chemoprevention of intestinal polyposis in the …...thritic dose, for the inhibition of colonic...

[CANCER RESEARCH 61, 1733–1740, February 15, 2001]

Chemoprevention of Intestinal Polyposis in theApcD716 Mouse by Rofecoxib, aSpecific Cyclooxygenase-2 Inhibitor

Masanobu Oshima,1 Naomi Murai(Hata), 1 Stacia Kargman,1 Meztli Arguello, Pauline Luk, Elizabeth Kwong,Makoto M. Taketo, and Jilly F. Evans2

Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd. (Merck), Tsukuba 300-2611, Japan [M. O., N. M. H.]; Merck Frosst Center for Therapeutic Research, Pointe-ClaireDorval, Quebec, Canada H9R 4P8 [S. K., M. A., P. L., E. K.]; University of Tokyo, Graduate School of Pharmaceutical Sciences, Laboratory of Biomedical Genetics, Tokyo113-0033 Japan [M. M. T.]; and Merck & Co., Inc., West Point, Pennsylvania 19486 [J. F. E.]

ABSTRACT

Mutations in the human adenomatous polyposis(APC) gene are causa-tive for familial adenomatous polyposis (FAP), a rare condition in whichnumerous colonic polyps arise during puberty and, if left untreated, leadto colon cancer. TheAPCgene is a tumor suppressor that has been termedthe “gatekeeper gene” for colon cancer. In addition to the 100% mutationrate in FAP patients, theAPC gene is mutated in>80% of sporadic colonand intestinal cancers. TheApc gene in mice has been mutated either bychemical carcinogenesis, resulting in the Min mouseApcD850, or by het-erologous recombination, resulting in theApcD716 or ApcD1368 mice (M.Oshima et al., Proc. Natl. Acad. Sci. USA,92: 4482–4486, 1995). AlthoughhomozygoteApc2/2 mice are embryonically lethal, the heterozygotes areviable but develop numerous intestinal polyps with loss ofApc heterozy-gosity within the polyps (M. Oshimaet al., Proc. Natl. Acad. Sci. USA,92:4482–4486, 1995). The proinflammatory, prooncogenic protein cyclooxy-genase (COX)-2 has been shown to be markedly induced in theApcD716

polyps at an early stage of polyp development (M. Oshimaet al., Cell,87:803–809, 1996). We demonstrate here that treatment with the specificCOX-2 inhibitor rofecoxib results in a dose-dependent reduction in thenumber and size of intestinal and colonic polyps in theApcD716mouse. Theplasma concentration of rofecoxib that resulted in a 55% inhibition ofpolyp number and an 80% inhibition of polyps >1 mm in size is compa-rable with the human clinical steady-state concentration of 25 mg rofe-coxib (Vioxx) taken once daily (A. Porraset al., Clin. Pharm. Ther., 67:137, 2000). Polyps from both untreated and rofecoxib- or sulindac-treatedApcD716 mice expressed COX-1 and -2, whereas normal epithelium fromall mice expressed COX-1 but minimal amounts of COX-2. Polyps fromeither rofecoxib- or sulindac-treated mice had lower rates of DNA repli-cation, expressed less proangiogenic vascular endothelial-derived growthfactor and more membrane-bound b-catenin, but showed unchangednuclear localization of this transcription factor. This study showing theinhibition of polyposis in the ApcD716 mouse suggests that the specificCOX-2 inhibitor rofecoxib (Vioxx) has potential as a chemopreventiveagent in human intestinal and colon cancer.

INTRODUCTION

The majority of epidemiological studies that included the use ofNSAIDs3 as a risk factor have demonstrated that constant use ofNSAIDs is associated with a significantly reduced risk of colon cancer(1). Although each NSAID has unique physical properties and phar-macokinetics, the mechanism of action common to all at clinicallyachievable drug concentrations is the inhibition of COX enzymaticconversion of the polyunsaturated fatty acid arachidonic acid to PGG2

(2). PGG2 is converted to protaglandin H2 by the peroxidase activity

of the COX enzyme, and then PGG2 may be converted to one ofseveral of the five biologically active prostanoids, PGE2, prosta-glandin D2, prostaglandin F2a, prostacyclin, or thromboxane (3).Elevated PGE2 has been measured in rodent and human colonictumors, and the inhibition of prostaglandin synthesis by NSAIDtreatment has been shown to inhibit tumor growth in animal models(4–6). On the basis of such observations, the NSAID sulindac wasstudied in FAP patients for prevention of polyp growth (7). Thisclinical trial showed that treatment with sulindac decreased polypnumber and size, and that when sulindac treatment was stopped, polypgrowth recurred (7).

