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2003;63:586-592. Cancer Res
Min Yao, Stacia Kargman, Eugene C. Lam, et al.
Mice
Growth and Metastatic Potential of Colorectal Carcinoma in
Inhibition of Cyclooxygenase-2 by Rofecoxib Attenuates the
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[CANCER RESEARCH 63, 586–592, February 1, 2003]
Inhibition of Cyclooxygenase-2 by Rofecoxib Attenuates the Growth and Metastatic
Potential of Colorectal Carcinoma in Mice
Min Yao,
1
Stacia Kargman,
1
Eugene C. Lam, Colleen R. Kelly, Yaxin Zheng, Pauline Luk, Elizabeth Kwong,
Jilly F. Evans, and M. Michael Wolfe
2
Section of Gastroenterology, Boston University School of Medicine and Boston Medical Center, Boston, Massachusetts 02118 [M. Y., E. C. L., C. R. K., Y. Z., M. M. W.]; Merck
Frosst Centre for Therapeutic Research, Pointe Claire, Dorval, Quebec, H9H 3L1 Canada [S. K., P. L., E. K.]; and Merck Research Laboratories, West Point, Pennsylvania
19486 [J. F. E.]
ABSTRACT
A large number of epidemiological studies have shown that regular use of
aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) results in a
40–50% reduced risk of colorectal cancer (CRC). Furthermore, NSAIDs
cause the regression of preexisting adenomas in patients with familial ade-
nomatous polyposis and significantly inhibit tumor growth in animal models
of CRC. To establish a CRC liver metastasis model, we implanted mouse
colon tumor MC-26 cells into the splenic subcapsule of BALB/c mice, after
which micewere giveneither standardchow orchow containingthe cyclooxy-
genase (COX)-2-specific inhibitor rofecoxib, alone or in combination with the
standard antineoplastic agents, 5-fluoruracil or irinotecan. After 14 days,
mice that were given rofecoxib or irinotecan, but not 5-fluoruracil, had
significantly smaller primary tumors and fewer metastases. Rofecoxib, at
clinical anti-inflammatory plasma concentrations, enhanced the effects of
both antineoplastic agents when used in combination. Biochemicalanalyses of
the primary splenic tumor in rofecoxib-treated mice showed no alteration in
COX-1 expression, but significant decreases in the expression of the tumor-
promoting proteins COX-2, cyclin D1, cytosolic

