Honours Thesis

Suppression of the chemokine receptor CXCR4 on the surface of colorectal cancer

cells by non-steroidal anti-inflammatory drugs (NSAIDs)

David Chiu

Supervisor: Dr. Jonathan Blay, PhDDepartment of Pharmacology

Faculty of MedicineDalhousie UniversityHalifax, Novas Scotia

ii

April 10, 2006Table of Contents

List of Figures and Tables iiiAbstract ivList of Abbreviations vAcknowledgements vi

IntroductionHistory of NSAIDs 1Colorectal Cancer – The Problem1Colorectal Cancer – Prevention 3NSAIDs inhibit cyclooxygenase enzymes 4CXCR4, a chemoreceptor implicated in tumour progression and metastasis 6Hypothesis 7Specific Objectives 7

Materials and Methods 8

ResultsValidation of the assay system 9COX1-selective NSAIDs produce significant but variable inhibition of cell-surface CXCR4 9COX2-selective NSAIDs produce significant but variable inhibition of cell-surface CXCR4 12Sulindac and its metabolites are potent inhibitors of CXCR4 expression 14Only sulfasalazine’s activated metabolite, 5-ASA, decreases CXCR4 expression 16

DiscussionThe down-regulation of CXCR4 by NSAIDs shows features of COX dependence 22The COX-related down-regulation of CXCR4 does not seem to be through either COX isoenzyme alone 26Possible COX-independent pathways 27Significance of findings 30Conclusion 31

References 32

iii

List of Figures and Tables

Figure 1 Genetic model of colorectal tumorigenesis 2Figure 2 Eicosanoid biosynthesis by cyclooxygenase enzymes 5Figure 3 COX-1 selective NSAIDs produce significant but variable

inhibition of cell-surface CXCR4. 11Figure 4 COX-2 selective NSAIDs produce significant but variable

inhibition of cell-surface CXCR4. 13Figure 5 Sulindac compounds are potent inhibitors of CXCR4 expression. 15Figure 6 Sulfasalazine and its metabolites have little effect on CXCR4 expression. 17Figure 7 Relation between COX and CXCR4 inhibition 20Figure 8 Relation between CXCR4 inhibition and relative COX selectivity 21Figure 9 Proposed mechanism by which Sulindac and its two metabolites

down-regulate CXCR4 25Figure 10 COX-independent action of NSAIDs 28

Table 1 Potency of NSAIDs in inhibiting cell-surface CXCR4 18Table 2 Comparison of CXCR4 inhibition to COX potency and selectivity 23

iv

Abbreviations

5-ASA 5-aminosalicylic acidAPC Adenomatosis polyposis coliBSA Bovine serum albumincpm Counts per minuteCOX CyclooxygenaseCRC Colorectal cancerCXCL12 CXC ligand 12CXCR4 CXC chemokine receptor 4DMSO Dimethyl sulfoxideFAP Familial adenomatous polyposisIC Inhibitory concentrationIg ImmunoglobulinLOX LipooxygenaseNCS Newborn calf serumNF Nuclear factorNS-398 N-[2-(Cyclohexyloxy)-4-nitrophenyl]methanesulfonamideNSAID Non-steroidal anti-inflammatory drugPBS Phosphate-buffered salinePG ProstaglandinPGI2 ProstacyclinPPAR Peroxisome proliferator-activated receptor

v

Acknowledgements

First and foremost, I would like to thank my supervisor, Dr. Jonathan Blay, for this immense

opportunity to learn and think and persist and laugh and rejoice. Your endless support and

encouragement has made this time seem less like work and more like playing, sometimes (or

rather often) in the literal sense.

The time would not have been quite the same without the Blayettes, Cynthia Richard, Erica

Lowthers and Susan Tyler (in no particular order of liking or disliking). Your equally endless

opposition and discouragement has made me the manly man that I am today.

In light of the above, I would like to thank Heather Sams for always had my back during the

rough and tough times.

And last but certainly not least, I would like to thank Ernest Tan, whose retirement from the lab

and the sport of tennis will forever be missed. In these two areas (and probably many more), you

taught me everything I know.

vi

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to have various anti-cancer

properties. These effects are thought to be mediated largely by their inhibition of prostaglandin

biosynthesis by preventing the action of cyclooxygenase (COX) enzymes. In preliminary studies,

our lab found that certain NSAIDs down-regulated the expression of cell-surface CXCR4, a

chemokine receptor implicated in various tumourigenic processes, on HT-29 colorectal cancer

(CRC) cells. It is thought that NSAIDs may be exerting their anti-cancer effects, at least in part,

through the down-regulation of CXCR4. Furthermore, this decrease may result from the

inhibition of COX. The present study expanded on these findings by considering whether

NSAIDs in general down-regulate CXCR4, and if so, whether it is a COX-dependent effect. Four

groups of broadly-acting and structurally distinct NSAIDs were assessed for their ability to

decrease cell-surface CXCR4 expression on HT-29 cells in vitro. Of the 12 compounds

examined eight produced consistent and significant reductions in CXCR4. The down-regulation

of CXCR4 was dose-dependent up to the highest (100 µM) concentration examined. Cell

number- and isotype-corrected values from four independent experiments were used to calculate

each compound’s IC25. Variations in CXCR4 inhibiting potencies did not seem to be a function

of the compound’s potency or selectivity in COX inhibition. These findings suggest that COX-

independent pathways may be partly or even mostly involved in the down-regulation of CXCR4.

vii

Introduction

History of NSAIDsOne hundred years after the advent of aspirin, non-steroidal anti-inflammatory drugs

(NSAIDs) have become some of the most commonly and regularly used drugs in the treatment of

inflammation, pain and fever. They are a group of broadly-acting and structurally distinct

compounds that are typically orally administered and easily absorbed in the intestine. Highly

bound to plasma proteins, NSAIDs circulate throughout the body and act on tissue only in their

free form. Most of the drugs are deactivated by enzymes in the liver while some are administered

as prodrugs which become physiologically activated by enzymes in certain areas of the body.

The effects of NSAIDs are by and large mediated through the inhibition of prostaglandin

biosynthesis by cyclooxygenase (COX) enzymes; however, due to the multifunctional nature of

COX, side effects have been linked to NSAIDs amongst the high risk population (Singh, 1998).

Conventional non-selective COX inhibitors such as aspirin are associated with gastrointestinal

disturbances such as peptic ulcers and gastrointestinal bleeding (Wolfe et al., 1999), while the

newer generation of selective COX-2 inhibitors such as celecoxib have in very rare instances

caused cardiovascular complications such as myocardial infarctions, strokes and heart failures

(Caldwell et al., 2006, Solomon et al., 2005).

Colorectal Cancer – The Problem

1

viii

Colorectal cancer (CRC) has been the focus of much research in recent history. We now

know that the progression to CRC involves a stepwise series of somatic or germline mutations,

each of which confers a proliferative advantage on the mutated cell (Vogelstein & Kinzler,

2004). This process of clonal expansion underlies the long latency period of 10 to 15 years

(Nowell, 2002) during which time normal tissue transforms into neoplastic adenomatous tissue,

and adenomatous tissue becomes a malignant carcinoma (Fig. 1).

Figure 1. The genetic model of colorectal tumorigenesis is relatively well characterized.

Tumorigenesis is a well-characterized, stepwise process often involving an initial mutation in the

adenomatosis polyposis coli (APC) gene which renders an individual prone to developing

intestinal polyps, or gastrointestinal ingrowths. The formation of polyps is a significant risk

factor leading to CRC. Subsequent mutations in oncogenes and tumor suppressor genes drive the

progression from adenoma to adenocarcinoma to carcinoma. From Brown and DuBois (2005).

