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Tumor Biology |
716 Mouse by Rofecoxib, a Specific Cyclooxygenase-2 Inhibitor
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd. (Merck), Tsukuba 300-2611, Japan [M. O., N. M. H.]; Merck Frosst Center for Therapeutic Research, Pointe-Claire Dorval, Quebec, Canada H9R 4P8 [S. K., M. A., P. L., E. K.]; University of Tokyo, Graduate School of Pharmaceutical Sciences, Laboratory of Biomedical Genetics, Tokyo 113-0033 Japan [M. M. T.]; and Merck & Co., Inc., West Point, Pennsylvania 19486 [J. F. E.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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850, or by heterologous
recombination, resulting in the
Apc716 or
Apc1368 mice (M. Oshima
et al., Proc. Natl. Acad. Sci. USA, 92:
4482–4486, 1995). Although homozygote
Apc-/- mice are embryonically lethal, the
heterozygotes are viable but develop numerous intestinal polyps with
loss of Apc heterozygosity within the polyps (M. Oshima
et al., Proc. Natl. Acad. Sci. USA, 92:
4482–4486, 1995). The proinflammatory, prooncogenic protein
cyclooxygenase (COX)-2 has been shown to be markedly induced in
the Apc716 polyps at an early
stage of polyp development (M. Oshima et al., Cell,
87: 803–809, 1996). We demonstrate here that treatment
with the specific COX-2 inhibitor rofecoxib results in a dose-dependent
reduction in the number and size of intestinal and colonic polyps in
the Apc716 mouse. The plasma
concentration of rofecoxib that resulted in a 55% inhibition of polyp
number and an 80% inhibition of polyps >1 mm in size is comparable
with the human clinical steady-state concentration of 25 mg rofecoxib
(Vioxx) taken once daily (A. Porras et al., Clin. Pharm.
Ther., 67: 137, 2000). Polyps from both untreated
and rofecoxib- or sulindac-treated
Apc716 mice expressed COX-1
and -2, whereas normal epithelium from all mice expressed COX-1 but
minimal amounts of COX-2. Polyps from either rofecoxib- or
sulindac-treated mice had lower rates of DNA replication, expressed
less proangiogenic vascular endothelial-derived growth factor
and more membrane-bound ß-catenin, but showed unchanged
nuclear localization of this transcription factor. This study showing
the inhibition of polyposis in the
Apc716 mouse suggests that
the specific COX-2 inhibitor rofecoxib (Vioxx) has potential as a
chemopreventive agent in human intestinal and colon cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, prostacyclin, or
thromboxane (3)
. Elevated
PGE2 has been measured in rodent and human
colonic tumors, and the inhibition of prostaglandin synthesis by NSAID
treatment has been shown to inhibit tumor growth in animal models
(4, 5, 6)
. On the basis of such observations, the NSAID
sulindac was studied in FAP patients for prevention of polyp growth
(7)
. This clinical trial showed that treatment with
sulindac decreased polyp number and size, and that when sulindac
treatment was stopped, polyp growth recurred (7)
. In the early 1990s, a second form of COX, termed prostaglandin G/H synthase-2 or, more commonly, COX-2, was identified that was 60% identical to the original COX-1 (8, 9, 10) . COX-2 mRNA and protein were highly inducible by inflammatory and growth factors, whereas COX-1 expression was constitutive in most tissues, including the GI tract (8, 9, 10, 11) . The discovery of the second COX isoform led to the hypothesis that COX-2-specific inhibitors would be as efficacious as nonspecific COX-1/COX-2-inhibitor NSAIDs with respect to prostaglandin-mediated pain and inflammation in arthritis, but with a much-improved GI safety margin (12) . Two specific COX-2 inhibitors, i.e., rofecoxib (Vioxx) and celecoxib (Celebrex), have been shown preclinically and clinically to have comparable efficacy to NSAIDs for relief of pain and inflammation in osteoarthritis, but to have decreased risk of GI damage (13, 14, 15, 16, 17, 18) .