In the early 1990s, a second form of COX, termed prostaglandinG/H synthase-2 or, more commonly, COX-2, was identified that was60% identical to the original COX-1 (8–10). COX-2 mRNA andprotein were highly inducible by inflammatory and growth factors,whereas COX-1 expression was constitutive in most tissues, includingthe GI tract (8–11). The discovery of the second COX isoform led tothe hypothesis that COX-2-specific inhibitors would be as efficaciousas nonspecific COX-1/COX-2-inhibitor NSAIDs with respect to pros-taglandin-mediated pain and inflammation in arthritis, but with amuch-improved GI safety margin (12). Two specific COX-2 inhibi-tors, i.e., rofecoxib (Vioxx) and celecoxib (Celebrex), have beenshown preclinically and clinically to have comparable efficacy toNSAIDs for relief of pain and inflammation in osteoarthritis, but tohave decreased risk of GI damage (13–18).

Given the epidemiology of NSAID protection for colon cancer, weand others investigated whether this chemopreventive effect might bespecifically through the inhibition of COX-2-produced prostaglan-dins. COX-2 mRNA and protein were shown to be markedly elevatedin human colon tumor tissue, whereas COX-1 expression remainedthe same or decreased (19, 20). COX-2 is also overexpressed inhuman colonic polyps and in macrophages in intimate contact withthese sporadic polyps (21, 22). The growth of human colon tumorcells expressing COX-2 can be inhibitedin vitro and in vivo bytreatment with COX-2 inhibitors (23, 24). Mechanistic studies haverevealed that this growth inhibition results from antiproliferative,proapoptopic, and antiangiogenic effects (23–27). Elevated concen-trations of COX-2 mRNA and protein have now been associated withesophageal, head and neck, breast, lung, prostate, and other cancers,and it has been suggested that COX-2 inhibitors may have benefit inmalignancies other than colon cancer (28).

A relevant animal model in which to test COX-2 inhibitors forprevention of the polyp precursors of adenocarcinomas is theApcD716

mouse, which develops hundreds of intestinal polyps from birththrough the first 3 months of development (29). Both the geneticdeletion of COX-2 expression and pharmacological inhibition withthe specific COX-2 inhibitor, MF-tricyclic, have been shown to mark-edly attenuate the number and size of polyps in theApcD716 mouse(30). The specific COX-2 inhibitor celecoxib (Celebrex) has beenshown to decrease polyp number and size in the chemically inducedApc mutant Min mouse (31). In clinical trials in FAP patients, cele-coxib has also shown moderate efficacy, at twice the approved ar-

Received 8/8/00; accepted 12/13/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

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: [email protected].

3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; COX,cyclooxygenase; PGG2, prostaglandin G2; PGE2, prostaglandin E2; FAP, familial ade-nomatous polyposis; GI, gastrointestinal; VEGF, vascular endothelial growth factor;HPLC, high-performance liquid chromatography; BrdUrd, bromodeoxyuridine.

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thritic dose, for the inhibition of colonic polyps (32). In the studydescribed here, we carefully investigated the efficacy of the specificCOX-2 inhibitor rofecoxib (Vioxx) for chemoprevention of intestinalpolyposis in theApcD716mouse. To profile the potential for long-termprophylactic use as a chemopreventive agent, we chose doses ofrofecoxib at or below the steady-state concentrations achieved at theclinical doses of Vioxx for arthritis. In addition, we monitored pa-rameters of COX expression, angiogenesis, and polyp proliferation tofurther our understanding of the potential mechanism of chemopre-vention by rofecoxib.

MATERIALS AND METHODS

Reagents and Chemicals.Mouse chow containing compounds, synthe-sized at Merck Research Laboratories, was prepared at Purina as describedpreviously (30). Full-length sheep seminal vesicle COX-1- and sheep placentalCOX-2-purified proteins were purchased from Cayman (Ann Arbor, MI) andwere used to generate rabbit polyclonal antibodies, as described previously(20). Under the immunoblotting conditions used in this study, the anti-COXantibodies demonstrated no significant cross-reactivity with the alternate ovineCOX isoform. VEGF antiserum (SC-507), purchased from Santa Cruz Bio-technology (Santa Cruz, CA), was an affinity-purified rabbit polyclonal anti-body raised against an epitope corresponding to amino acids 1–140 of humanorigin. The antiserum recognizes all splice variants of VEGF and cross-reactswith mouse VEGF.b-catenin antisera was purchased from Sigma (St. Louis,MO). All other reagents, if not specifically noted, were of highest reagentgrade.

HPLC Quantitation of Rofecoxib and Sulindac and Its Metabolites andin ApcD716 Mouse Plasma.Drug concentrations were measured in terminal-bleed plasma samples taken from all mice after they were killed at 12 weeksof age. Blood was collected in heparinized tubes and the plasma separated andfrozen at270°C before preparation for HPLC analysis. For the samples from0.0075% w/w rofecoxib and 0.015% w/w sulindac animals, 100ml of plasmawere mixed with an equal volume of acetonitrile, centrifuged at 10,0003 g for15 min, and a 25-ml aliquot of the supernatant was analyzed by reverse-phaseHPLC separation on a HP1090 system (Hewlett-Packard, Palo Alto, CA) withan Eclipse XDB-C18 rapid resolution column (753 4.6 mm, 3.5mm; HewlettPackard) for rofecoxib, a Symmetry C18 column (1503 3.9 mm, 5mm;Waters, Milford, MA) for sulindac, or an Inertsil phenyl column (1003 3 mm,5 mm; MetaChem Technologies, Inc., CA) for sulindac sulfide and sulfone,using a 65:35 (aqueous, 0.1%trifluroacetic acid: acetonitrile, 0.1% trifluoro-acetic acid) solvent at a flow rate of 1 ml/min monitoring at 220 nm forrofecoxib and at 330 nm for sulindac and its metabolites. Drug concentrationswere determined in comparison with standard curves constructed for eachcompound, separated under identical conditions. For the 0.0025% w/w rofe-coxib plasma samples the plasma was concentrated 3-fold using a SpeedVacPlus SC210A (Savant Instruments, Inc., Holbrook, NY) drier before HPLCseparation.