-catenin, matrix metallo-
proteinases-2 and -9, and vascular endothelial cell- derived growth factor.
Rofecoxib also decreased growth-enhancing prostaglandin E
2
and tumor-
suppressive interleukin-10, whereas antineoplastic interleukin-12 was in-
creased. Two separate survival studies were performed. When mice were fed
chow containing 0.01% rofecoxib beginning on day 0 after tumor cell im-
plantation, which achieved clinical anti-inflammatory plasma concentrations,
survival time was significantly longer compared with mice given control
chow. After 30 days, mortality in the control group was 90%, whereas only
one mouse (5%) treated with rofecoxib had died after 30 days. In the second
survival study, all of the mice were initially fed with regular chow after tumor
cell implantation. On day 7, mice were randomly divided into three dietary
groups: control chow, low-dose (0.01%) rofecoxib chow, and high-dose
(0.025%) rofecoxib chow. After 28 days, mortality was 100%, 20%, and 10%
in control, low-, and high-dose rofecoxib fed groups, respectively. These
studies demonstrate that rofecoxib decreases the growth and metastatic po-
tential of CRC in mice through multiple mechanisms. These studies in mice
also provide important information that supports the benefit of COX-2
inhibition, not only in the prevention of CRC, but also potentially in the
treatment of this common malignancy. Clinical trials will be necessary to
assess the utility of COX-2 inhibitors as adjuvant therapy for early-stage
disease and as potential agents, either alone or in combination, with more
established drugs, for the treatment of refractory CRC.
INTRODUCTION
CRC
3
is second only to lung cancer as a cause of death from
malignant disease in the United States (1), with one-half of patients
incurable at their presentation (2). Nearly 130,000 new cases of CRC
were diagnosed in the United States in 1999, resulting in 56,600
associated deaths (3). Approximately 6% of Americans will develop
CRC during their lifetime, and 2.6% will die from this disease (4).
Chemotherapeutic modalities to treat refractory CRC, although asso-
ciated with significant toxicity, have provided only minimal benefit in
improving survival (5, 6). A large number of epidemiological studies
have shown that regular use of aspirin or other NSAIDs results in a
40–50% reduced risk of CRC (7, 8). Furthermore, NSAIDs cause the
regression of preexisting adenomas in patients with familial adenom-
atous polyposis (9) and significantly inhibit tumor growth in animal
models of CRC (8, 10).
The mechanism of inhibition of COX activity by aspirin and
NSAIDs was first described by Vane in 1971 (11). This observation
led to the hypothesis that both the toxicity and efficacy of NSAIDs are
mediated through the inhibition of COX-mediated prostaglandin syn-
thesis (12). In the early 1990s, several groups reported the discovery
of two COX isoforms, a constitutively expressed isoform, COX-1, and
a strongly inducible form, COX-2, which is involved in growth
proliferation and the inflammatory response (13, 14). Despite ⬃60%
amino acid identity, the two COX isoenzymes are encoded by distinct
genes and differ significantly with regard to tissue-specific distribu-
tion (12). COX-1 appears to function as a physiological regulatory
enzyme in most tissues, including the gastric mucosa, the kidney, and
platelets, whereas COX-2 is nearly undetectable in most (but not all)
tissues under normal physiological conditions (12). COX-2 mRNA
expression and protein was found to be enhanced in human colorectal
adenomas and adenocarcinomas, leading to the hypothesis that selec-
tive COX-2 inhibition might thereby reduce the development of CRC
(15–17). Specific COX-2 inhibition, either by targeted knockout of
the COX-2 gene or by pharmacological intervention, has been shown
to effectively decrease the growth of murine intestinal adenomas (17–
19). In a rat model of chemical-induced CRC, the COX-2 selective
inhibitor celecoxib suppressed the formation of azoxymethane-
induced aberrant crypt foci (20). Celecoxib also inhibited the inci-
dence and multiplicity of colon tumors by ⬃93 and 97%, respectively,
and diminished the overall rat colon tumor burden by ⬃87% (21). A
recent human study demonstrated that selective COX-2 inhibition
with celecoxib in 77 individuals with familial adenomatous polyposis
decreased the mean number of colonic polyps and the polyp burden by
28.0 and 30.7%, respectively (9).
No published studies have reported the benefits of specific COX-2
inhibitors in treating metastatic cancer. However, one clinical study
involving the use of the potent nonselective COX inhibitor indo-
methacin demonstrated that the treatment of end-stage metastatic
patients (mainly CRC and liver, pancreas, and gastric primary can-
cers) resulted in increased survival (250–510 days), less pain, and
diminished use of other analgesics (22, 23).
In the present study, we sought to determine whether rofecoxib, a
specific inhibitor of COX-2, could reduce primary tumor growth and
liver metastatic potential of mouse colon tumors in vivo. We used
BALB/c mice, in which MC-26 cells, a transplantable mouse CRC
Received 4/30/02; accepted 11/27/02.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
M. Y. and S. K. contributed equally to this work.
2
To whom requests for reprints should be addressed, at Boston Medical Center,
Section of Gastroenterology, 650 Albany Street, Boston, MA 02118. Phone: (617)
638-8330; Fax: (617) 638-7785; E-mail: michael.wolfe@bmc.org.
3
The abbreviations used are: CRC, colorectal cancer; NSAID, nonsteroidal anti-
inflammatory drug; COX, cyclooxygenase; 5-FU, 5-fluoruracil; LV, leucovorin; MMP,
matrix metalloproteinase; VEGF, vascular endothelial growth factor; IL, interleukin;
HPLC, high-performance liquid chromatography; PGE
2
, prostaglandin E
2
; CPT-11, iri
-
notecan.
586
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on May 31, 2013. © 2003 American Association for Cancercancerres.aacrjournals.org Downloaded from
cell line expressing COX-2 protein, were implanted surgically into the
splenic capsule. Mice were then administered either standard chow or
chow containing the COX-2-specific inhibitor rofecoxib alone, or in
combination with standard antineoplastic agents. Our initial hypoth-
esis was that rofecoxib alone would have minimal effect but would
have additive or synergistic effects with the standard antineoplastic
agents. Our studies demonstrate that COX-2 inhibition by rofecoxib
alone decreases the growth and liver metastatic potential of CRC in
mice, in addition to augmenting the antineoplastic properties of the
standard chemotherapeutic agents.
MATERIALS AND METHODS
Preparation of Rofecoxib and Other Antineoplastic Agents