2

ix

CRC is the fourth most commonly diagnosed form of cancer in Canada but the second

leading cause of cancer mortality (Canadian Cancer Society, 2006). Current treatment options

include surgery, chemotherapy and radiation therapy, each of which is an attempt to either

remove the cancerous tissue or slow its uncontrolled growth. Despite some success, our

treatment options are few and for the most part ineffective for individuals with advanced or

metastatic forms of CRC. The five year survival rate for patients with non-metastatic CRCs is

90% compared to a grim 19% for those whose tumour cells have gained the ability to metastasize

to distant sites in the body (Edwards et al., 2005). As cases of CRC are often not diagnosed at the

pre-metastatic stage, this results in the high mortality rate for CRC.

Colorectal Cancer – PreventionGiven the challenges in diagnosing CRC in its early stages and the resulting high

mortality rate, an increasing focus has been directed towards the prevention of CRC. Three

approaches exist in this regard.

First and foremost, a healthy lifestyle is well-recognized in lowering an individual’s

chance of developing many types of cancers (Martinez, 2005). Specific risk factors include

smoking, alcohol consumption, physical inactivity and a poor diet. Underscoring these factors is

the belief that the discrepancy in cancer incidence between North America and many Asian

countries is due to differences in dietary habits (Parkin, 2001). This presumption is supported by

epidemiological and clinical evidence suggesting that an increased dietary intake of vitamin A

and carotenoids, compounds often found in fruits and vegetables, significantly lowers the

formation of intestinal polyps (Nkondjock & Ghadirian, 2004, Steck-Scott et al., 2004).

A second form of prevention involves regular screening and treatment. Individuals with

familial adenomatous polyposis (FAP) have a germline, autosomal dominant mutation in the

3

x

APC gene (Galiatsatos & Foulkes, 2006). This disease is characterized by the formation of

premalignant intestinal polyps, and if left untreated, will inevitably lead to the development of

CRC by the individual’s third or fourth decade of life. Polypectomies, or the removal of

premalignant polyps, are very effective in keeping patients with FAP cancer free (Smith et al.,

2006). Unfortunately, regular screening procedures like colonoscopies and flexible

sigmoidoscopies are undergone by only 50% of Americans. In addition, the issue of economic

feasibility presents a problem for many health care professionals (Winawer, 2005).

The great potential of chemoprevention has been receiving more serious attention within

the scientific community. Agents found to have anti-cancer properties in vitro include folate

(Lamprecht & Lipkin, 2003, Song et al., 2000), retinoids (Suzui et al., 2006), calcium ((Govers

et al., 1996, Wallace et al., 2004) and hormones such as androgen and estrogen (Algarte-Genin

et al., 2004, Limer & Speirs, 2004).

Of particular interest has been the chemoprevention of CRC by NSAIDs (Ulrich et al.,

2006). Initial studies revealed that chemically induced tumour growth in mice was inhibited by

indomethacin, a potent and non-selective NSAID (Kudo et al., 1980, Narisawa et al., 1981). This

foreshadowed the landmark epidemiological study by Kune in 1988 (Kune et al., 1988) which

found that the regular use of aspirin reduced the risk of CRC in humans. Since then, various

experimental and clinical studies have been carried out with evidence clearly pointing towards

the anti-cancer properties of NSAIDs in not only CRC but also cancers of the lung (Holick et al.,

2003), oesophagus (Corley et al., 2003), breast (Harris et al., 2003, Terry et al., 2004), prostate

(Mahmud et al., 2004) and stomach (Wang et al., 2003) .

Despite these beneficial effects, the potentially serious side effects of NSAIDs limits their

use in high risk patients (Becker, 2005, Singh, 1998). This concern has directed recent scientific

investigation into the molecular mechanism by which NSAIDs exert their desirable effects, with

the goal of designing drugs with reduced unwanted effects and enhanced therapeutic profiles.

Several major target pathways have been identified including the COX, lipooxygenase (LOX),

NF-κB and peroxisome proliferator-activated receptor (PPAR) pathways (Kashfi & Rigas, 2005).

NSAIDs inhibit cyclooxygenase enzymes

4

xi

The anti-inflammatory, anti-pyretic and analgesic effects of NSAIDs are for the most part

the result of COX inhibition. There are two important isoenzymes, the COX-1 and the COX-2

isoenzyme (Figure 2).

5

xii

Figure 2. Eicosanoid biosynthesis by cyclooxygenase enzymes

Both COX isoenzymes play key roles in the formation of eicosanoids, or products of arachidonic

acid metabolism. The enzymes convert arachidonic acid into prostaglandin (PG) G and

subsequently to PGH2, a precursor to all eicosanoids. Modified from Rang et al. (2003).

The COX-1 isoenzyme is widely and constitutively expressed with a prominent role in

body homeostasis (Dubois et al., 1998). It is the major COX isoenzyme in red blood cells and in

this respect converts prostaglandin (PG) H2 into thromboxane A2, a key factor in platelet

functioning, blood clotting (Hankey & Eikelboom, 2006) and vasoconstriction. The latter effect

of thromboxane is counterbalanced by prostacyclin (PGI2) produced by COX-1 in endothelial

cells. Thus, the basal expression of COX-1 regulates blood flow within the body.

The COX-2 isoenzyme is only constitutively expressed in certain areas of the body.

COX-2 in the kidney produces PGs that modulate water and electrolyte homeostasis (Harris et

al., 1994). In the brain, PGs produced by COX-2 induce fevers (Cao et al., 1997). The inhibition

of PG synthesis in the brain then is the basis for the anti-pyretic activity of NSAIDs. During

inflammation, COX-2 expression is induced resulting in the production of local mediators of the

inflammatory response such as PGE2 and PGI2 (Anderson et al., 1996).

COX expression is often up-regulated in CRC (Eberhart et al., 1994) as well as many

other cancers (Soslow et al., 2000). It is not surprising then that the production of PGE2, a

principal COX product, is dramatically increased in tumour tissue compared to normal adjacent

mucosa (Pugh & Thomas, 1994, Rigas et al., 1993). Although the role of COX in cancer

progression has not been completely elucidated, its importance is evident.

CXCR4, a chemoreceptor implicated in tumour progression and metastasisCXCR4 is a G-protein-coupled chemokine receptor whose only known ligand is

CXCL12 (stromal cell-derived factor 1 – SDF-1α), a growth factor and chemoattractant. The

CXCR4-CXCL12 axis has a major role in directing cells throughout the body (Tachibana et al.,

6

xiii

1998, Zou et al., 1998). Hematopoietic stem cells from the bone marrow, for example, home

towards the high levels of CXCL12 secreted by liver cells post-injury (Dalakas et al., 2005).

CXCR4 is highly expressed in various cancers including those of the breast, prostate,

lung, esophagus and stomach (Darash-Yahana et al., 2004, Kaifi et al., 2005, Oda et al., 2006,

Salvucci et al., 2005, Yasumoto et al., 2006). In CRC patient samples, CXCR4 is more highly

expressed than in surrounding normal tissue (Dwinell et al., 1999, Jordan et al., 1999, Kim et al.,

2005). CXCR4 is also the most consistently expressed of the chemokine receptors. In CRC, high

CXCR4 expression is implicated in tumour cell proliferation, protection from apoptosis and

metastasis (Richard et al., 2006, Zeelenberg et al., 2003). In endothelial cells, high CXCR4

expression promotes tumour angiogenesis, or vascular growth (Guleng et al., 2005). As

expected, antagonizing CXCR4 or inhibiting its expression decreases these tumourigenic

processes (Chen et al., 2003, Liang et al., 2004, Marchesi et al., 2004) as well as the tumour

burden in murine models (Rubin et al., 2003).

Given all of these findings, it is not surprising that increased CXCR4 in tumours is

associated with poor prognosis in patients with CRC (Kim et al., 2005) as well as other cancers

(Kaifi et al., 2005, Laverdiere et al., 2005). Clearly then, CXCR4 is a good target for cancer

therapies.

Preliminary studies in our lab suggested that select NSAIDs could decrease cell-surface

CXCR4 expression on HT-29 cells. Perhaps then, one mechanism by which NSAIDs exert their

anti-cancer effects is by down-regulating CXCR4 expression in tumour cells. It is this possibility

that has led to my current investigation.