ApcD716 Knockout Mice Construction and Inhibitor Study Protocol.The construction of theApcD716 knockout mice was as described previously(29). ApcD716 knockout mice were prepared byin vitro fertilization using twoC57BL/6 backgroundApcD716 male mice and C57BL/6 female mice. Progenywere genotyped by PCR assay to determine wild type or heterozygote for theApc allele. Five or sixApcD716 heterozygote mice, randomized from eightlitters, were used for each treatment group. After weaning at 3 weeks of age,mice were fedad 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 accordancewith the amount of chow eaten. Final dose achieved for rofecoxib in chow at0.0025% w/w was 4.7 mg/kg/day, for rofecoxib in chow at 0.0075% w/w was14.7 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 orsulindac treatments.

Intestinal Polyp Scoring. At 12 weeks of age, mice were killed by CO2

inhalation, blood samples were taken, and the number and size of polyps wereassessed according to the method described previously (29). Briefly, the wholeGI tract of each mouse was excised and rinsed gently in PBS using a syringebefore being opened longitudinally. The number and size of polyps werecounted under a dissection microscope at315–60 magnification by an indi-vidual blinded to the treatment status of each animal.

Histological Analysis and Immunohistochemistry. After polyp scoringand sampling for Western analysis, intestinal samples were fixed in 10%formaldehyde-PBS, embedded in paraffin, and sectioned at 4-mm thickness.For immunohistochemistry, sections were treated with 3% H2O2 for 1 h andincubated with 10% goat serum-3% BSA in PBS at 37°C for 1 h to blocknonspecific binding. The specimens were then incubated with primary anti-body [1:500 diluted rabbit polyclonal antibody against the COOH terminus ofhumanb-catenin, which cross-reacts with mouseb-catenin (Sigma), in 10%goat serum-3% BSA-PBS] for 60 min at room temperature, and with thesecondary antibody (biotinylated goat antirabbit IgG; Vector Research), andthen incubated with avidin-biotin-peroxidase complex (Vector Research) la-beled with peroxidase and colored with diaminobenzidine substrate.

BrdUrd Incorporation Analysis. A BrdUrd detection kit (BoehringerMannheim) was used according to the manufacturer’s protocol. One represent-ative mouse from each group was inoculatedi.v. with 300 ml of the BrdUrdsolution and sacrificed 4 h later. After polyp scoring, intestinal samples werefixed with 70% ethanol at 4°C overnight, dehydrated, embedded in paraffin,and sectioned serially at 5-mm thickness. Immunostaining of BrdUrd incorpo-ration in nucleus was performed according to the manufacturer’s protocol.Serial sections were stained with hemotoxylin. Three to four independentpolyps from each group were photographed, and the total number of cells andthe number of BrdUrd-positive cells were counted. The labeling indices weredetermined by dividing the number of the labeled nuclei by the number of totalnuclei (about 5000 nuclei for each group).

Preparation of Microsomal Membranes from Intestinal Tissues.Nor-mal mouse intestinal mucosa and autologous polyp tissue were excised, frozenimmediately in liquid N2, and stored at270°C. Frozen tissues were thawed inice-cold homogenization buffer [50 mM potassium phosphate (pH 7.1) con-taining 0.1M NaCl, 2 mM EDTA, 0.4 mM phenylmethylsulfonyl fluoride, 60mg/ml soybean trypsin inhibitor, 2mg/ml leupeptin, 2mg/ml aprotinin, and 2mg/ml pepstatin; all from Sigma Chemical Co., St. Louis, MO]. Tissues weredisrupted twice, for 15 s each, on ice using a tissue-tearer (IKA Labortechnik,Germany). Samples were homogenized by sonication at 4°C using a ColeParmer 4710 series ultrasonic homogenizer (Cole Parmer Instrument Co.,Chicago, IL). Debris was removed by centrifugation at 1,0003 g for 15 minat 4°C, and the resultant supernatants were subjected to centrifugation at100,0003 g for 45 min at 4°C. Membrane fractions were resuspended inhomogenization buffer, and then sonicated to obtain a homogenous membranesuspension. Protein concentrations were determined for each sample using aprotein assay kit (Bio-Rad, Mississauga, Ontario, Canada).