We purchased 5-FU and LV from Sigma Chemical Co. (St. Louis, MO).
The working solution was prepared by adding 5-FU and LV to 0.9% NaCl
adjusted to pH 8.6 with 0.01 M NaOH, after which the solution was sonicated
for 5 min and then filtered through a 0.22
m filter. We obtained CPT-11 from
Pharmacia (Kalamazoo, MI) and prepared the working solution by diluting 20
mg/ml of the agent into a 2 mg/ml concentration with 5% glucose. 5-FU, LV,
and CPT-11 were injected i.p. at doses of 30 mg/kg, 50 mg/kg, and 30 mg/kg,
respectively. We prepared 0.01% rofecoxib chow, equivalent to a dose of 20
mg/kg/day, for use in these experiments.
Cell Line
We obtained MC-26 cells, a transplantable mouse colon cancer cell line
(24), from Dr. K. K. Tanabe Massachusetts General Hospital, (Boston, MA)
and maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD), sup-
plemented with 10% FBS and antibiotics (100 units/ml penicillin and 100
g/ml streptomycin), at 37°C in a humidified atmosphere of 95% air/5% CO
2
.
Animal Preparation, Treatment, and Surveillance
We purchased 6-to-10-week-old male BALB/c mice from Taconic (Ger-
mantown, NY). We harvested MC-26 cells from subconfluent cultures using
trypsin-EDTA (Life Technologies, Inc.), followed by centrifugation at 300 ⫻ g
for 15 min at room temperature. We then resuspended cells in serum-free
DMEM or HBSS (Life Technologies, Inc.), and the cell number was adjusted
to a final concentration of 200,000 cells/ml. A 1-cm incision was made in the
mice under i.p. anesthesia with pentobarbital (Abbott Laboratories, North
Chicago, IL) at 65 mg/kg body weight. Using a 27-gauge needle and a 1-ml
syringe, we injected 100
l of tumor cell suspension (2 ⫻ 10
4
) into the
subsplenic capsule of mice. All of the animal studies were conducted using a
protocol approved by the Institutional Animal Care and Use Committee at
Boston University Medical Center.
On day 0, after complete recovery from surgery, we randomly divided mice
into eight groups: Group 1, control (n ⫽ 20), received control chow plus 0.9%
NaCl or 5% glucose i.p. injection on days 4, 5, 7, 8, 11, and 12; Group 2,
rofecoxib alone (n ⫽ 20), received rofecoxib 0.01% chow, and 0.9% NaCl or
5% glucose i.p. injection on days 4, 5, 7, 8, 11, and 12; Group 3, 5-FU/LV
alone (n ⫽ 11), received control chow plus 5-FU 30 mg/kg and LV 50 mg/kg
on days 5, 7, and 12. Injection of 5-FU was performed 1 h after LV injection.
Five % glucose i.p. injection was given on days 4, 8, and 11; Group 4, CPT-11
alone (n ⫽ 11), received control chow plus CPT-11 30 mg/kg on days 4, 8, and
11, and 0.9% NaCl i.p. injection on days 5, 7, and 12; Group 5, rofecoxib/5-
FU/LV (n ⫽ 11), received rofecoxib chow plus 5-FU 30 mg/kg and LV 50
mg/kg on days 5, 7 and 12, and 5% glucose i.p. injection on days 4, 8, and 11.
Group 6, rofecoxib/CPT-11 (n ⫽ 11), received rofecoxib chow plus CPT-11 30
mg/kg on days 4, 8, and 11, and 0.9% NaCl i.p. injection on days 5, 7, and 12;
Group 7, 5-FU/LV/CPT-11 (n ⫽ 11), received control chow plus 5-FU 30
mg/kg and LV 50 mg/kg on days 5, 7, and 12, and CPT-11 30 mg/kg on days
4, 8, and 11; Group 8, rofecoxib/5-FU/LV/CPT-11 (n ⫽ 11); received rofe-
coxib chow plus 5-FU 30 mg/kg and LV 50 mg/kg on days 5, 7, and 12, and
CPT-11 30 mg/kg on days 4, 8, and 11.
On day 14, blood samples were obtained, after which all of the mice were
sacrificed. During the dissection, primary (splenic) tumor size was determined
by weighing and then measuring the longest and shortest diameters of the
tumor. Tumor volume (mm
3
) was calculated using the standard formula: tumor
volume ⫽ (shortest diameter)
2
⫻ (longest diameter) ⫻ 0.5. We fixed a portion
of the tumor tissue in 10% formalin for subsequent histological examination
and then flash-froze the remaining tissue in liquid nitrogen and stored it at
⫺70°C. Metastatic nodules in the liver were detected macroscopically and
confirmed microscopically, and the percentage of mice with hepatic metastasis
was recorded.
In a separate experiment, to identify the effect of rofecoxib on liver weight,
five mice without tumor cell injection were fed 0.01% rofecoxib chow, and
another five mice without tumor cell injection were fed control chow for 14
days. All of the mice in both groups were sacrificed on day 14, and liver
weights were measured.
Survival Studies
Two separate experiments were performed to assess survival.
Protocol 1. On day 0, after complete recovery from surgery, mice were
randomly divided into two groups (n ⫽ 20 in each group): a control group, in
which mice were fed with regular chow; and a rofecoxib group, in which mice
were fed with rofecoxib 0.01% chow (⬃20 mg/kg mouse weight, which
achieved a plasma concentration of 0.26
g/ml). This survival study was
terminated on day 30.
Protocol 2. On day 0, after complete recovery from surgery, all of the mice
were fed regular chow. On day 7, mice were randomly divided into three
groups (n ⫽ 20 in each group): a control group, which received regular chow;
a low-dose rofecoxib group, which received chow containing rofecoxib 0.01%
(which achieved a plasma concentration of 0.26
g/ml); and a high-dose
rofecoxib group, which received chow containing rofecoxib 0.025% (⬃50
mg/kg mouse weight, which achieved a plasma concentration of 0.55
g/ml).
The survival study was terminated when mortality in the control group reached
100%.
Preparation of Microsomal Membranes from Splenic Tumor Tissue
Splenic tumor tissue was excised, immediately frozen in liquid N
2
, and
stored at ⫺70°C. Frozen tissues were thawed in ice-cold homogenization
buffer [50 m
M potassium phosphate (pH 7.1), containing 0.1 M NaCl, 2 mM
EDTA, 0.4 mM phenylmethylsulfonyl fluoride, 60 g/ml soybean trypsin inhib-
itor, 2 g/ml leupeptin, 2 g/ml aprotinin, and 2 g/ml pepstatin, all from Sigma
Chemical Co.]. Tissues were disrupted twice, for 10 s each, on ice using a
tissue tearer (IKA Labortechnik, Staufen, Germany). Samples were homoge-
nized by sonication at 4°C using a Cole Parmer 4710 series ultrasonic homog-
enizer (Cole Parmer Instrument Co., Chicago, IL). Debris was removed by
centrifugation at 1,000 ⫻ g for 15 min at 4°C, and the resultant supernatants
were subjected to centrifugation at 100,000 ⫻ g for 30 min at 4°C. Membrane
fractions were resuspended in homogenization buffer and then sonicated to
obtain a homogeneous membrane suspension. Protein concentrations were
determined for each sample using a protein assay kit (Bio-Rad, Mississauga,
Ontario, Canada), and equal gel loading was determined by Coomassie Blue
staining (Sigma).
SDS-PAGE and Immunoblot Analysis
Microsomal and cytosolic fractions were mixed with 0.5 volume of SDS
sample buffer [20 m
M Tris-HCl (pH 6.8), containing 0.4% (w/v) SDS, 4%
glycerol, 0.24 M