Given the epidemiology of NSAID protection for colon cancer, we and others investigated whether this chemopreventive effect might be specifically through the inhibition of COX-2-produced prostaglandins. COX-2 mRNA and protein were shown to be markedly elevated in human colon tumor tissue, whereas COX-1 expression remained the same or decreased (19 , 20) . COX-2 is also overexpressed in human colonic polyps and in macrophages in intimate contact with these sporadic polyps (21 , 22) . The growth of human colon tumor cells expressing COX-2 can be inhibited in vitro and in vivo by treatment with COX-2 inhibitors (23 , 24) . Mechanistic studies have revealed that this growth inhibition results from antiproliferative, proapoptopic, and antiangiogenic effects (23, 24, 25, 26, 27) . Elevated concentrations of COX-2 mRNA and protein have now been associated with esophageal, head and neck, breast, lung, prostate, and other cancers, and it has been suggested that COX-2 inhibitors may have benefit in malignancies other than colon cancer (28) .
A relevant animal model in which to test COX-2 inhibitors for
prevention of the polyp precursors of adenocarcinomas is the
Apc716 mouse,
which develops hundreds of intestinal polyps from birth through the
first 3 months of development (29)
. Both the genetic
deletion of COX-2 expression and pharmacological inhibition with the
specific COX-2 inhibitor, MF-tricyclic, have been shown to
markedly attenuate the number and size of polyps in the
Apc716 mouse
(30)
. The specific COX-2 inhibitor celecoxib (Celebrex)
has been shown to decrease polyp number and size in the chemically
induced Apc mutant Min mouse (31)
. In clinical
trials in FAP patients, celecoxib has also shown moderate efficacy, at
twice the approved arthritic dose, for the inhibition of colonic polyps
(32)
. In the study described here, we carefully
investigated the efficacy of the specific COX-2 inhibitor rofecoxib
(Vioxx) for chemoprevention of intestinal polyposis in the
Apc716 mouse. To
profile the potential for long-term prophylactic use as a
chemopreventive agent, we chose doses of rofecoxib at or below the
steady-state concentrations achieved at the clinical doses of Vioxx for
arthritis. In addition, we monitored parameters of COX
expression, angiogenesis, and polyp proliferation to further our
understanding of the potential mechanism of chemoprevention by
rofecoxib.
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MATERIALS AND METHODS |
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HPLC Quantitation of Rofecoxib and Sulindac and Its Metabolites
and in Apc716 Mouse Plasma.
Drug concentrations were measured in terminal-bleed plasma samples
taken from all mice after they were killed at 12 weeks of age.
Blood was collected in heparinized tubes and the plasma separated and
frozen at -70°C before preparation for HPLC analysis. For the
samples from 0.0075% w/w rofecoxib and 0.015% w/w sulindac animals,
100 µl of plasma were mixed with an equal volume of acetonitrile,
centrifuged at 10,000 x g for 15 min, and a
25-µl aliquot of the supernatant was analyzed by reverse-phase HPLC
separation on a HP1090 system (Hewlett-Packard, Palo Alto, CA) with an
Eclipse XDB-C18 rapid resolution column (75 x 4.6 mm,
3.5 µm; Hewlett Packard) for rofecoxib, a Symmetry C18 column
(150 x 3.9 mm, 5 µm; Waters, Milford, MA) for
sulindac, or an Inertsil phenyl column (100 x 3 mm, 5
µm; MetaChem Technologies, Inc., CA) for sulindac sulfide and
sulfone, using a 65:35 (aqueous, 0.1%trifluroacetic acid:
acetonitrile, 0.1% trifluoroacetic acid) solvent at a flow rate of 1
ml/min monitoring at 220 nm for rofecoxib and at 330 nm for sulindac
and its metabolites. Drug concentrations were determined in comparison
with standard curves constructed for each compound, separated under
identical conditions. For the 0.0025% w/w rofecoxib plasma samples the
plasma was concentrated 3-fold using a SpeedVac Plus SC210A (Savant
Instruments, Inc., Holbrook, NY) drier before HPLC separation.
Apc716 Knockout Mice
Construction and Inhibitor Study Protocol.
The construction of the
Apc716 knockout
mice was as described previously (29)
.