SDS-PAGE and Immunoblot Analysis. Membrane fractions were mixedwith 0.5 volume of SDS sample buffer [20 mM Tris-HCl (pH 6.8) containing0.4% (w/v) SDS, 4% glycerol, 0.24M b-mercaptoethanol, and 0.5% bromphe-nol blue], boiled for 5 min and analyzed by SDS-PAGE on 93 10-cm precast4–20% Tris-glycine acrylamide gels (NOVEX, San Diego, CA) according tothe method of Laemmli (18). Proteins were electrophoretically transferredto nitrocellulose membranes as described previously (19). Primary antisera toCOX-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) andb-catenin(Sigma) were used at final dilutions of 1:500 and 1:5000 according to themanufacturer’s instructions. The secondary horseradish peroxidase-linked goatantirabbit IgG antibody (Santa Cruz Biotechnology) was used at dilutions of1:3000–1:6000 for COX-1 and -2, 1:2000–1:3000 for VEGF and 1:5000 forb-catenin. Immunodetection was performed using enhanced chemilumines-cence according to the manufacturer’s instructions (Amersham). Protein bandswere visualized using a FUJI LAS-1000-plus Luminescent Image AnalyzerFig. 1. Protocol for rofecoxib and sulindac treatment ofApcD716 mice.

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COX-2 INHIBITION AND CHEMOPREVENTION



(Fuji Photo Film Company, Japan) and quantitated using Fuji Film ImageGauge version 3.122. The volumes of absorbance corresponding to the purifiedCOX isoform (Cayman) or a purifiedMr 46,000-tagged fusion protein corre-sponding to amino-terminal amino acids 1–147 of human VEGF orb-catenin(a thioredoxin fusion protein, a kind gift of Astrid Kral, Merck ResearchLaboratories, West Point, PA) proteins were used to calculate the quantity ofCOX, VEGF, orb-catenin protein in normal and polyp intestinal tissues.

RESULTS

Rofecoxib Is a Specific COX-2 Inhibitor in in Vitro HumanCOX-2 Recombinant Whole Cell and ex Vivo Human WholeBlood Assays.We have shown previously, in both human recombi-nant COX-2-expressing cell lines (33) and in human whole-bloodassays (14, 34, 35), that rofecoxib is a highly selective inhibitor ofCOX-2 with minimal activity against COX-1 (Table 1). Sulindac is aprodrugin vivo that is inactive as a COX inhibitor until it is convertedto the metabolite sulindac sulfide, which is a potent inhibitor of bothCOX-1- and COX-2-derived prostaglandin production inin vitrohuman recombinant COX-1 and COX-2 whole-cell assays and also isactive in human whole blood assays (Table 1). The oxidative metab-olite of sulindac,i.e., sulindac sulfone, does not inhibit COX-1 orCOX-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 notavailable.

Steady-state Concentrations of Rofecoxib and Sulindac and ItsMetabolites Sulindac Sulfide and Sulindac Sulfone in theApcD716

Mouse and in Clinical Studies.Concentrations of rofecoxib, sulin-dac, and its metabolites sulindac sulfide and sulindac sulfone weremeasured by HPLC in terminal-bleed plasma samples (Table 2). Therange of drug concentrations in individual mice converted to micro-molar concentrations is compared with the peak and trough concen-trations of steady-state clinical dosing of rofecoxib (Vioxx) and su-lindac (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 somewhatvariable from mouse to mouse (Table 3).

Altered Morphology of Polyps in the ApcD716 Mouse afterTreatment with Rofecoxib or Sulindac. Fig. 2A depicts the mor-phology of polyps in the untreatedApcD716 mice as was observedpreviously (38). Both the rofecoxib and sulindac-treated mice intes-tines had many fewer polyps, and those present were much smallerand flatter than those in the untreated mice intestines (Fig. 2,B andC).

In Vivo Inhibition of Polyp Number and Size in the ApcD716

Mouse by Rofecoxib and Sulindac.As depicted in Fig. 3, bothrofecoxib and sulindac treatment resulted in a marked diminution ofpolyp number in theApcD716 mice. The average inhibition of numberof 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, therofecoxib- and sulindac-treated mice had the greatest decreases innumbers of the larger-sized polyps, as shown in Fig. 4 and in Table 4.

Effect of Rofecoxib and Sulindac Treatment on the Expressionof COX-1, COX-2, VEGF, and b-Catenin Expression.Samples ofboth normal intestine and polyps from three mice from each treatmentgroup were assessed for expression of COX-1, COX-2, VEGF, andb-catenin proteins by specific immunoblot analyses, as shown by arepresentative set of immunoblots in Fig. 5 and Fig. 7. COX-1 andCOX-2 protein are microsomal membrane-bound proteins, and ex-pression was investigated in 100,0003 g pellet fractions only. COX-1was expressed in both normalApcD716 intestine and in polyps from allsamples, with no significant changes with rofecoxib or sulindac treat-ment (Fig. 5 and Fig. 6). COX-2 was expressed at low or undetectablelevels in normal intestine samples, but the COX-2 protein was mark-edly up-regulated in polyp samples (Figs. 5. and 6). In some mice,treatment with rofecoxib or sulindac slightly enhanced the proteinamount of COX-1 and COX-2 in polyps, possibly through stabiliza-tion of the protein by decreased proteolysis as has been reported byothers for COX-1 in the presence of indomethacin (39).