HypothesisNSAIDs down-regulate cell-surface CXCR4 expression on colorectal cancer cells in vitro

through a COX-dependent mechanism.

Specific Objectives To verify that NSAIDs in general cause a decrease in cell-surface CXCR4 expression;

To establish whether the effect is mediated by inhibiting COX; and

To explore whether either of the two COX isoenzymes play an exclusive role in this

effect.

7

xiv

I selected four groups of broadly-acting and structurally distinct NSAIDs and examined their

effect on CXCR4 in an in vitro system. My findings suggest that NSAIDs down-regulation

CXCR4 and that the pattern shows features of both COX-dependence and COX-independence.

Materials and Methods

MaterialsThe HT-29 human colorectal carcinoma cell line was from the American Type Culture

Collection (Manassas, VA). Media, sera and culture vessels (Nunc) were from Invitrogen

Canada (Burlington, Ontario, Canada). Adenosine, piroxicam, indomethacin, aspirin, diclofenac,

meloxicam, NS-398, sulindac, sulindac sulfide, sulindac sulfone, sulfasalazine, 5-aminosalicylic

acid and sulfapyridine were from Sigma Chemical Co. (St. Louis, MO). Mouse anti-human

CXCR4 monoclonal antibody (clone 12G5) and anti-mouse IgG2a isotype control antibodies

(clone G155-178) were from BD Pharmingen (San Diego, CA). 125I-labeled sheep anti-mouse

IgG, F(ab')2 fragment was obtained from PerkinElmer Life Sciences (NEN, Boston, MA).

Cell cultureCells were cultured in DMEM with 5% (v/v) newborn calf serum (NCS). For binding assays,

cells were seeded with 10% v/v NCS into 48-well plates at 50,000 cells/well. In all culture

situations, cells were first allowed to attach for 48 h. The medium was then replaced with

DMEM containing 1% NCS, and after a further 48 h the cultures were treated with drugs at

concentrations from 1 to 100 μM or with vehicle controls. Control treatments always included

the appropriate solvent control, which in this case was a dimethyl sulfoxide (DMSO)

concentration of no greater than 0.05% (v/v). Binding assays were performed after a 48-h drug

treatment.

8

xv

Assay for cell-surface CXCR4An indirect radioantibody binding assay that provides quantitative measurement of proteins

exposed on cultured cell monolayers (Tan et al., 2004) was used to measure cell-surface CXCR4

protein levels. All steps were performed at 4°C. Monolayer cultures were washed with

phosphate-buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) and then

incubated with 125 µL PBS containing 1% BSA and 1 µg/mL of anti-CXCR4 or isotype control.

After a 60-min incubation, the cells were washed twice and further incubated with 125 µL PBS

containing 1% BSA and 1 μCi/mL 125I-labeled goat anti-mouse IgG for 60 minutes. The

monolayers were then washed three times and solubilized in 0.5 M NaOH, followed by counting

of radioactivity. The CXCR4-specific radioactivity was determined by subtracting the result for

the corresponding isotype control. Cell counts were performed using a Coulter® Model

ZM30383 particle counter (Beckman Coulter, Mississauga, Ontario, Canada), and results were

corrected to counts per minute per 100,000 cells.

Statistical AnalysisEach figure shows a representative result from a series of experiments done on at least four

independent occasions. Data were analyzed using Students t-test and are indicated as such if

significant at the P < 0.05 (*, #) or P < 0.01 (**, ##) level.

Inhibitory Concentration ValuesCell number- and isotype-corrected data from four independent experiments were used to

calculate IC25 values as shown in Table 1.

Results

Validation of the assay systemAdenosine, a purine nucleotide found in high concentrations within the tumour

microenvironment (Blay et al., 1997), was used to show that CXCR4 could be positively

9

xvi

regulated in these cells as expected (Richard et al., 2006). A significant up-regulation was

produced at concentrations as low as 3 μM while an increasing trend was still evident at 300 μM.

COX1-selective NSAIDs produce significant but variable inhibition of cell-surface CXCR4

The use of non-selective (i.e. relatively COX-1 selective) NSAIDs like piroxicam,

indomethacin and aspirin in CRC chemoprevention has been supported by various murine model

studies (Reddy & Rao, 2005, Ulrich et al., 2006). In the current study, piroxicam and

indomethacin were found to produce dose-dependent decreases in CXCR4 while aspirin

interestingly had no effect up to 100 µM (Fig. 3). Indomethacin had the greatest effect at the

highest concentration examined (100 µM), inhibiting CXCR4 expression from 50 to 100%. Its

high potency was further reflected in its IC25 value, which was eight times lower than that of

piroxicam (Fig. 1. Panel D).

10

xvii

Figure 3. COX-1 selective NSAIDs produce significant but variable inhibition of cell-

surface CXCR4.

(a) (b)

(c) (d)

11

xviii

48-h after the addition of (a) piroxicam, (b) indomethacin and (c) aspirin at concentrations

between 0 and 100 μM, cells were assayed for cell-surface CXCR4 (mean ± S.E.) using an

indirect radioantibody binding assay. Cell number- and isootype-corrected values from four

independent experiments were used to calculate the IC25 value for each drug (d).

COX2-selective NSAIDs produce significant but variable inhibition of cell-surface CXCR4

The newer COX-2 selective inhibitors have been the focus of much attention as evidence

suggests the COX-2 isoform has a major role in tumorigenesis. Studies have found that the

COX-2 isoform is highly expressed in CRC patient tumours (Eberhart et al., 1994) while mice

prone to developing CRC but not expressing the COX-2 gene show reduced polyp formation and

better prognosis (Oshima et al., 1996).

Given these findings, the effect of COX-2 selective inhibitors on CXCR4 was also

assessed. Both diclofenac and meloxicam produced reliable dose-dependent decreases in CXCR4

(Fig. 4). Meloxicam produced a more gradual decline over the concentrations examined while

diclofenac had little effect sometimes up to 30µM before a sharp decline was observed.

Interestingly, NS-398 seemed to produce a modest decrease (Fig. 4. Panel C) but after values

were corrected for non-specific radioactivity and cell number, no effect was seen (Fig. 4. Panel

D).

12

xix

Figure 4. COX-2 selective NSAIDs produce significant but variable inhibition of cell-

surface CXCR4.

(a) (b)

(c) (d)

13

xx

48-h after the addition of (a) diclofenac, (b) meloxicam and (c) NS-398 at concentrations

between 0 and 100 μM, cells were assayed for cell-surface CXCR4 (mean ± S.E.) using an

indirect radioantibody binding assay. Cell number- and isootype-corrected values from four

independent experiments were used to calculate the IC25 value for each drug (d).

Sulindac and its metabolites are potent inhibitors of CXCR4 expressionSulindac, a prodrug, is itself without marked direct effects on tissue physiology.

Interestingly, in the present study, it produced a significant and reliable decrease in CXCR4. In

the body, the prodrug is absorbed by the intestinal epithelium and passes into the liver where it is

either reversibly converted to the physiologically active sulindac sulfide (which is known to have

a 500-fold increase in potency), or irreversibly oxidized to the inactive sulindac sulfone (Duggan

et al., 1978). These compounds are then secreted back into the intestinal lumen along with the

bile. It was interesting to note that both metabolites produced a greater decrease in CXCR4 than

the parent drug at 100 µM (Fig. 5. Panel B and C), though notably the activated sulfide was

almost 10 times more potent (Fig. 5. Panel D).

14

xxi

Figure 5. Sulindac compounds are potent inhibitors of CXCR4 expression.

(a) (b)

(c) (d)

15

xxii

48-h after the addition of (a) sulindac, (b) sulindac sulfide and (c) sulindac sulfone at

concentrations between 0 and 100 μM, cells were assayed for cell-surface CXCR4 (mean ± S.E.)

using an indirect radioantibody binding assay. Cell number- and isootype-corrected values from

four independent experiments were used to calculate the IC25 value for each drug (d).