-mercaptoethanol, and 0.5% bromphenol blue], boiled for 5
min and analyzed by SDS-PAGE on 9 ⫻ 10 cm precast 4–20% Tris-glycine
acrylamide gels (NOVEX, San Diego, CA), according to the method of
Laemmli (25). Proteins were electrophoretically transferred to nitrocellulose
membranes, as described previously (26). Primary antisera to COX-1 (MF241;
Merck-Frosst, Montreal, Canada), COX-2 (MF243; Merck-Frosst),

-catenin
(Sigma), cyclin D1 (BD Transduction), MMP-2 (Oncogene), MMP-9 (Onco-
gene), and VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) were used at
final dilutions of 1:3,000; 1:7,500; 1:5,000; 1:1,000; 1:1,000; 1:1,000, and
1:200, respectively, according to the manufacturers’ instructions. The second-
ary horseradish peroxidase-linked goat antirabbit or goat antimouse IgG anti-
bodies (Santa Cruz) were used at dilutions of 1:2,000 to 1:6,000. Immunode-
tection was performed using enhanced chemiluminescence according to the
manufacturer’s instructions (Amersham, Elk Grove Village, IL). Protein bands
were visualized using a Fuji LAS-1000 plus Luminescent Image Analyzer
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COX-2 INHIBITION BY ROFECOXIB IN THE TREATMENT OF CRC
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(Fuji Photo Film Company, Tokyo, Japan) and quantitated using Fuji Film
Image Gauge version 3.122. The volumes of absorbance corresponding to the
purified COX isoform (Cayman), a purified M
r
46,000 tagged fusion protein
corresponding to NH
2
-terminal amino acids 1–147 of human VEGF or