VEGF has been shown to be associated with membranes andcytoplasm and also has been shown to be secreted from cells. Usingan antisera that recognizes all forms of VEGF, we investigated the

Table 1 COX-1 and -2 inhibition potencies in human recombinant whole cell andwhole blood assays

Compound

Human recombinantChinese hampster ovary cell IC50 (mM)a,b,c

Human whole bloodIC50 (mM)d

COX-1 COX-2 COX-1 COX-2

Rofecoxib .20.00 0.02 19 0.5Sulindac .100 .100Sulindac sulfide 0.028 0.004 1.02 10.43Sulindac sulfone .100 .100

a IC50, concentration at which 50% inhibition of prostaglandin product occurs in eachassay.

b Ref. 21.c Ref. 39.d Ref. 40.

Table 2 Plasma concentrations of rofecoxib and sulindac and its metabolites inApcD716 mice

GroupSample

no.Inhibitor(mg/ml)

Average inhibitor(mg/ml) SD

Vioxx, 0.0075% 2-1 0.19814.7 mg/kg/day 2-2 0.217

2-3 0.2222-4 0.1692-5 0.2292-6 0.328 0.23 0.05

Vioxx, 0.0025% 3-1 0.0244.7 mg/kg/day 3-2 0.059

3-3 0.0733-4 0.0433-5 0.017 0.04 0.02

Sulindac, 0.015% 4-1 2.48532.6 mg/kg/day 4-2 0.726Sulindac 4-3 0.266

4-4 2.4764-5 2.057 1.6 1.04

Sulindac, 0.015% 4-1 3.00Sulindac sulfide 4-2 1.29

4-3 1.544-4 1.484-5 1.57 1.78 0.69

Sulindac, 0.015% 4-1 15.66Sulindac sulfone 4-2 6.11

4-3 4.874-4 12.804-5 12.56 10.4 4.67

Table 3 Drug concentrations in ApcD716 mice or in humans at clinicalsteady-state conditions

Plasma concentration (mM)

Rofecoxib Sulindac Sulindac sulfide Sulindac sulfone

ApcD716 miceRofecoxib 0.0025% 0.05–0.22Rofecoxib 0.0075% 0.5–1.0Sulindac 0.015% 0.8–7.5 4–9 15–47Human steady-stateVioxx 25 mg q.d.a 0.3–1.0Clinoril 200 mgb.i.d.b 15 21 8

a Ref. 7 (trough-peak).q.d., every day.b Ref. 41.b.i.d., twice a day.

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expression of cellular VEGF in both 100,0003 g pellets (membranes)and supernatants (cytosol and nucleoplasm) from both normal intes-tine and polyp samples (Figs. 5 and 6). Under our SDS-PAGEconditions, VEGF remained a dimer at approximatelyMr 42,0000,although this appeared as a doublet in the membrane fractions (Fig. 5).VEGF was markedly up-regulated in polyps in comparison with thematching normal epithelium in both membrane and cytosolic fractions(Figs. 5 and 6). Normal intestinal concentrations of VEGF weresubtracted from polyp amounts in each mouse (for three mice fromeach group), and the mean cytosolic or membrane VEGF was calcu-lated for each group. Then the VEGF concentrations in each groupwere compared, and although there was high variability in the in-creases in control polyps there was 68% decrease in membrane-boundVEGF (P 5 0.053; t test) for the rofecoxib-treated (0.0075% w/w)group versuscontrol. The mice treated with the lower rofecoxibconcentration (0.0025% w/w) and the sulindac-treated animalsshowed a;35% and;45% decrease, respectively, in membrane-bound VEGF, but with the high control variation, this was not statis-tically significant. For cytosolic VEGF concentrations, there was a

nonstatistically significant trend for decreases in polyps of all treat-ment groups compared with control polyps.

A representative set ofb-catenin gels and the quantitation of datafrom three mice from each group is shown in Fig. 7. There was anincrease in cytosolicb-catenin expression in the polyp as comparedwith normal intestine in all samples: control and rofecoxib- andsulindac-treated. However, in agreement with the nuclear localizationseen by immunohistochemical analysis, treatment did not alter signif-icantly the amount ofb-catenin in the cytoplasmic/nucleoplasmicfractions (Fig. 8). The total amount of supernatantb-catenin in thenormal tissues was at least 100-fold less than the total amount ofb-catenin in the membrane/microsomal 100,0003 g-pelleted fraction(Fig. 7). In comparison to normal intestine from the same animal,control polyps all had greatly reduced membrane-boundb-catenin

Fig. 3. Inhibition of polyp number by rofecoxib or sulindac treatment ofApcD716mice.ApcD716mice were treated with rofecoxib or sulindac in the chow for 8 weeks. Mice werekilled at 11 weeks of age and polyp number assessed as described in “Materials andMethods.” The mean polyp number for each group is given with one SD given inparentheses.