Only sulfasalazine’s activated metabolite, 5-ASA, decreases CXCR4 expressionSulfasalazine is used in the treatment of non-specific inflammatory bowel diseases like

ulcerative colitis and Crohn’s disease, both substantial risk factors for CRC (Cheng &

Desreumaux, 2005, van Staa et al., 2005). In the present system however, sulfasalazine had no

effect on CXCR4 expression. I further examined the effects of its two metabolites, 5-

aminosalicylic acid (5-ASA) and sulfapyridine, which result from the breakdown of sulfasalazine

by intestinal bacterial enzymes. Although sulfapyridine had no effects, 5-ASA produced a small

but significant decrease in CXCR4 levels in all experiments (Fig. 4. Panel B). The decrease was

usually significant by 30 µM.

IC25 values for all 12 compounds are shown in Table 1.

16

xxiii

(a) (b)

(c) (d)

17

xxiv

Figure 6. Sulfasalazine and its metabolites have little effect on CXCR4 expression.

48-h after the addition of (a) sulfasalazine, (b) 5-aminosalicylic acid and (c) sulfapyridine at

concentrations between 0 and 100 μM, cells were assayed for cell-surface CXCR4 (mean ± S.E.)

using an indirect radioantibody binding assay. Cell number- and isootype-corrected values from

four independent experiments were used to calculate the IC25 value for each drug (d).

Compound

Potency

CXCR4IC25

(µM)COX-1

SelectivePiroxicam 57.8 ±16.6

Indomethacin 8.05 ±1.78

Aspirin >100

COX-2 Selective

Diclofenac 27.2 ±8.8

Meloxicam 36.5 ±7.8

NS-398 >100

Activated via the liver

Sulindac 31.7 ±12.1

Sulindac sulfide 4.25 ±0.45

Sulindac sulfone 24.2 ±6.7

Activated in the colon

Sulfasalazine >100

5-Aminosalicylic acid

98.2 ±28.6

Sulfapyridine >100

18

xxv

Table 1. Potency of NSAIDs in inhibiting cell-surface CXCR4

Cell number- and isotype-corrected values from four independent experiments were used to

calculate each compound’s IC25 value. This reflects their relative potency in the down-regulation

of CXCR4. Some compounds had no effects on CXCR4 up to 100 μM. This is indicated by

>100.

To assess the link between CXCR4 down-regulation and the inhibition of COX by

NSAIDs, I compared each compound’s IC25 value to its known COX-1- and COX-2-inhibiting

potency (Fig. 7) and to its relative COX-selectivity (Fig. 8). These comparisons in general

showed non-linear relationships with R2 values substantially below 1.

19

xxvi

Figure 7. Relation between COX and CXCR4 inhibition

Scatter plots were generated comparing each compound’s IC25 value to its potency towards (a)

COX-1 and (b) COX-2 inhibition. IC50:COX values were derived from a study by Warner et al.

(1999). R2 values for both relationships were substantially below 1.

(a) (b)

R2 = 0.1376

R2 = 0.273

20

xxvii

Figure 8. Relation between CXCR4 inhibition and relative COX selectivity

Each compound’s IC25 value was compared to its relative COX selectivity. COX selectivity was

calculated by taking the log of the IC50:COX-1 to IC50:COX-2 ratios as derived from Warner et al.

(1999). The R2 value for this relationship was substantially below 1.

R2 = 0.166

21

xxviii

Discussion

CXCR4 is a protein often found to be over-expressed in CRCs (Dwinell et al., 1999,

Jordan et al., 1999, Kim et al., 2005) as well as many other cancers (Darash-Yahana et al., 2004,

Kaifi et al., 2005, Oda et al., 2006, Salvucci et al., 2005, Yasumoto et al., 2006). In this context,

it has been implicated in tumour cell proliferation, survival and metastasis. Studies in murine

models have also shown CXCR4 to be responsible for tumour growth through promoting

angiogenesis (Guleng et al., 2005). Preliminary experiments in our lab suggested that select

NSAIDs decrease cell-surface CXCR4 expression in vitro on the HT-29 CRC cell line. This

might relate, at least in part, to the known chemopreventative and chemotherapeutic effects of

NSAIDs. This investigation expanded upon our previous work by asking the following three

questions:

(1) Do NSAIDs in general cause a decrease in CXCR4 expression?

(2) Is the effect mediated by inhibiting the COX enzyme?

(3) Do either of the two COX isoforms play an exclusive role?

The down-regulation of CXCR4 by NSAIDs shows features of COX dependence

22

xxix

Prostaglandin E2 (PGE2), a major product of COX, was recently found to up-regulate the

expression of CXCR4 in an endothelial cell model (Salcedo et al., 2003). Although our lab was

unable to reproduce this PGE2 stimulatory effect in the cancerous epithelial HT-29 cell line, it

may be hypothesized that COX enzymes synthesize various eicosanoids which contribute to an

increase in CXCR4. This suggests that the inhibition of eicosanoid biosynthesis by NSAIDs may

contribute to the down-regulation of CXCR4.

My findings provide further support for this line of thought. Both indomethacin and

diclofenac are potent COX inhibitors (Warner et al., 1999) and as expected, both were very

potent and efficacious in the down-regulation of CXCR4 (Table 2). Aspirin produced no change

in CXCR4. This is quite consistent with aspirin’s known clinical potency. It is only from a very

large oral dose (between 1200 to 1500mg) that aspirin produces anti-inflammatory effects

(Katzung & Furst, 1998). This equates to a steady state plasma concentration on the order of 1 to

10 mM (Schwertner et al., 2005) which was not examined in the present study.

Compound

Potency Selectivity

CXCR4 COX-1 COX-2 IC50 ratioIC25

(µM)IC50

(µM)IC50

(µM)log

[COX-1/COX-2]*COX-1

SelectivePiroxicam 57.8 ±16.6 2.4 7.9 -0.517

Indomethacin 8.05 ±1.78 0.013 1 -1.89

Aspirin >100 1.7 >100 **

COX-2 Selective

Diclofenac 27.2 ±8.8 0.075 0.038 0.295

Meloxicam 36.5 ±7.8 5.7 2.1 0.434

NS-398 >100 6.9 0.35 1.29

Activated via the liver

Sulindac 31.7 ±12.1 >100 >100 ---

Sulindac sulfide 4.25 ±0.45 1.9 55 -1.46

Sulindac sulfone 24.2 ±6.7 --- -- ---

Activated in the

Sulfasalazine >100 3242 2507 0.111

23

xxx

colon 5-Aminosalicylic acid

98.2 ±28.6 410 61 0.827

Sulfapyridine >100 --- --- ---

Table 2. Comparison of CXCR4 inhibition to COX potency and selectivity

IC25 values for each compound are compared to their known COX potencies and selectivity.

Values for COX were derived from a study by Warner et al. (1999).

* Positive values reflect relative COX-2 selectivity. Negative values reflect relative COX-1

selectivity.

** Aspirin is COX-1 selective at low doses.

The link between the inhibition of COX and CXCR4 is further supported by examining

data for the NSAIDs activated in the liver and colon. Sulindac and sulfasalazine each produce

two metabolites, one of which has increased COX-inhibiting activity over its respective parent

drug whereas the other metabolite in each case has no COX activity. This ability to inhibit COX

parallels the potency towards CXCR4 repression, further suggesting that a decrease in CXCR4 is

dependent on COX inhibition (Fig. 5 and 6).

The sulindac family of compounds in general was found to have substantial activity on

the down-regulation of CXCR4. This is interesting because both sulindac and sulindac sulfone

have negligible if any COX-inhibiting activity. Despite this, both compounds produced

significant and reliable declines in CXCR4 levels. This strikingly contrasts the little to no effect

obtained by aspirin and NS-398, both of which are COX inhibitors.