-cate
-
nin (a thioredoxin fusion protein; a kind gift of Astrid Kral, MRL, West Point,
PA) proteins were used to calculate the quantity of COX or VEGF protein in
splenic tumor samples. Immunoblot analyses of the above proteins were
performed using the same nitrocellulose membrane by stripping the membrane
with a buffered solution containing 62.5 m
M Tris-HCl, 2% SDS, and 100 nM
2-mercaptoenthenol.
ELISA and Measurement of Plasma Rofecoxib Levels
The concentrations of IL-10, IL-12, and PGE
2
in splenic tumor cytosolic
fractions were determined by quantitative ELISA, according to the manufac-
turer’s directions. IL-10 and IL-12 immunoassay kits were purchased from
Biosource International (Camillaro, CA), and PGE
2
kits were purchased from
Assay Designs, Inc. (Ann Arbor, MI).
Plasma rofecoxib levels were measured by HPLC. A set of four rofecoxib
standards, ranging from 0.1 to 5.0
g/ml (R
2
⬎ 0.9999), were prepared in
acetonitrile. We added 100
l of blank mouse plasma to each 100-
l aliquot
of the standards and then mixed the standards on an automatic vortex mixer for
10 min. The sample was centrifuged at 14,000 rpm for 15 min, and the
supernatant was transferred to a HPLC vial for injection. Sample preparation
was identical to the standard preparation procedure with the exception of a
100-
l aliquot of acetonitrile added to 100
l of plasma sample. An HP1090
system equipped with an UV detector was fitted with an Eclipse XDB-C18
Rapid 74Resolution (4.6 ⫻ 75 mm, 3.5
m) analytical column. We used a flow
rate of 1.0 ml/min with a solvent ratio of 65% aqueous (0.1% trifluoracetic acid
in water) to 35% organic (0.1% trifluoracetic acid in acetonitrile), an oven
temperature of 40°C, and an injection volume of 25
l. Rofecoxib was
detected at a wavelength of 275 nm.
Statistical Analysis
Using SAS 8.0, we performed one-way ANOVA to compare tumor volume
and liver weight and densitometric values of Western blot bands among the
different animal groups, followed by Tukey’s procedure for paired compari-
sons. We used the Fisher Exact
2
test for categorical data such as the
incidence rate of liver metastasis. We performed survival analysis using the
Kaplan-Meier method and log-rank tests. We assigned statistical significance
if P ⬍ 0.05.
RESULTS
Plasma Rofecoxib Concentrations. Rofecoxib could be detected
in the plasma of all of the groups treated with rofecoxib alone or in
combination with standard antineoplastic agents. Plasma drug con-
centrations in mice fed with 0.01% rofecoxib chow were similar to
trough levels achieved at steady-state with once daily 25-mg dosing in
a 70-kg human (27), and no significant differences were detected
among the various groups (Fig. 1).
Effect of Rofecoxib Treatment on the Expression of COX-1,
COX-2, Cyclin D1,