Fig. 4. Inhibition of polyp size by rofecoxib or sulindac treatment ofApcD716 mice.ApcD716mice were treated with rofecoxib or sulindac in the chow for 8 weeks. Mice werekilled at 11 weeks of age and polyp size and number were assessed as described in“Materials and Methods.”

Table 4 Inhibition of polyp size in ApcD716 mice treated with rofecoxib or sulindac

Polyp Size(mm)

% Inhibition of polyps of specific sizes

Rofecoxib(0.0075% w/w)

Rofecoxib(0.0025% w/w)

Sulindac(0.015% w/w)

,1 39 14 21–2 80 57 762–3 97 91 80.3 100 0 0

Fig. 2. Histology ofApcD716 mice. Hematoxylin and eosin staining histology of rolledintestinal tract from control untreated mouse (A), rofecoxib-treated (0.0075% w/w) mouse(B), and sulindac-treated (0.015% w/w) mouse (C).Arrows, polyps.Bars, 1 mm.

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(Fig. 7). However, the concentration of membrane-boundb-catenin incontrol polyps was still 10-fold higher than the cytosolic concentra-tion. Treatment with rofecoxib and sulindac tended to increase themembrane-boundb-catenin in polyps, with aP valueP 5 0.059t testfor the 0.0075% rofecoxib dose (Fig. 8).

Unchanged Nuclear Localization of Polypb-Catenin in theApcD716 Mouse after Treatment with Rofecoxib or Sulindac. Asshown in Fig. 8, immunohistochemical staining forb-catenin wasobserved in both the cytoplasm and the nucleus of all treatment groupswith no apparent inhibition in nuclear localization by any of the drugtreatments.

Decreased DNA Replication inApcD716 Mouse Polyps afterTreatment with Rofecoxib or Sulindac. We investigated an indexof cellular proliferation,i.e., BrdUrd-labeling in polyps from controland rofecoxib- and sulindac-treated mice. In comparison to controlBrdUrd-labeling, the rofecoxib (0.0075% w/w and 0.0025% w/w) andsulindac (0.015% w/w) showed a 35%, 50%, and 35% decrease inBrdUrd labeling, respectively (Fig. 9).

DISCUSSION

We show here that treatment with either rofecoxib or sulindacresults in a diminution in the number and size of intestinal polyps inthe ApcD716 mouse. Rofecoxib at 0.0075% w/w in chow, a dose thatgave mouse plasma concentrations comparable with those achieved inhumans at clinical steady-state with a 25-mg tablet taken once daily,decreasesApcD716 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 insize. We used sulindac as our positive COX-1/COX-2 nonselectiveinhibitor control because this compound had been shown previouslyto inhibit polyp number in the Min mouse model (6). There wasgreater variability in the plasma concentrations of sulindac and itsmetabolites in our study in comparison with rofecoxib, but an average38% inhibition of polyp number and.75% inhibition of polyps.1mm in size was observed after treatment with sulindac at 0.015% w/w

Fig. 6. Quantitation of COX-1, COX-2, and VEGF protein expression in normal mouseintestinal mucosa and autologous polyp tissue. Microsomal COX-1 protein (A), microso-mal COX-2 protein (B), microsomal VEGF standards (C), and cytosoli VEGF protein (D).Aliquots of COX-1, COX-2, or VEGF standards and of microsomal or supernatant proteinfrom normal (N) or polyp (P) tissue were separated by SDS-PAGE, transferred tonitrocellulose, and blotted with COX-1, COX-2, or VEGF antisera, with detection byenhanced chemiluminescence. The amounts of COX-1, COX-2, or VEGF protein weredetermined by image quantitation using a charge-coupled device camera as indicated in“Materials and Methods.” The area of absorbance for known quantities of COX-1,COX-2, and VEGF proteins were used to assess approximate amounts of COX-1 andCOX-2 and VEGF protein in microsomal or cytosolic samples from the normal mucosaand polyp samples. Values represent the mean6 SE.

Fig. 5. Representative immunoblot analysis of COX-1 and -2 and VEGF proteinexpression in normal mouse intestinal mucosa and autologous polyp tissue. Immunoblotanalysis of matched normal (N) and polyp (P) tissue using an anti-COX-1 antiserum (A),a COX-2 antiserum (B), and a VEGF antiserum (CandD). Purified COX standards andmicrosomal protein samples (50mg/lane and 20mg/lane, COX and VEGF, respectively)were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted withanti-COX-1, anti-COX-2, or anti-VEGF antisera, with detection by chemiluminescence.1and6, protein standards, in ng.2–5 (4 representative mice of 12 mice examined), fromuntreated 0.0075% rofecoxib-treated, 0.0025% rofecoxib-treated, or 0.015% sulindac-treated mice, respectively. Purified COX-1 and COX-2 standards are shown at theleft andright of blots A andB, respectively. Purified VEGF-fusion protein standards, in ng, areshown to theleft of blotsC andD. The apparent molecular weight of the protein standardsis indicated on theright. Values represent the mean6 SEM.