The potency of sulindac, the prodrug, may be due to the HT-29 cell line expressing

enzymes required to generate the active metabolite. The equilibrium between sulindac and the

sulfide is maintained physiologically by the enzymes sulindac oxidase and sulindac reductase

(Fig. 9) which are expressed predominantly in the liver but also in minute quantities throughout

the body (Duggan et al., 1980). If the sulfide compound can be generated in the in vitro system,

24

Oxidase

Reductase

- -

Oxidase

xxxi

then the potency in CXCR4 inhibition attributed to sulindac could be explained. Despite this

possibility, it is evident that the down-regulation of CXCR4 by NSAIDs cannot be explained

solely by COX-dependent mechanisms. Because sulindac sulfone is irreversibly generated, it

must act through COX-independent means to produce a decrease in CXCR4.

Sulindac sulfide Sulindac Sulindac sulfone

COX CXCR4

Figure 9. Proposed mechanism by which Sulindac and its two metabolites down-regulate

CXCR4

-

?

25

xxxii

In the body, sulindac and its physiologically active metabolite, sulindac sulfide, are in

equilibrium while the COX-independent metabolite, sulindac sulfone, is irreversibly formed. It is

possible that HT-29 cells express the enzymes which convert one sulindac compound into the

other. In this way, sulindac may be attributed COX-inhibiting activity by first becoming reduced

to its active sulfide form. However, the effect of the inactive sulfone on CXCR4 must still be

accounted for by some COX-independent pathway.

The COX-related down-regulation of CXCR4 does not seem to be through either COX isoenzyme alone

Numerous studies have implicated the inducible and pro-inflammatory COX-2 isoform in

cancer progression (Samoha & Arber, 2005). Examining CRC tissue samples from patients,

COX-2 was found to be overexpressed in 45% of colon adenomas and 85% of colon carcinomas,

while no change in COX-1 was found (Eberhart et al., 1994). COX-2 up-regulation has been

noted in tumours of the breast and lungs as well (Soslow et al., 2000). Furthermore, the mere

overexpression of the COX-2 gene in mice is sufficient to produce mammary gland tumours (Liu

et al., 2001), while human FAP equivalent mice without COX-2 gene expression have

dramatically fewer and smaller polyps than mice that did express COX-2 (Oshima et al., 1995,

Oshima et al., 1996). The increase of COX-2 mRNA in stool has even been explored as a

biomarker for diagnosing CRC (Kanaoka et al., 2004). These and countless other studies have

steered basic and clinical investigations towards the use of COX-2 selective inhibitors in the

chemoprevention and treatment of NSAIDs. Despite rare cases of cardiovascular complications

in high risk individuals, clinical trials with COX-2 inhibitors have proven effective in reducing

26

xxxiii

polyp formation in patients with FAP (Hallak et al., 2003, Phillips et al., 2002, Steinbach et al.,

2000).

It is important to realize, however, that these findings do not preclude the involvement of

the COX-1 isoform in CRC progression. As a follow up to Oshima’s 1996 COX-2 knockout

studies in mice, Chulada found that the likelihood of polyp formation was reduced in mice that

were unable to express COX-1 (Chulada et al., 2000). Furthermore, two large scale

chemoprevention studies have demonstrated that regular use of aspirin, a COX-1 selective

NSAID, effectively lowers the likelihood of polyp formation in patients with previous adenomas

or carcinomas (Baron et al., 2003, Sandler et al., 2003). Taken together, these thoughts are

consistent with the finding that the combined use of COX-1 and COX-2 selective NSAIDs in

APC gene deficient mice is more effective in the prevention of polyp formation than either alone

(Kitamura et al., 2004).

Data from my own findings did not definitively suggest that the COX-related down-

regulation of CXCR4 was dependent on the inhibition of either COX isoenzyme alone.

Comparison of the IC25 values for both COX-1 and COX-2 selective NSAIDs revealed no

apparent difference between groups. This was confirmed in scatter plots of each compound’s

known COX-inhibiting potency and relative selectivity as a function of its IC25 value (Fig. 7 and

8). In fact, the non-linear relationship of these plots, which have R2 values substantially below

one, provides objective evidence for the involvement of COX-independent pathways. The down-

regulation of CXCR4 by NSAIDs cannot simply be linked to the inhibition of COX. Indeed, the

effect on CXCR4 may at least in part – or even mostly – be due to the action of NSAIDs on

COX-independent targets.

Possible COX-independent pathwaysIf the HT-29 cell line lacks the expression of enzymes which convert sulindac into its

metabolites, then the potent effect of sulindac, the prodrug with negligible COX-activity, would

also be unexplained by the effect of COX-inhibition alone. Although the prodrug and its active

metabolite are separated by a 500-fold difference in potency, there was only a ten fold difference

in potency in reducing cell-surface CXCR4 expression. This argues against a completely COX-

dependent effect.

27

xxxiv

This reasoning is supported by the substantial decrease in CXCR4 produced by sulindac

sulfone. Given that the sulfone is irreversibly formed, it cannot subsequently be converted into

the COX-inhibiting sulfide. Not only does this finding argue against a completely COX-

dependent effect, but the decrease in CXCR4, in this case, must be accounted for entirely by

COX-independent pathways.

A number of pathways have been identified as possible mediators in the anti-cancer

effects of sulindac and other NSAIDs (Fig. 10). For example, sulindac, its metabolites and

aspirin are all able to inhibit the transcription-promoting activity of NF-κB, a factor implicated in

tumourigenesis (Yamamoto et al., 1999, Yin et al., 1998). This inhibition results in the decreased

proliferation of colon cancer cells in vitro.

xxxv

Figure 10. COX-independent action of NSAIDs

NSAIDs have several actions that are COX-independent including the inhibition of NF-κB, a

factor involved in survival, and the activation of the caspase pathway, which leads to

programmed cell death. Modified from Ricchi et al. (2003).

28

xxxvi

Sulindac sulfide and sulfone also act on other cellular proteins. In CRC cell lines, both

sulindac metabolites inhibit the expression of β-catenin (Chang et al., 2005), a transcription

factor inducer which is normally under the control of the unmutated APC tumour suppressor

gene. This inhibition is thought to result from the induction of caspase pathways by sulindac

sulfide and sulfone which leads to the degradation of β-catenin (Rice et al., 2003).

Studies in humans using sulindac sulfone have validated the role of COX-independent

pathways in the control of cancer progression. Clinical trials of the COX-inactive sulfone in the

chemoprevention of colorectal polyps in patients with FAP (Arber et al., 2006, van Stolk et al.,

2000) and chemotherapy of advanced solid tumours (Witta et al., 2004) have shown promise and

require further studies.

It is possible that the activation or inhibition of one of these pathways may also have lead

to the decrease in cell-surface CXCR4 observed in the present in vitro system. Such a conclusion

is consistent with the finding that NF-κB promotes breast cancer cell metastasis through inducing

the expression of CXCR4 (Helbig et al., 2003). Perhaps then, sulindac, which has negligible

COX-inhibiting activity, decreases cell-surface CXCR4 in the HT-29 cell line by inhibiting the

action of NF-κB.

This, of course, may represent the molecular basis by which only sulindac and its

metabolites produce a down-regulation in cell-surface CXCR4. The non-linear appearance of

Figures 7 and 8 seems to provide objective evidence for the partial or even predominant

involvement of COX-independent pathways in the down-regulation of CXCR4 by those NSAIDs

assessed in this study. Overall, the effects of these compounds on CXCR4 expression are not

exclusively a function of their potency towards COX inhibition.

Consider the potent inhibition of CXCR4 produced by sulindac sulfone compared to the

little or no effect produced by aspirin, NS-398 and 5-ASA (Table 2). If it were to be concluded

that CXCR4 expression is mediated solely through the inhibition of COX, then a decrease should

have been produced by these COX-acting compounds.