-Catenin, MMP-2, MMP-9, and VEGF. We
examined the effect of rofecoxib treatment on the expression of mouse
splenic tumor proteins involved in proliferation, cell cycle regulation,
angiogenesis, and metastasis. Splenic tumor samples from rofecoxib-
treated or control groups were assessed for expression of COX-1,
COX-2,

-catenin, cyclin D1, MMP-2, MMP-9, and VEGF proteins
by specific immunoblot analyses. Mice treated with rofecoxib had a
statistically significant reduction in splenic tumor levels of COX-2,
cyclin D1,

-catenin, MMP-2, and VEGF protein, in comparison with
mice treated with control chow (Fig. 2, B–E, G). Mice treated with
rofecoxib showed a trend toward reduction of MMP-9 protein expres-
sion; however, this reduction did not reach statistical significance
(Fig. 2F).
Effect of Rofecoxib Treatment on the Expression of PGE
2
,
IL-10, and IL-12. Splenic tumor samples from rofecoxib-treated and
control mice were assessed for PGE
2
, IL-10, and IL-12 concentrations
by quantitative ELISA, as described above. The concentration of
growth-proliferative, immunosuppressive PGE
2
was reduced in the
splenic tumors of mice treated with rofecoxib in comparison with
control animals (Fig. 3A). This reduction in PGE
2
is consistent with
the reduction in the COX-2 enzyme in the tumors of rofecoxib-treated
animals (Fig. 2B). The tumor promoting IL-10 cytokine was signifi-
cantly reduced in tumor samples of rofecoxib-treated animals as
compared with controls (Fig. 3B). In contrast, levels of the potent
proinflammatory tumor suppressive cytokine IL-12 were markedly
increased in rofecoxib-treated splenic tumors as compared with con-
trol mouse splenic tumors (Fig. 3C).
Effect of Rofecoxib Treatment on Tumor Growth and Metas-
tasis and on the Survival of Mice with Implanted Splenic CRC
Cells. Rofecoxib alone, or in combination with standard antineoplas-
tic agents, significantly inhibited in situ colon cancer growth and the
incidence of spontaneous liver metastasis (Fig. 4). Primary splenic
tumor volume was significantly decreased in all of the groups, al-
though to a lesser extent in those mice treated with 5-FU/LV (Fig.
5A). Moreover, rofecoxib potentiated the effects of both 5-FU/LV and
CPT-11 on tumor volume (Fig. 5A). Liver weight (Fig. 5B) and the
incidence of liver metastasis (Fig. 5C) were not significantly affected
in mice treated with 5-FU/LV alone. In contrast, both rofecoxib and
CPT-11 decreased liver weight and metastasis significantly, and the
effects of rofecoxib and CPT-11 were additive (Figs. 5, B and C). We
did not detect metastases in any of mice treated with both rofecoxib
and CPT-11 (Fig. 5C). Liver weight and metastasis were diminished
in mice treated with a combination of rofecoxib and 5-FU/LV com-
pared with the latter alone but were increased compared with mice
treated with rofecoxib alone.
In the initial survival study, in which mice were fed with 0.01%
rofecoxib chow on day 0, the survival time in mice treated with
rofecoxib chow was significantly longer than in mice given control
chow. After 30 days, mortality in the control group was 90%, whereas
only one mouse (5%) treated with rofecoxib had died after 30 days
(P ⬍ 0.00001; Fig. 6A). In the second survival study, mice were fed
with regular chow on recovery from surgery, and on day 7, they were
randomly divided into three groups. All of the mice fed with control
chow died by day 28, and mortality rates were 100, 20, and 10%,
respectively, in the control, 0.01 and 0.025% rofecoxib groups
(P ⬍ 0.0001 for both treatment groups; Fig. 6B).
Fig. 1. Plasma rofecoxib concentrations in different groups of mice receiving rofe-
coxib-containing chow. Blood was obtained from mice at day 14, and after centrifugation
and separation, plasma rofecoxib levels were measured by HPLC, as described in
“Materials and Methods.” Values represent the mean ⫾ SE of nine samples in each group.
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DISCUSSION
A large body of experimental evidence supports the premise that
COX-2 expression may contribute to tumorigenesis and that COX-2
inhibitors might be useful in the prevention of intestinal polyposis and
colon cancer (9, 22, 23). Increased levels of COX-2 mRNA and
COX-2 protein have been found in colonic tumors and polyps, in
comparison with normal colonic epithelium (15–17). Several mecha-
nisms have been supported for why COX-2 expression in neoplastic
tissues enhances tumor growth. There is evidence for amplification of
tumor cell proliferation by COX-2-produced PGE
2
and inhibition of
tumor cell apoptosis, enhancement of stromal cell angiogenesis, and
decreased immune surveillance of tumor cells. Recently, Dormond et
al. (28) demonstrated an important functional link among COX-2,
integrin
␣
V