Apc716 knockout
mice were prepared by in vitro fertilization using two
C57BL/6 background
Apc716 male mice
and C57BL/6 female mice. Progeny were genotyped by PCR assay to
determine wild type or heterozygote for the Apc allele. Five
or six Apc716
heterozygote mice, randomized from eight litters, were used for each
treatment group. After weaning at 3 weeks of age, mice were fed
ad libitum with diet either containing drug or without drug
(control) for 8 weeks as shown in Fig. 1
. Food intake and body weights were monitored every week, and the actual
drug doses were calculated in accordance with the amount of chow eaten.
Final dose achieved for rofecoxib in chow at 0.0025% w/w was 4.7
mg/kg/day, for rofecoxib in chow at 0.0075% w/w was 14.7 mg/kg/day,
and for sulindac in chow at 0.015% w/w was 32.6 mg/kg/day. There was
no inhibition or increase in animal weight by either rofecoxib or
sulindac treatments.
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Histological Analysis and Immunohistochemistry.
After polyp scoring and sampling for Western analysis, intestinal
samples were fixed in 10% formaldehyde-PBS, embedded in paraffin, and
sectioned at 4-µm thickness. For immunohistochemistry, sections were
treated with 3% H2O2 for
1 h and incubated with 10% goat serum-3% BSA in PBS at 37°C
for 1 h to block nonspecific binding. The specimens were then
incubated with primary antibody [1:500 diluted rabbit polyclonal
antibody against the COOH terminus of human ß-catenin, which
cross-reacts with mouse ß-catenin (Sigma), in 10% goat serum-3%
BSA-PBS] for 60 min at room temperature, and with the secondary
antibody (biotinylated goat antirabbit IgG; Vector Research), and then
incubated with avidin-biotin-peroxidase complex (Vector Research)
labeled with peroxidase and colored with diaminobenzidine substrate.
BrdUrd Incorporation Analysis.
A BrdUrd detection kit (Boehringer Mannheim) was used according to the
manufacturer’s protocol. One representative mouse from each group was
inoculated i.v. with 300 µl of the BrdUrd solution and
sacrificed 4 h later. After polyp scoring, intestinal samples were
fixed with 70% ethanol at 4°C overnight, dehydrated, embedded in
paraffin, and sectioned serially at 5-µm thickness. Immunostaining of
BrdUrd incorporation in nucleus was performed according to the
manufacturer’s protocol. Serial sections were stained with
hemotoxylin. Three to four independent polyps from each group were
photographed, and the total number of cells and the number of
BrdUrd-positive cells were counted. The labeling indices were
determined by dividing the number of the labeled nuclei by the number
of total nuclei (about 5000 nuclei for each group).
Preparation of Microsomal Membranes from Intestinal Tissues.
Normal mouse intestinal mucosa and autologous polyp tissue were
excised, frozen immediately in liquid N2, and
stored at -70°C. Frozen tissues were thawed in ice-cold
homogenization buffer [50 mM potassium phosphate (pH 7.1)
containing 0.1 M NaCl, 2 mM EDTA, 0.4
mM phenylmethylsulfonyl fluoride, 60 µg/ml soybean
trypsin inhibitor, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 2
µg/ml pepstatin; all from Sigma Chemical Co., St. Louis, MO].
Tissues were disrupted twice, for 15 s each, on ice using a
tissue-tearer (IKA Labortechnik, Germany). Samples were
homogenized by sonication at 4°C using a Cole Parmer 4710 series
ultrasonic homogenizer (Cole Parmer Instrument Co., Chicago, IL).
Debris was removed by centrifugation at 1,000 x g for 15 min at 4°C, and the resultant supernatants were
subjected to centrifugation at 100,000 x g
for 45 min at 4°C. Membrane fractions were resuspended in
homogenization buffer, and then sonicated to obtain a homogenous
membrane suspension. Protein concentrations were determined for each
sample using a protein assay kit (Bio-Rad, Mississauga, Ontario,
Canada).
SDS-PAGE and Immunoblot Analysis.
Membrane fractions were mixed with 0.5 volume of SDS sample buffer [20
mM Tris-HCl (pH 6.8) containing 0.4% (w/v) SDS, 4%
glycerol, 0.24 M ß-mercaptoethanol, and 0.5% bromphenol
blue], boiled for 5 min and analyzed by SDS-PAGE on 9 x 10-cm precast 4–20% Tris-glycine acrylamide gels (NOVEX, San
Diego, CA) according to the method of Laemmli (18)
.