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in chow. Rofecoxib and sulindac both resulted in a marked sizereduction and a flattened morphology of the remaining polyps. Oursulindac dosing achieved concentrations of the active COX-1/COX-2inhibitor sulindac sulfide of 4–9mM, which should have resulted insignificant inhibition of both COX-1 and COX-2 (Tables 1 and 3). In

Fig. 7. Representative immunoblot analysis and quantification ofb-catenin proteinexpression in normal mouse intestinal mucosa and autologous polyp tissue. Aliquots of athioredoxin-b-catenin fusion standard and of normal (N) and matched polyp (P) micro-somal and supernatant protein were separated by SDS-PAGE, transferred to nitrocellu-lose, and blotted withb-catenin antiserum, with detection and quantitation as described in“Materials and Methods.”A, 100,0003 g, pellet; B, 100,0003 g, supernatant;C,quantitation ofA; andD, quantitation ofB. 1, MCF7 mammary adenocarcinoma wholecell lysate control.2, b-catenin fusion protein standards.3–6, samples from untreated,0.0075% rofecoxib-treated, 0.0025% rofecoxib, and 0.015% sulindac-treated mice, re-spectively. Values inC andD, mean6 SE.

Fig. 8. Representativeb-catenin immunohistochemis-try in polyps fromApcD716mice. Serial sections of typicalpolyps from control untreated mouse (Aand B), rofe-coxib-treated (0.0075% w/w) mouse (CandD), and su-lindac-treated (0.015% w/w) mouse (EandF). Hematox-ylin and eosin staining (A,C, andE) and immunostainingwith anti-b-catenin antisera (B,D, andF). In polyps fromall mice, b-catenin is localized to the nucleus, whereasbasolateral staining is observed in normal epithelial cells(arrow headsin B, D, andF). A–C,E, G, andH, bars, 200mm; D, F, andI, bars, 40mm.

Fig. 9. BrdUrd-labeling in polyps fromApcD716 mice. Serial sections of typical polypsfrom control untreated mouse (AandB), rofecoxib-treated (0.0075% w/w) mouse (CandD), and sulindac-treated (0.015% w/w) mouse (EandF). A, C, andE, hematoxylin andeosin staining;B, D, andF, BrdUrd immunostaining.

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our mouse study, in comparison with humans, there is a greater conver-sion of sulindac to sulindac sulfone, and this metabolite is approximatelysix times the concentration of sulindac itself or of sulindac sulfide (Table3). In our study, the terminal bleed-plasma concentration of sulindacsulfone was between 15–50mM, equivalent to about four times thatachieved in humans given sulindac at a 200-mg twice-daily dosing (37).It has been shown previously that sulindac sulfone at equivalent concen-trations to sulindac sulfide (0.5 mg/day in drinking water) was relativelyineffective at inhibiting polyp number in the Min mouse (40). However,no terminal drug blood concentrations were determined in the latterstudy. In a Phase 1 trial of sulindac sulfone (Exsulind) as a chemopre-ventive in patients with FAP, doses of 200 mg and 300 mg twice dailywere tolerated and achieved peak plasma concentrations of;15–30mM

sulindac sulfone (41). However, in the latter study, even at these rela-tively high drug concentrations, there was a very modest clinical effectwith a nonsignificant trend toward an increase in apoptosis but with nodecrease in polyp number or cellular proliferation (41). Therefore, weassume that the majority of polyp-number and -size inhibition seen withsulindac and its metabolites in our mouse study is attributable to theinhibition of COX-2 by sulindac sulfide; but there may be a minorcontribution of sulindac sulfone at another target. With regard to sulindacitself, one study has shown an effect of the parent compound at$100-mM

concentrationsin vitro on peroxisome proliferator activated receptordtranscription (42). Given the blood levels of,10–20mM at steady stateachieved in our mouse study or for clinical doses of sulindac (Table 3),it is unlikely that this mechanism would be relevant either in our mousestudy or with sulindac in clinical use in humans.

COX-1 protein was observed in all samples of normal epitheliumand polyp, with a weak trend for increase in concentration in thesulindac-treated mice. It seems that COX-1 activation is not linkedwith the process of polyp formation inasmuch as COX-1 proteinconcentration is equivalent in the normal intestine and in the polyp,and rofecoxib shows marked inhibitory growth effects at concentra-tions far below any possible COX-1 inhibition (Table 1). COX-2concentrations were either unchanged or slightly increased in normalintestine or polyps from the rofecoxib- and sulindac-treated mice,possibly because of the inhibitor stabilization of protein, as has beenreported previously (38). We see no evidence for a transcriptionaldecrease in the concentration of COX-2 by rofecoxib.

VEGF protein was markedly elevated in all polyps from each groupin comparison with the normal intestinal epithelium control tissue.VEGF concentrations in 100,0003 g pellet and 100,0003 g super-natant fractions of rofecoxib- or sulindac-treated polyps showed adecreasing trend, with less membrane-bound VEGF in the higher-doserofecoxib-treated polyps. This probably reflects both the decreasedvasculature of smaller-sized drug-treated polyps and also the down-regulation of VEGF production within these polyps. Previous studieshave shown that the overexpression of COX-2 causes increased cel-lular VEGF, and thatin vivo tumors are less vascularized and growmore slowly in a COX-2-negative host (26, 43).