3, and the small GTPases Cdc42 and Rac. Using human
umbilical vein endothelial cells, they found that both NS-398 and
indomethacin, but not the specific COX-1 inhibitor SC-560, sup-
pressed the activity of integrin
␣
V

3 (an adhesion receptor critically
involved in mediating tumor angiogenesis) and prevented activation
of Cdc42 and Rac, which are critically involved in regulating cell
migration after integrin engagement (28).
COX-2-produced prostaglandins also increase the metastatic poten-
tial of human CRC cells by enhancing proteolysis of the basement
membrane (15). Tsujii et al. (15) programmed Caco-2 cells to con-
stitutively express COX-2, which increased invasiveness compared
with control cells. This phenotypic change was associated with an
increase in the activation of MMP-2, as determined using gelatin
zymography, and an increase in MMP-1 mRNA. Both effects were
reversed by the nonspecific NSAID metabolite sulindac sulfide (15).
Tomozawa et al. (10) injected MC-26 cells into the tail vein of
BALB/c mice and demonstrated that the i.p. injection of JTE-522, a
selective COX-2 inhibitor, decreased the number of lung metastases.
The authors concluded that selective COX-2 inhibition might be
effective in decreasing the hematogenous metastasis of CRC (10).
Using a cell invasion/migration assay that employs Matrigel-coated
Fig. 2. Effect of rofecoxib treatment on mouse
splenic tumor COX-1 and -2, cyclin D1,

-catenin,
MMP-2 and -9, and VEGF expression. Splenic
tumors from rofecoxib-treated and control mice
were homogenized, separated by SDS-PAGE,
transferred to nitrocellulose, and blotted with
COX-1, COX-2, cyclin D1,

-catenin, MMP-2 and
-9, and VEGF antisera, with detection by enhanced
chemiluminescence. The amounts of COX-1,
COX-2,

-catenin, and VEGF were determined by
image quantification using a charge-coupled device
camera as indicated in “Materials and Methods.”
The area of absorbance for known quantities of
COX-1, COX-2,

-catenin, and VEGF proteins
were used to assess approximate amounts of these
proteins in splenic tumor samples. Values represent
the mean ⫾ SE for nine animals in each treatment
group for COX-1 (A), COX-2 (B), cytoplasmic

-catenin (D), and cytoplasmic VEGF (G), and for
five animals in each group for Cyclin D1 (C) and
MMP (E and F) proteins. To the left of each bar
graph pair, a representative immunoblot analysis
from five animals in each treatment group for each
protein examined, as well as a representative im-
munoblot of