This conclusion is reiterated by studies where cell lines that do not express the COX

genes still show modified cancer kinetics in response to NSAIDs. The anti-proliferative and anti-

mitogenic effects of celecoxib, a COX-2 inhibitor, is no different in both in vitro and in vivo

models regardless of whether the cells express the COX-2 gene (Grosch et al., 2001, Maier et al.,

2005).

29

xxxvii

The involvement of COX-independent pathways in the down-regulation of CXCR4 in

CRC must be further clarified. Additionally, by using COX knockouts, we may definitively

determine if the inhibition of COX is a required step in the down-regulation of CXCR4. In the

end, perhaps both COX-dependent and COX-independent mechanisms contribute to the anti-

cancer effects of NSAIDs (Marx, 2001).

Significance of findingsThe conclusions of in vitro studies such as this one are often challenged. Findings may

require the administration of clinically unattainable levels of a given compound to produce a

statistically significant cellular change (Marx, 2001). For example, 12.5mg diclofenac-K tablets

can be orally administered twice a day for pain relief. This would result in a plasma

concentration of roughly 0.1 to 1 µM (Hinz et al., 2005).

In response to this concern, it may be helpful to remember that NSAIDs are by and large

administered orally. In CRCs, this would entail their direct access to their target in the

gastrointestinal epithelium. Studies have often compared drug concentrations at various sites of

the body after oral or topical administration and have always found local concentrations to be

higher than circulating plasma concentrations (Duggan et al., 1980, Mills et al., 2005).

In this regard, the use of orally administered NSAIDs in the chemoprevention of CRC is

entirely appealing. Drug concentrations in the gastrointestinal lumen are often substantially

higher than those obtained in the blood (Katzung & Furst, 1998). This high concentration may

then result in the sufficient down-regulation of CXCR4 to inhibit is many tumour promoting

effects. In fact, a mere 2 fold increase in cell-surface CXCR4 on HT-29 cells is sufficient to

induce its chemotactic migration towards an increasing concentration of CXCL12 in vitro

(Richard et al., 2006). This small change in CXCR4 has substantial implications in CRC

metastasis.

Ultimately then, my findings need to be confirmed in in vivo systems.

30

xxxviii

ConclusionThe current investigation focused on the possible link between the inhibition of the COX

isoforms by NSAIDs and the expression of CXCR4, a chemokine receptor implicated in tumour

progression and metastasis. My findings confirm that NSAIDs in general down-regulate cell-

surface CXCR4 expression in the HT-29 cell line. Although CXCR4 down-regulation seems to

exhibit features of COX-dependence, COX-independent pathways are clearly involved. Further

studies using COX knockouts will be required to definitively determine the role of COX in

CXCR4 regulation.

31

xxxix

References

Algarte-Genin, M., O. Cussenot, and P. Costa. 2004. Prevention of prostate cancer by androgens: experimental paradox or clinical reality. Eur. Urol. 46:285-94; discussion 294-5.

Anderson, G. D., S. D. Hauser, K. L. McGarity, M. E. Bremer, P. C. Isakson, and S. A. Gregory. 1996. Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. J. Clin. Invest. 97:2672-2679.

Arber, N., S. Kuwada, M. Leshno, R. Sjodahl, R. Hultcrantz, and D. Rex. 2006. Sporadic adenomatous polyp regression with exisulind is effective but toxic: a randomised, double blind, placebo controlled, dose-response study. Gut 55:367-373.

Baron, J. A., B. F. Cole, R. S. Sandler, R. W. Haile, D. Ahnen, R. Bresalier, G. McKeown-Eyssen, R. W. Summers, R. Rothstein, C. A. Burke, D. C. Snover, T. R. Church, J. I. Allen, M. Beach, G. J. Beck, J. H. Bond, T. Byers, E. R. Greenberg, J. S. Mandel, N. Marcon, L. A. Mott, L. Pearson, F. Saibil, and R. U. van Stolk. 2003. A randomized trial of aspirin to prevent colorectal adenomas. N. Engl. J. Med. 348:891-899.

Becker, R. C. 2005. COX-2 inhibitors. Tex. Heart Inst. J. 32:380-383.

Blay, J., T. D. White, and D. W. Hoskin. 1997. The extracellular fluid of solid carcinomas contains immunosuppressive concentrations of adenosine. Cancer Res. 57:2602-2605.

Brown, J. R. and R. N. DuBois. 2005. COX-2: a molecular target for colorectal cancer prevention. J. Clin. Oncol. 23:2840-2855.

Caldwell, B., S. Aldington, M. Weatherall, P. Shirtcliffe, and R. Beasley. 2006. Risk of cardiovascular events and celecoxib: a systematic review and meta-analysis. J. R. Soc. Med. 99:132-140.

Canadian Cancer Society. 2006. Colorectal cancer stats. 2006:1.

Cao, C., K. Matsumura, K. Yamagata, and Y. Watanabe. 1997. Involvement of cyclooxygenase-2 in LPS-induced fever and regulation of its mRNA by LPS in the rat brain. Am. J. Physiol. 272:R1712-25.

Chang, W. C., L. C. Everley, G. R. Pfeiffer 2nd, H. S. Cooper, A. Barusevicius, and M. L. Clapper. 2005. Sulindac Sulfone Is Most Effective in Modulating β-Catenin-Mediated Transcription in Cells with Mutant APC. Ann. N. Y. Acad. Sci. 1059:41-55.

Chen, Y., G. Stamatoyannopoulos, and C. Z. Song. 2003. Down-regulation of CXCR4 by inducible small interfering RNA inhibits breast cancer cell invasion in vitro. Cancer Res. 63:4801-4804.

32

xl

Cheng, Y. and P. Desreumaux. 2005. 5-aminosalicylic acid is an attractive candidate agent for chemoprevention of colon cancer in patients with inflammatory bowel disease. World J. Gastroenterol. 11:309-314.

Chulada, P. C., M. B. Thompson, J. F. Mahler, C. M. Doyle, B. W. Gaul, C. Lee, H. F. Tiano, S. G. Morham, O. Smithies, and R. Langenbach. 2000. Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min mice. Cancer Res. 60:4705-4708.

Corley, D. A., K. Kerlikowske, R. Verma, and P. Buffler. 2003. Protective association of aspirin/NSAIDs and esophageal cancer: a systematic review and meta-analysis. Gastroenterology 124:47-56.

Dalakas, E., P. N. Newsome, D. J. Harrison, and J. N. Plevris. 2005. Hematopoietic stem cell trafficking in liver injury. FASEB J. 19:1225-1231.

Darash-Yahana, M., E. Pikarsky, R. Abramovitch, E. Zeira, B. Pal, R. Karplus, K. Beider, S. Avniel, S. Kasem, E. Galun, and A. Peled. 2004. Role of high expression levels of CXCR4 in tumor growth, vascularization, and metastasis. FASEB J. 18:1240-1242.

Dubois, R. N., S. B. Abramson, L. Crofford, R. A. Gupta, L. S. Simon, L. B. Van De Putte, and P. E. Lipsky. 1998. Cyclooxygenase in biology and disease. FASEB J. 12:1063-1073.

Duggan, D. E., K. F. Hooke, and S. S. Hwang. 1980. Kinetics of the tissue distributions of sulindac and metabolites. Relevance to sites and rates of bioactivation. Drug Metab. Dispos. 8:241-246.

Duggan, D. E., K. F. Hooke, R. M. Noll, H. B. Hucker, and C. G. Van Arman. 1978. Comparative disposition of sulindac and metabolites in five species. Biochem. Pharmacol. 27:2311-2320.

Dwinell, M. B., L. Eckmann, J. D. Leopard, N. M. Varki, and M. F. Kagnoff. 1999. Chemokine receptor expression by human intestinal epithelial cells. Gastroenterology 117:359-367.

Eberhart, C. E., R. J. Coffey, A. Radhika, F. M. Giardiello, S. Ferrenbach, and R. N. DuBois. 1994. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 107:1183-1188.