Proteins were electrophoretically transferred to
nitrocellulose membranes as described previously (19)
.
Primary antisera to COX-1 and COX-2 were used at a final dilution of
1:3,000 and 1:5,000–10,000, respectively. Primary antiserum to VEGF
(Santa Cruz) and ß-catenin (Sigma) were used at final dilutions of
1:500 and 1:5000 according to the manufacturer’s instructions. The
secondary horseradish peroxidase-linked goat antirabbit IgG antibody
(Santa Cruz Biotechnology) was used at dilutions of 1:3000–1:6000 for
COX-1 and -2, 1:2000–1:3000 for VEGF and 1:5000 for
ß-catenin. Immunodetection was performed using enhanced
chemiluminescence according to the manufacturer’s instructions
(Amersham). Protein bands were visualized using a FUJI LAS-1000-plus
Luminescent Image Analyzer (Fuji Photo Film Company, Japan) and
quantitated using Fuji Film Image Gauge version 3.122. The
volumes of absorbance corresponding to the purified COX isoform
(Cayman) or a purified Mr
46,000-tagged fusion protein corresponding to amino-terminal
amino acids 1–147 of human VEGF or ß-catenin (a thioredoxin fusion
protein, a kind gift of Astrid Kral, Merck Research
Laboratories, West Point, PA) proteins were used to calculate
the quantity of COX, VEGF, or ß-catenin protein in normal and polyp
intestinal tissues.
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RESULTS |
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. Sulindac is a prodrug in vivo that is inactive as a COX
inhibitor until it is converted to the metabolite sulindac sulfide,
which is a potent inhibitor of both COX-1- and COX-2-derived
prostaglandin production in in vitro human recombinant COX-1
and COX-2 whole-cell assays and also is active in human whole blood
assays (Table 1)
. The oxidative metabolite of sulindac,
i.e., sulindac sulfone, does not inhibit COX-1 or COX-2
enzyme activity in human whole-blood assays (Table 1)
. Mouse
whole-blood assays have not been able to be developed, therefore exact
activity of the COX inhibitors in mouse blood is not available.
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716 Mouse and in Clinical
Studies.
. The range of drug concentrations in individual mice converted to
micromolar concentrations is compared with the peak and trough
concentrations of steady-state clinical dosing of rofecoxib (Vioxx) and
sulindac (Clinoril) and its metabolites (Table 3
and Refs. 36
and 37
). Although all mice ate very
consistent amounts of chow (
4 g/day), the terminal-bleed plasma
concentrations of sulindac were somewhat variable from mouse to mouse
(Table 3)
.
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716 Mouse after Treatment with Rofecoxib
or Sulindac.
depicts the morphology of polyps in the untreated
Apc716 mice as was
observed previously (38)
. Both the rofecoxib and
sulindac-treated mice intestines had many fewer polyps, and those
present were much smaller and flatter than those in the untreated mice
intestines (Fig. 2, B and C)
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716 Mouse by Rofecoxib and Sulindac.
, both rofecoxib and sulindac treatment resulted in a marked diminution
of polyp number in the
Apc716 mice. The
average inhibition of number of polyps for rofecoxib at 0.0025% w/w or
4.7 mg/kg/day was 36%, for rofecoxib at 0.0075% w/w or 14.7 mg/kg/day
was 55%, and for sulindac 0.015% w/w or 32.6 mg/kg/day was 38%. In
addition, the rofecoxib- and sulindac-treated mice had the greatest
decreases in numbers of the larger-sized polyps, as shown in Fig. 4
and in Table 4
.
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and Fig. 7
. COX-1 and COX-2 protein are microsomal membrane-bound
proteins, and expression was investigated in 100,000 x g pellet fractions only. COX-1 was expressed in both normal
Apc716 intestine
and in polyps from all samples, with no significant changes with
rofecoxib or sulindac treatment (Fig. 5
and Fig. 6
). COX-2 was expressed at low or undetectable levels in normal intestine
samples, but the COX-2 protein was markedly up-regulated in polyp
samples (Figs. 5
. and 6). In some mice, treatment with rofecoxib or
sulindac slightly enhanced the protein amount of COX-1 and COX-2 in
polyps, possibly through stabilization of the protein by decreased
proteolysis as has been reported by others for COX-1 in the presence of
indomethacin (39)
.
