of angiogenesis. We found that
celecoxib, a compound with ⬎300-fold in vitro selectivity for COX-2
versus COX-1 (5) was a very potent inhibitor of angiogenesis with an
ED50 around 0.3 mg/kg/day, a dose in the range of efficacy against
inflammation (8, 16). The effect of celecoxib was dose-dependent and
specific for the target enzyme because, at all doses tested, it did not
inhibit serum TXB2 derived from platelet COX-1. The potent antiangiogenic effect of celecoxib seems to be derived from its capacity to
inhibit PG production via COX-2 because neither an inactive isomer
of celecoxib, nor a COX-1 inhibitor was able to block the development of angiogenesis in the rat cornea. Indomethacin, a potent inhibitor of both COX-1 and -2, inhibited both angiogenesis and platelet
COX-1 activity, and at higher doses caused gastrointestinal toxicity
and death. Taken together, these results suggest that PGs produced by
FGF-2 induction of COX-2 are essential to neovascularization. These
new findings contrast somewhat with a recently published study by
Tsujii et al. (22). Using a coculture system of endothelial cells with
colon cancer cells they report that COX-1, but not COX-2, is the
enzyme necessary for the endothelial cells to form tubes in vitro. Tube
formation by capillary endothelial cells in vitro may closely reflect
quiescent established capillaries in vivo that, in fact, express COX-1.
Our results are different perhaps because angiogenic endothelial cells
and the supporting cells associated with them are neither quiescent nor
established, do express COX-2, and are growth inhibited by celecoxib.
Importantly, they also show that COX-2 from human colon cancer
cells induces the production of angiogenic growth factors and that this
production is curtailed by COX-2 inhibition. In contrast to human
tumors, we cannot detect COX-2 in the cancer cells used in the animal
models of cancer, except in the neovasculature. However, the pronounced expression of COX-2 in human neoplastic epithelium, as
well as associated human angiogenic vessels, supports the hypothesis
by Tsujii et al. (22) that COX-2 inhibition in human cancer would
inhibit the production of angiogenic growth factors by the tumor cells.
In addition, we have shown that COX-2 inhibition in the presence of
growth factor FGF-2 has a direct antiangiogenic effect on the neo-
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vasculature. Thus, in human cancer, inhibition of COX-2 may prove
effective in two ways: by blunting the tumor cell production of
angiogenic growth factors and by inhibiting the growth of the neovascular cells themselves.
The contribution of inflammatory mediators to the angiogenesis
of tumors and their growth is becoming evident. Our observation
that FGF-2-induced angiogenesis is dependent on the expression of
COX-2 strongly suggests that COX-2 plays an important role in the
generation of tumor blood supply. This was further demonstrated
with the presence of COX-2 in the neovasculature of most human
tumors and in metastases such as in the liver. On the basis of the
role of COX-2 in angiogenesis, together with the expression of the
enzyme in human tumors and the remarkable efficacy observed by
inhibitors of this pathway in animal models, we postulate that
inhibition of COX-2 should produce antiangiogenic and antitumor
results in the clinic. This hypothesis is further supported by studies
indicating a role for COX-2 in the production of angiogenic factors
by colon cancer cells (22), increased cell proliferation (23), prevention of apoptosis (24), increased metastatic potential (25), and
the inhibition of immune surveillance (26). Finally, anti-inflammatory treatment using indomethacin, a nonselective inhibitor of
COX-1 and -2, or prednisolone, a steroid known to inhibit the
synthesis of COX-2 protein, prolonged survival in patients with
metastatic solid tumors (27). In light of these data, a clinical trial
designed to assess the use of celecoxib in the therapy of human
cancer seems justified.
ACKNOWLEDGMENTS
We thank Drs. Nasir Khan, Peter Humphrey, and Robert Soslow for expert
opinions as pathologists for the immunohistology slides. We also thank Drs.
William Catalona, Cristian Ramos, Andrew Dannenberg, Joseph Portanova,
Catherine Tripp, Geetha Vasanthakumar, Christopher J. Smith, and Dowdy
Jackson for suggestions in preparing the manuscript; and Steven D. Williams
for help preparing the figures.
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Antiangiogenic and Antitumor Activities of Cyclooxygenase-2
Inhibitors
Jaime L. Masferrer, Kathleen M. Leahy, Alane T. Koki, et al.
Cancer Res 2000;60:1306-1311.
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