Edwards, B. K., M. L. Brown, P. A. Wingo, H. L. Howe, E. Ward, L. A. Ries, D. Schrag, P. M. Jamison, A. Jemal, X. C. Wu, C. Friedman, L. Harlan, J. Warren, R. N. Anderson, and L. W. Pickle. 2005. Annual report to the nation on the status of cancer, 1975-2002, featuring population-based trends in cancer treatment. J. Natl. Cancer Inst. 97:1407-1427.

Galiatsatos, P. and W. D. Foulkes. 2006. Familial adenomatous polyposis. Am. J. Gastroenterol. 101:385-398.

33

xli

Govers, M. J., D. S. Termont, J. A. Lapre, J. H. Kleibeuker, R. J. Vonk, and R. Van der Meer. 1996. Calcium in milk products precipitates intestinal fatty acids and secondary bile acids and thus inhibits colonic cytotoxicity in humans. Cancer Res. 56:3270-3275.

Grosch, S., I. Tegeder, E. Niederberger, L. Brautigam, and G. Geisslinger. 2001. COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib. FASEB J. 15:2742-2744.

Guleng, B., K. Tateishi, M. Ohta, F. Kanai, A. Jazag, H. Ijichi, Y. Tanaka, M. Washida, K. Morikane, Y. Fukushima, T. Yamori, T. Tsuruo, T. Kawabe, M. Miyagishi, K. Taira, M. Sata, and M. Omata. 2005. Blockade of the stromal cell-derived factor-1/CXCR4 axis attenuates in vivo tumor growth by inhibiting angiogenesis in a vascular endothelial growth factor-independent manner. Cancer Res. 65:5864-5871.

Hallak, A., L. Alon-Baron, R. Shamir, M. Moshkowitz, B. Bulvik, E. Brazowski, Z. Halpern, and N. Arber. 2003. Rofecoxib reduces polyp recurrence in familial polyposis. Dig. Dis. Sci. 48:1998-2002.

Hankey, G. J. and J. W. Eikelboom. 2006. Aspirin resistance. Lancet 367:606-617.

Harris, R. C., J. A. McKanna, Y. Akai, H. R. Jacobson, R. N. Dubois, and M. D. Breyer. 1994. Cyclooxygenase-2 is associated with the macula densa of rat kidney and increases with salt restriction. J. Clin. Invest. 94:2504-2510.

Harris, R. E., R. T. Chlebowski, R. D. Jackson, D. J. Frid, J. L. Ascenseo, G. Anderson, A. Loar, R. J. Rodabough, E. White, A. McTiernan, and Women's Health Initiative. 2003. Breast cancer and nonsteroidal anti-inflammatory drugs: prospective results from the Women's Health Initiative. Cancer Res. 63:6096-6101.

Helbig, G., K. W. Christopherson 2nd, P. Bhat-Nakshatri, S. Kumar, H. Kishimoto, K. D. Miller, H. E. Broxmeyer, and H. Nakshatri. 2003. NF-kappaB promotes breast cancer cell migration and metastasis by inducing the expression of the chemokine receptor CXCR4. J. Biol. Chem. 278:21631-21638.

Hinz, B., J. Chevts, B. Renner, H. Wuttke, T. Rau, A. Schmidt, I. Szelenyi, K. Brune, and U. Werner. 2005. Bioavailability of diclofenac potassium at low doses. Br. J. Clin. Pharmacol. 59:80-84.

Holick, C. N., D. S. Michaud, M. F. Leitzmann, W. C. Willett, and E. Giovannucci. 2003. Aspirin use and lung cancer in men. Br. J. Cancer 89:1705-1708.

Jordan, N. J., G. Kolios, S. E. Abbot, M. A. Sinai, D. A. Thompson, K. Petraki, and J. Westwick. 1999. Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells. J. Clin. Invest. 104:1061-1069.

Kaifi, J. T., E. F. Yekebas, P. Schurr, D. Obonyo, R. Wachowiak, P. Busch, A. Heinecke, K. Pantel, and J. R. Izbicki. 2005. Tumor-cell homing to lymph nodes and bone marrow and CXCR4 expression in esophageal cancer. J. Natl. Cancer Inst. 97:1840-1847.

34

xlii

Kanaoka, S., K. Yoshida, N. Miura, H. Sugimura, and M. Kajimura. 2004. Potential usefulness of detecting cyclooxygenase 2 messenger RNA in feces for colorectal cancer screening. Gastroenterology 127:422-427.

Kashfi, K. and B. Rigas. 2005. Non-COX-2 targets and cancer: expanding the molecular target repertoire of chemoprevention. Biochem. Pharmacol. 70:969-986.

Katzung, B. G. and D. E. Furst. 1998. Nonsteroidal Anti-Inflammatory Drugs; Disease-Modifying Antirheumatic Drugs; Nonopioid Analgesics; Drugs Used in Gout, p. 578-602. In B. G. Katzung (ed.), Basic & Clinical Pharmacology. Appleton & Lange, Stamford, Connecticut.

Kim, J., H. Takeuchi, S. T. Lam, R. R. Turner, H. J. Wang, C. Kuo, L. Foshag, A. J. Bilchik, and D. S. Hoon. 2005. Chemokine receptor CXCR4 expression in colorectal cancer patients increases the risk for recurrence and for poor survival. J. Clin. Oncol. 23:2744-2753.

Kitamura, T., M. Itoh, T. Noda, M. Matsuura, and K. Wakabayashi. 2004. Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice. Int. J. Cancer 109:576-580.

Kudo, T., T. Narisawa, and S. Abo. 1980. Antitumor activity of indomethacin on methylazoxymethanol-induced large bowel tumors in rats. Gann 71:260-264.

Kune, G. A., S. Kune, and L. F. Watson. 1988. Colorectal cancer risk, chronic illnesses, operations, and medications: case control results from the Melbourne Colorectal Cancer Study. Cancer Res. 48:4399-4404.

Lamprecht, S. A. and M. Lipkin. 2003. Chemoprevention of colon cancer by calcium, vitamin D and folate: molecular mechanisms. Nat. Rev. Cancer. 3:601-614.

Laverdiere, C., B. H. Hoang, R. Yang, R. Sowers, J. Qin, P. A. Meyers, A. G. Huvos, J. H. Healey, and R. Gorlick. 2005. Messenger RNA expression levels of CXCR4 correlate with metastatic behavior and outcome in patients with osteosarcoma. Clin. Cancer Res. 11:2561-2567.

Liang, Z., T. Wu, H. Lou, X. Yu, R. S. Taichman, S. K. Lau, S. Nie, J. Umbreit, and H. Shim. 2004. Inhibition of breast cancer metastasis by selective synthetic polypeptide against CXCR4. Cancer Res. 64:4302-4308.

Limer, J. L. and V. Speirs. 2004. Phyto-oestrogens and breast cancer chemoprevention. Breast Cancer Res. 6:119-127.

Liu, C. H., S. H. Chang, K. Narko, O. C. Trifan, M. T. Wu, E. Smith, C. Haudenschild, T. F. Lane, and T. Hla. 2001. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J. Biol. Chem. 276:18563-18569.

Mahmud, S., E. Franco, and A. Aprikian. 2004. Prostate cancer and use of nonsteroidal anti-inflammatory drugs: systematic review and meta-analysis. Br. J. Cancer 90:93-99.

35

xliii

Maier, T. J., A. Janssen, R. Schmidt, G. Geisslinger, and S. Grosch. 2005. Targeting the beta-catenin/APC pathway: a novel mechanism to explain the cyclooxygenase-2-independent anticarcinogenic effects of celecoxib in human colon carcinoma cells. FASEB J. 19:1353-1355.

Marchesi, F., P. Monti, B. E. Leone, A. Zerbi, A. Vecchi, L. Piemonti, A. Mantovani, and P. Allavena. 2004. Increased survival, proliferation, and migration in metastatic human pancreatic tumor cells expressing functional CXCR4. Cancer Res. 64:8420-8427.