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and 6)
. Under our SDS-PAGE
conditions, VEGF remained a dimer at approximately
Mr 42,0000, although this appeared as
a doublet in the membrane fractions (Fig. 5)
. VEGF was markedly
up-regulated in polyps in comparison with the matching normal
epithelium in both membrane and cytosolic fractions (Figs. 5
and 6)
.
Normal intestinal concentrations of VEGF were subtracted from polyp
amounts in each mouse (for three mice from each group), and the mean
cytosolic or membrane VEGF was calculated for each group. Then the VEGF
concentrations in each group were compared, and although there
was high variability in the increases in control polyps there was 68%
decrease in membrane-bound VEGF (P = 0.053;
t test) for the rofecoxib-treated (0.0075% w/w)
group versus control. The mice treated with the lower
rofecoxib concentration (0.0025% w/w) and the sulindac-treated animals
showed a 35% and 45% decrease, respectively, in membrane-bound
VEGF, but with the high control variation, this was not statistically
significant. For cytosolic VEGF concentrations, there was a
nonstatistically significant trend for decreases in polyps of all
treatment groups compared with control polyps.
A representative set of ß-catenin gels and the quantitation of data
from three mice from each group is shown in Fig. 7
. There was an increase in cytosolic ß-catenin expression in the polyp
as compared with normal intestine in all samples: control and
rofecoxib- and sulindac-treated. However, in agreement with the nuclear
localization seen by immunohistochemical analysis, treatment did not
alter significantly the amount of ß-catenin in the
cytoplasmic/nucleoplasmic fractions (Fig. 8)
. The total amount of supernatant ß-catenin in the normal
tissues was at least 100-fold less than the total amount of ß-catenin
in the membrane/microsomal 100,000 x g-pelleted fraction (Fig. 7)
. In comparison to normal
intestine from the same animal, control polyps all had greatly reduced
membrane-bound ß-catenin (Fig. 7)
. However, the concentration of
membrane-bound ß-catenin in control polyps was still 10-fold higher
than the cytosolic concentration. Treatment with rofecoxib and sulindac
tended to increase the membrane-bound ß-catenin in polyps, with a
P value P = 0.059 t
test for the 0.0075% rofecoxib dose (Fig. 8)
.
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716 Mouse after Treatment
with Rofecoxib or Sulindac.
, immunohistochemical staining for ß-catenin was
observed in both the cytoplasm and the nucleus of all treatment groups
with no apparent inhibition in nuclear localization by any of the drug
treatments.
Decreased DNA Replication in
Apc716 Mouse Polyps after
Treatment with Rofecoxib or Sulindac.
We investigated an index of cellular proliferation, i.e.,
BrdUrd-labeling in polyps from control and rofecoxib- and
sulindac-treated mice. In comparison to control BrdUrd-labeling, the
rofecoxib (0.0075% w/w and 0.0025% w/w) and sulindac (0.015% w/w)
showed a 35%, 50%, and 35% decrease in BrdUrd labeling, respectively
(Fig. 9)
.
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DISCUSSION |
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716 mouse.