Membrane-boundb-catenin was reduced;5-fold in the polyps ofcontrol mice in comparison with normal intestinal tissue. This is a novelfinding and the first quantitative measurement ofb-catenin and its intra-cellular distribution in mouse polyps. Although there was a small increasein the cytoplasmic/nucleoplasmicb-catenin in polyps, this was,5% ofthe concentration ofb-catenin lost from the membrane fraction. There-fore the dramatic reduction in membrane-boundb-catenin in controlpolyps must reflect a decrease in transcription, translation, or stabilizationof the protein. A quantitatively small amount ofb-catenin, which possi-bly is qualitatively important as a transcriptional activator, is localized tothe soluble fraction in the control polyps but not in normal intestine.Rofecoxib treatment partially restored to normal intestinal levels theconcentration of membrane-boundb-catenin in polyps. The rofecoxib-

treated polyps may be more differentiated, and hence, may maintain amore normal complement ofb-catenin-E-cadherin complexes. Free andbound intracellular pools of catenins have been shown previously to be indynamic equilibrium (44). A trend for loss ofb-catenin complexes atcell-cell junctions as has been reported in primary colorectal tumors andin the corresponding liver metastases (45). It is possible that the loss ofmembraneb-catenin expression may be important in early polyp growth,and subsequent up-regulation of cytoplasmic/nucleoplasmicb-cateninmay be important in a later adenoma stage. Progression to adenocarci-noma may involve both the loss ofb-catenin from membranes and thenuclear localization of transcriptionally activeb-catenin.

We showed that both rofecoxib and sulindac treatment of micedecreased DNA replication within polyps, as demonstrated by de-creased BrdUrd incorporation (although this was not dose-dependentfor rofecoxib). We did not investigate apoptosis in this study, althoughothers have shown that sulindac sulfide increases enterocyte apoptosisin Min mice (40). We assume that rofecoxib- or sulindac sulfide-inhibition of COX-2-produced PGE2 results in decreased proliferationand perhaps increased apoptosis through EP prostanoid receptor-mediated changes in second-messenger signaling.

The potential effects of COX-2 inhibition on immune surveillancewere not investigated in the present study, but COX-2 has beenlocalized to macrophages inApcD716 and Min mouse polyps and inhuman sporadic polyps (22, 30, 46). In addition, enhanced secretion ofPGE2 has been shown by tissue-fixed macrophages in colon carcino-mas (47). Specific inhibition of COX-2 in a murine Lewis lungcarcinoma model restores host antitumor reactivity by decreasing theimmune suppressor cytokine interleukin 10 and increasing the antitu-mor cytokine interleukin 12 (48). Given the potential for inhibition ofCOX-2 in tumor, stromal, and immune cells, it is not surprising thatcombination therapy of COX-2 inhibitors with antiproliferative agentsand radiation therapy result in synergistic benefits in tumor regression(49–51).

In conclusion, we present here the first demonstration of the che-mopreventive efficacy of the specific COX-2 inhibitor rofecoxib inthe Apc D716 mouse model at blood levels comparable with thoseachieved in humans with a clinical anti-inflammatory dose. We dem-onstrate the reduction in both number and size of polyps by rofecoxibtreatment, and that this is associated with a decrease in membrane-bound VEGF. In addition, we make the novel observation of a markeddecrease in membrane-boundb-catenin in control polypsversusnor-mal intestine, and that rofecoxib treatment partially restores thismembrane localization. On the basis of the data presented here, wesuggest that the specific COX-2 inhibitor rofecoxib (Vioxx) may havea therapeutic benefit in colorectal cancer.

ACKNOWLEDGMENTS

We thank Emilie Beskar and Drs. Tony Ford-Hutchinson, Robert Gould,Astrid Kral, Nancy Kohl, Ken Thomas, and David Heimbrook (Merck, WestPoint, PA); Dr. Jules Schwartz (Merck, Rahway, NJ); Christine Brideau, Drs.Elizabeth Vadas, Denis Riendeau, Chi Chan, Joseph Mancini, and GaryO’Neill (Merck Frosst, Montreal, Canada); and Dr. Mitsuaki Yoshida (Banyu-Merck, Tsukuba) for their support. We also thank Dr. Thomas Hugh (RoyalNorth Shore Hospital, Sydney, Australia) for helpful discussions onb-cateninlocalization, and Sandy Camburn for her outstanding administrative assistance.

This paper is dedicated to Lison Bastien and her ongoing battle with cancer.

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2001;61:1733-1740. Cancer Res Masanobu Oshima, Naomi Murai(Hata), Stacia Kargman, et al. Mouse by Rofecoxib, a Specific Cyclooxygenase-2 Inhibitor

716∆ApcChemoprevention of Intestinal Polyposis in the

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