-actin. O.D., absorbance.
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chambers, we have recently reported that the COX-2 inhibitor NS-398
decreased MC-26 cell migration by ⬃60%, indicating that COX-2
inhibition may decrease the metastatic potential of CRC, at least in
part, by inhibiting cellular invasion (29).
As stated previously, CRC usually presents at an advanced stage
and is the second leading cause of cancer deaths in the United States.
Up to 50% of patients will die of their disease within 5 years from
diagnosis (4). Whereas the 5-year survival of early-stage CRC (Dukes
A or B) is relatively high, only ⬃40% of those individuals with
advanced stages (Dukes C and D) will be alive 5 years after the initial
diagnosis (4, 6). In a recent study by Saltz et al. (5), of 683 patients
with refractory CRC, a combination of CPT-11 and 5-FU/LV im-
proved survival by only 2.2 months over either agent alone, from a
median of 12.6 months to 14.8 months. Although the addition of
CPT-11 to 5-FU/LV did not compromise the quality of life of partic-
ipants in this study, subsequent studies (30, 31) have reported signif-
icant toxicity associated with the use of this intense parenteral drug
regimen. In contrast, as p.o. administered drugs, COX-2 inhibitors are
convenient and relatively well-tolerated by most individuals and have
been reported to incite fewer upper gastrointestinal-related events,
such as ulcer-associated hemorrhage (32, 33).
In the present study, we examined the effect of rofecoxib treatment
on the growth and metastatic potential of colorectal carcinoma in a
mouse model of colorectal metastases. The rofecoxib plasma concen-
trations in drug-treated mice were equivalent to the trough levels in a
human 25-mg anti-inflammatory dose. Remarkably, this relatively
low dose of rofecoxib not only decreased the size of the primary CRC
tumor but also highly significantly decreased the rate and extent of
liver metastasis. A striking difference in mortality was also evident in
rofecoxib-treated mice when compared with control mice. We also
sought to determine molecular mechanisms that might account for the
beneficial effects of rofecoxib observed in this in vivo study by
examining factors shown in various in vitro models to be affected by
COX-2 overexpression or inhibition, such as cell proliferation, me-
tastasis, angiogenesis, and immune modulation. Immunoblot analysis
of the primary tumors demonstrated decreases in the protein expres-
sion of COX-2,

-catenin, cyclin D1, VEGF, MMP-2, MMP-9, and
IL-10. Interestingly, a significant increase in IL-12 protein was de-
tected in the primary tumors of mice treated with rofecoxib. Recent
studies (34–36) have suggested that these two important cytokines
play an important role in modulating tumor activity and that COX-2
Fig. 3. Effect of rofecoxib treatment on mouse splenic tumor PGE
2
(A), IL-10 (B), and
IL-12 (C) expression. Splenic tumors from rofecoxib-treated and control animals were
homogenized, as described in “Materials and Methods.” The concentrations of splenic
tumor PGE2, IL-10, and IL-12 were determined by quantitative ELISA. Values represent
mean ⫾ SE for nine animals in each treatment group for PGE
2
and IL-12 and for four
animals in each treatment group for IL-10.
Fig. 4. Primary tumor and hepatic metastasis at
day 14 in a representative control mouse (left) and
in one mouse treated with rofecoxib (right).
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COX-2 INHIBITION BY ROFECOXIB IN THE TREATMENT OF CRC
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may alter their balance in vivo. IL-10 has been found to be tumor
promoting, whereas IL-12 appears to function as a tumor-suppressing
cytokine (36). Huang et al. (34) found that PGE
2
production by
non-small cell lung cancer was dependent on COX-2 expression and
that an increase in PGE
2
was associated with the induction of IL-10
and the suppression of IL-12. COX-2 inhibition reversed the balance
of these cytokines in the lung cancer microenvironment (34). More
recent studies using a murine Lewis lung carcinoma model have
corroborated these observations (36).
The prolonged survival of rofecoxib-treated animals in our mouse
model of CRC metastasis is most likely the result of multiple mech-
anisms, including antiproliferative and antiangiogenic effects and
enhanced immune surveillance. Reductions in primary tumor size and
metastatic potential are associated with decreases not only in COX-2
expression and PGE
2
synthesis but also in the lowering of various
angiogenic factors and a restoration of the balance between cytokines
that favors tumor suppression. These studies in mice provide impor-
tant information that support the benefit of COX-2 inhibition not only
in the prevention on CRC but also in the treatment of this common
malignancy. Clinical trials will be necessary to assess the utility of
COX-2 inhibitors as adjuvant therapy for early-stage disease and as
potential agents, either alone or in combination with more established
drugs, for the treatment of refractory CRC.
ACKNOWLEDGMENTS
We are grateful to Merck and Co., USA, for a Vioxx Medical School Grant
and to Dr. Ian Rodger for his thoughtful suggestions during the course of these
studies and in the preparation of the manuscript.
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