Rofecoxib at 0.0075% w/w in chow, a dose that gave mouse plasma
concentrations comparable with those achieved in humans at clinical
steady-state with a 25-mg tablet taken once daily, decreases
Apc716 polyp
number by 55% and inhibits 80% of polyps >1 mm in size. Rofecoxib at
0.0025% w/w in chow resulted in a 36% inhibition of polyp number and
a 57% inhibition of polyps >1 mm in size. We used sulindac as our
positive COX-1/COX-2 nonselective inhibitor control because this
compound had been shown previously to inhibit polyp number in the Min
mouse model (6)
. There was greater variability in the
plasma concentrations of sulindac and its metabolites in our study in
comparison with rofecoxib, but an average 38% inhibition of polyp
number and >75% inhibition of polyps >1 mm in size was observed
after treatment with sulindac at 0.015% w/w in chow. Rofecoxib and
sulindac both resulted in a marked size reduction and a flattened
morphology of the remaining polyps. Our sulindac dosing achieved
concentrations of the active COX-1/COX-2 inhibitor sulindac sulfide of
4–9 µM, which should have resulted in
significant inhibition of both COX-1 and COX-2 (Tables 1
and 3)
. In
our mouse study, in comparison with humans, there is a greater
conversion of sulindac to sulindac sulfone, and this metabolite is
approximately six times the concentration of sulindac itself or of
sulindac sulfide (Table 3)
. In our study, the terminal bleed-plasma
concentration of sulindac sulfone was between 15–50
µM, equivalent to about four times that
achieved in humans given sulindac at a 200-mg twice-daily
dosing (37)
. It has been shown previously that sulindac
sulfone at equivalent concentrations to sulindac sulfide (0.5 mg/day in
drinking water) was relatively ineffective at inhibiting polyp number
in the Min mouse (40)
. However, no terminal drug blood
concentrations were determined in the latter study. In a Phase 1 trial
of sulindac sulfone (Exsulind) as a chemopreventive in patients with
FAP, doses of 200 mg and 300 mg twice daily were tolerated and achieved
peak plasma concentrations of 15–30 µM
sulindac sulfone (41)
. However, in the latter study, even
at these relatively high drug concentrations, there was a very modest
clinical effect with a nonsignificant trend toward an increase in
apoptosis but with no decrease in polyp number or cellular
proliferation (41)
. Therefore, we assume that the majority
of polyp-number and -size inhibition seen with sulindac and its
metabolites in our mouse study is attributable to the inhibition of
COX-2 by sulindac sulfide; but there may be a minor contribution of
sulindac sulfone at another target. With regard to sulindac
itself, one study has shown an effect of the parent compound at
100-µM concentrations in vitro on
peroxisome proliferator activated receptor 
transcription
(42)
. Given the blood levels of <10–20
µM at steady state achieved in our mouse study
or for clinical doses of sulindac (Table 3)
, it is unlikely that this
mechanism would be relevant either in our mouse study or with sulindac
in clinical use in humans.
COX-1 protein was observed in all samples of normal epithelium and
polyp, with a weak trend for increase in concentration in the
sulindac-treated mice. It seems that COX-1 activation is not linked
with the process of polyp formation inasmuch as COX-1 protein
concentration is equivalent in the normal intestine and in the polyp,
and rofecoxib shows marked inhibitory growth effects at concentrations
far below any possible COX-1 inhibition (Table 1)
. COX-2 concentrations
were either unchanged or slightly increased in normal intestine or
polyps from the rofecoxib- and sulindac-treated mice, possibly because
of the inhibitor stabilization of protein, as has been reported
previously (38)
. We see no evidence for a transcriptional
decrease in the concentration of COX-2 by rofecoxib.
VEGF protein was markedly elevated in all polyps from each group in comparison with the normal intestinal epithelium control tissue. VEGF concentrations in 100,000 x g pellet and 100,000 x g supernatant fractions of rofecoxib- or sulindac-treated polyps showed a decreasing trend, with less membrane-bound VEGF in the higher-dose rofecoxib-treated polyps. This probably reflects both the decreased vasculature of smaller-sized drug-treated polyps and also the down-regulation of VEGF production within these polyps. Previous studies have shown that the overexpression of COX-2 causes increased cellular VEGF, and that in vivo tumors are less vascularized and grow more slowly in a COX-2-negative host (26 , 43) .
Membrane-bound ß-catenin was reduced 5-fold in the polyps of
control mice in comparison with normal intestinal tissue. This is a
novel finding and the first quantitative measurement of ß-catenin and
its intracellular distribution in mouse polyps. Although there was a
small increase in the cytoplasmic/nucleoplasmic ß-catenin in polyps,
this was <5% of the concentration of ß-catenin lost from the
membrane fraction. Therefore the dramatic reduction in membrane-bound
ß-catenin in control polyps must reflect a decrease in transcription,
translation, or stabilization of the protein. A quantitatively
small amount of ß-catenin, which possibly is qualitatively important
as a transcriptional activator, is localized to the soluble
fraction in the control polyps but not in normal intestine. Rofecoxib
treatment partially restored to normal intestinal levels the
concentration of membrane-bound ß-catenin in polyps.
The rofecoxib-treated polyps may be more differentiated, and hence, may
maintain a more normal complement of ß-catenin-E-cadherin complexes.
Free and bound intracellular pools of catenins have been shown
previously to be in dynamic equilibrium (44)
. A trend for
loss of ß-catenin complexes at cell-cell junctions as has been
reported in primary colorectal tumors and in the corresponding liver
metastases (45)
. It is possible that the loss of membrane
ß-catenin expression may be important in early polyp growth, and
subsequent up-regulation of cytoplasmic/nucleoplasmic ß-catenin may
be important in a later adenoma stage. Progression to adenocarcinoma
may involve both the loss of ß-catenin from membranes and the nuclear
localization of transcriptionally active ß-catenin.
We showed that both rofecoxib and sulindac treatment of mice decreased DNA replication within polyps, as demonstrated by decreased BrdUrd incorporation (although this was not dose-dependent for rofecoxib). We did not investigate apoptosis in this study, although others have shown that sulindac sulfide increases enterocyte apoptosis in Min mice (40) . We assume that rofecoxib- or sulindac sulfide-inhibition of COX-2-produced PGE2 results in decreased proliferation and perhaps increased apoptosis through EP prostanoid receptor-mediated changes in second-messenger signaling.
The potential effects of COX-2 inhibition on immune surveillance were
not investigated in the present study, but COX-2 has been localized to
macrophages in
Apc716 and Min
mouse polyps and in human sporadic polyps (22
, 30
, 46)
. In
addition, enhanced secretion of PGE2 has been
shown by tissue-fixed macrophages in colon carcinomas
(47)
. Specific inhibition of COX-2 in a murine Lewis lung
carcinoma model restores host antitumor reactivity by decreasing the
immune suppressor cytokine interleukin 10 and increasing the
antitumor cytokine interleukin 12 (48)
. Given the
potential for inhibition of COX-2 in tumor, stromal, and immune cells,
it is not surprising that combination therapy of COX-2 inhibitors with
antiproliferative agents and radiation therapy result in synergistic
benefits in tumor regression (49, 50, 51)
.
In conclusion, we present here the first demonstration of the
chemopreventive efficacy of the specific COX-2 inhibitor rofecoxib in
the Apc 716 mouse
model at blood levels comparable with those achieved in humans with a
clinical anti-inflammatory dose. We demonstrate the reduction in both
number and size of polyps by rofecoxib treatment, and that this is
associated with a decrease in membrane-bound VEGF. In addition, we make
the novel observation of a marked decrease in membrane-bound
ß-catenin in control polyps versus normal intestine, and
that rofecoxib treatment partially restores this membrane localization.
On the basis of the data presented here, we suggest that the specific
COX-2 inhibitor rofecoxib (Vioxx) may have a therapeutic benefit in
colorectal cancer.
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ACKNOWLEDGMENTS |
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This paper is dedicated to Lison Bastien and her ongoing battle with cancer.
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FOOTNOTES |
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1 Joint first authors; these authors contributed
equally to this work. 
2 To whom requests for reprints should be addressed, at
Department of Pharmacology, Merck & Co., Inc., WP26A-3000, 770
Sumneytown Pike, West Point, PA 19486. Phone: (215) 652-1254; Fax:
(215) 993-4007; E-mail: jilly_evans{at}merck.com
3 The abbreviations used are: NSAID, nonsteroidal
anti-inflammatory drug; COX, cyclooxygenase; PGG2,
prostaglandin G2; PGE2, prostaglandin
E2; FAP, familial adenomatous polyposis; GI,
gastrointestinal; VEGF, vascular endothelial growth factor; HPLC,
high-performance liquid chromatography; BrdUrd,
bromodeoxyuridine.
Received 8/ 8/00. Accepted 12/13/00.
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716 knockout mice by inhibition of cyclooxygenase 2 (COX-2).. Cell, 87: 803-809, 1996.[Medline]
716 knockout mice.. Cancer Res., 57: 1644-1647, 1997. is an APC-regulated target of nonsteroidal anti-inflammatory drugs.. Cell, 99: 335-345, 1999.[Medline]
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K. Yang, K. Fan, N. Kurihara, H. Shinozaki, B. Rigas, L. Augenlicht, L. Kopelovich, W. Edelmann, R. Kucherlapati, and M. Lipkin Regional response leading to tumorigenesis after sulindac in small and large intestine of mice with Apc mutations Carcinogenesis, March 1, 2003; 24(3): 605 - 611. [Abstract] [Full Text] [PDF]
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M. Yao, S. Kargman, E. C. Lam, C. R. Kelly, Y. Zheng, P. Luk, E. Kwong, J. F. Evans, and M. M. Wolfe Inhibition of Cyclooxygenase-2 by Rofecoxib Attenuates the Growth and Metastatic Potential of Colorectal Carcinoma in Mice Cancer Res., February 1, 2003; 63(3): 586 - 592. [Abstract] [Full Text] [PDF]
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S. Arico, S. Pattingre, C. Bauvy, P. Gane, A. Barbat, P. Codogno, and E. Ogier-Denis Celecoxib Induces Apoptosis by Inhibiting 3-Phosphoinositide-dependent Protein Kinase-1 Activity in the Human Colon Cancer HT-29 Cell Line J. Biol. Chem., July 26, 2002; 277(31): 27613 - 27621. [Abstract] [Full Text] [PDF]
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J. G. Edwards, S. P. Faux, S. M. Plummer, K. R. Abrams, R. A. Walker, D. A. Waller, and K. J. O'Byrne Cyclooxygenase-2 Expression Is a Novel Prognostic Factor in Malignant Mesothelioma Clin. Cancer Res., June 1, 2002; 8(6): 1857 - 1862. [Abstract] [Full Text] [PDF]
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C. Waskewich, R. D. Blumenthal, H. Li, R. Stein, D. M. Goldenberg, and J. Burton Celecoxib Exhibits the Greatest Potency amongst Cyclooxygenase (COX) Inhibitors for Growth Inhibition of COX-2-negative Hematopoietic and Epithelial Cell Lines Cancer Res., April 1, 2002; 62(7): 2029 - 2033. [Abstract] [Full Text] [PDF]
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M. J. Thun, S. J. Henley, and C. Patrono Nonsteroidal Anti-inflammatory Drugs as Anticancer Agents: Mechanistic, Pharmacologic, and Clinical Issues J Natl Cancer Inst, February 20, 2002; 94(4): 252 - 266. [Abstract] [Full Text] [PDF]
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 M. C. Specht, O. N. Tucker, M. Hocever, D. Gonzalez, L. Teng, and T. J. Fahey III Cyclooxygenase-2 Expression in Thyroid Nodules J. Clin. Endocrinol. Metab., January 1, 2002; 87(1): 358 - 363. [Abstract] [Full Text] [PDF]
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Y. Cao, K. B. Dave, T. P. Doan, and S. M. Prescott Fatty Acid CoA Ligase 4 Is Up-Regulated in Colon Adenocarcinoma Cancer Res., December 1, 2001; 61(23): 8429 - 8434. [Abstract] [Full Text] [PDF]
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J. J. Keller, G. J. A. Offerhaus, P. Drillenburg, E. Caspers, A. Musler, A. Ristimaki, and F. M. Giardiello Molecular Analysis of Sulindac-resistant Adenomas in Familial Adenomatous Polyposis Clin. Cancer Res., December 1, 2001; 7(12): 4000 - 4007. [Abstract] [Full Text] [PDF]
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G. Lal, C. Ash, K. Hay, M. Redston, E. Kwong, B. Hancock, T. Mak, S. Kargman, J. F. Evans, and S. Gallinger Suppression of Intestinal Polyps in Msh2-deficient and Non-Msh2-deficient Multiple Intestinal Neoplasia Mice by a Specific Cyclooxygenase-2 Inhibitor and by a Dual Cyclooxygenase-1/2 Inhibitor Cancer Res., August 1, 2001; 61(16): 6131 - 6136. [Abstract] [Full Text] [PDF]
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 M. E. Burleigh, V. R. Babaev, J. A. Oates, R. C. Harris, S. Gautam, D. Riendeau, L. J. Marnett, J. D. Morrow, S. Fazio, and M. F. Linton Cyclooxygenase-2 Promotes Early Atherosclerotic Lesion Formation in LDL Receptor-Deficient Mice Circulation, April 16, 2002; 105(15): 1816 - 1823. [Abstract] [Full Text] [PDF]
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