Martinez, M. E. 2005. Primary prevention of colorectal cancer: lifestyle, nutrition, exercise. Recent Results Cancer Res. 166:177-211.

Marx, J. 2001. Cancer research. Anti-inflammatories inhibit cancer growth--but how? Science 291:581-582.

Mills, P. C., B. M. Magnusson, and S. E. Cross. 2005. Penetration of a topically applied nonsteroidal anti-inflammatory drug into local tissues and synovial fluid of dogs. Am. J. Vet. Res. 66:1128-1132.

Narisawa, T., M. Sato, M. Tani, T. Kudo, T. Takahashi, and A. Goto. 1981. Inhibition of development of methylnitrosourea-induced rat colon tumors by indomethacin treatment. Cancer Res. 41:1954-1957.

Nkondjock, A. and P. Ghadirian. 2004. Dietary carotenoids and risk of colon cancer: case-control study. Int. J. Cancer 110:110-116.

Nowell, P. C. 2002. Tumor progression: a brief historical perspective. Semin. Cancer Biol. 12:261-266.

Oda, Y., H. Yamamoto, S. Tamiya, S. Matsuda, K. Tanaka, R. Yokoyama, Y. Iwamoto, and M. Tsuneyoshi. 2006. CXCR4 and VEGF expression in the primary site and the metastatic site of human osteosarcoma: analysis within a group of patients, all of whom developed lung metastasis. Mod. Pathol.

Oshima, M., J. E. Dinchuk, S. L. Kargman, H. Oshima, B. Hancock, E. Kwong, J. M. Trzaskos, J. F. Evans, and M. M. Taketo. 1996. Suppression of intestinal polyposis in Apc Δ716

knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell 87:803-809.

Oshima, M., H. Oshima, K. Kitagawa, M. Kobayashi, C. Itakura, and M. Taketo. 1995. Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc. Natl. Acad. Sci. U. S. A. 92:4482-4486.

Parkin, D. M. 2001. Global cancer statistics in the year 2000. Lancet Oncol. 2:533-543.

Phillips, R. K., M. H. Wallace, P. M. Lynch, E. Hawk, G. B. Gordon, B. P. Saunders, N. Wakabayashi, Y. Shen, S. Zimmerman, L. Godio, M. Rodrigues-Bigas, L. K. Su, J. Sherman, G. Kelloff, B. Levin, G. Steinbach, and FAP Study Group. 2002. A randomised, double blind, placebo controlled study of celecoxib, a selective cyclooxygenase 2 inhibitor, on duodenal polyposis in familial adenomatous polyposis. Gut 50:857-860.

36

xliv

Pugh, S. and G. A. Thomas. 1994. Patients with adenomatous polyps and carcinomas have increased colonic mucosal prostaglandin E2 . Gut 35:675-678.

Rang, H. P., M. M. Dale, J. M. Ritter, and P. K. Moore. 2003. Chemical Mediators, p. 122-261. In L. Hunter (ed.), Pharmacology. Elsevier Science Limited, London.

Reddy, B. S. and C. V. Rao. 2005. Chemoprophylaxis of colon cancer. Curr. Gastroenterol. Rep. 7:389-395.

Ricchi, P., R. Zarrilli, A. Di Palma, and A. M. Acquaviva. 2003. Nonsteroidal anti-inflammatory drugs in colorectal cancer: from prevention to therapy. Br. J. Cancer 88:803-807.

Rice, P. L., J. Kelloff, H. Sullivan, L. J. Driggers, K. S. Beard, S. Kuwada, G. Piazza, and D. J. Ahnen. 2003. Sulindac metabolites induce caspase- and proteasome-dependent degradation of beta-catenin protein in human colon cancer cells. Mol. Cancer. Ther. 2:885-892.

Richard, C. L., E. Y. Tan, and J. Blay. 2006. Adenosine upregulates CXCR4 and enhances the proliferative and migratory responses of human carcinoma cells to CXCL12/SDF-1α. Int J Cancer (in press).

Rigas, B., I. S. Goldman, and L. Levine. 1993. Altered eicosanoid levels in human colon cancer. J. Lab. Clin. Med. 122:518-523.

Rubin, J. B., A. L. Kung, R. S. Klein, J. A. Chan, Y. Sun, K. Schmidt, M. W. Kieran, A. D. Luster, and R. A. Segal. 2003. A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors. Proc. Natl. Acad. Sci. U. S. A. 100:13513-13518.

Salcedo, R., X. Zhang, H. A. Young, N. Michael, K. Wasserman, W. H. Ma, M. Martins-Green, W. J. Murphy, and J. J. Oppenheim. 2003. Angiogenic effects of prostaglandin E2 are mediated by up-regulation of CXCR4 on human microvascular endothelial cells. Blood 102:1966-1977.

Salvucci, O., A. Bouchard, A. Baccarelli, J. Deschenes, G. Sauter, R. Simon, R. Bianchi, and M. Basik. 2005. The role of CXCR4 receptor expression in breast cancer: a large tissue microarray study. Breast Cancer Res. Treat. 1-9.

Samoha, S. and N. Arber. 2005. Cyclooxygenase-2 inhibition prevents colorectal cancer: from the bench to the bed side. Oncology 69 Suppl 1:33-37.

Sandler, R. S., S. Halabi, J. A. Baron, S. Budinger, E. Paskett, R. Keresztes, N. Petrelli, J. M. Pipas, D. D. Karp, C. L. Loprinzi, G. Steinbach, and R. Schilsky. 2003. A randomized trial of aspirin to prevent colorectal adenomas in patients with previous colorectal cancer. N. Engl. J. Med. 348:883-890.

Schwertner, H. A., D. McGlasson, M. Christopher, and A. C. Bush. 2005. Effects of different aspirin formulations on platelet aggregation times and on plasma salicylate concentrations. Thromb. Res.

37

xlv

Singh, G. 1998. Recent considerations in nonsteroidal anti-inflammatory drug gastropathy. Am. J. Med. 105:31S-38S.

Smith, R. A., V. Cokkinides, and H. J. Eyre. 2006. American Cancer Society guidelines for the early detection of cancer, 2006. CA Cancer. J. Clin. 56:11-25; quiz 49-50.

Solomon, S. D., J. J. McMurray, M. A. Pfeffer, J. Wittes, R. Fowler, P. Finn, W. F. Anderson, A. Zauber, E. Hawk, and M. Bertagnolli. 2005. Cardiovascular risk associated with celecoxib in a clinical trial for colorectal adenoma prevention. N. Engl. J. Med. 352:1071-1080.

Song, J., A. Medline, J. B. Mason, S. Gallinger, and Y. I. Kim. 2000. Effects of dietary folate on intestinal tumorigenesis in the apcMin mouse. Cancer Res. 60:5434-5440.

Soslow, R. A., A. J. Dannenberg, D. Rush, B. M. Woerner, K. N. Khan, J. Masferrer, and A. T. Koki. 2000. COX-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer 89:2637-2645.

Steck-Scott, S., M. R. Forman, A. Sowell, C. B. Borkowf, P. S. Albert, M. Slattery, B. Brewer, B. Caan, E. Paskett, F. Iber, W. Kikendall, J. Marshall, M. Shike, J. Weissfeld, K. Snyder, A. Schatzkin, and E. Lanza. 2004. Carotenoids, vitamin A and risk of adenomatous polyp recurrence in the polyp prevention trial. Int. J. Cancer 112:295-305.

Steinbach, G., P. M. Lynch, R. K. Phillips, M. H. Wallace, E. Hawk, G. B. Gordon, N. Wakabayashi, B. Saunders, Y. Shen, T. Fujimura, L. K. Su, and B. Levin. 2000. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N. Engl. J. Med. 342:1946-1952.

Suzui, M., N. Sunagawa, I. Chiba, H. Moriwaki, and N. Yoshimi. 2006. Acyclic retinoid, a novel synthetic retinoid, induces growth inhibition, apoptosis, and changes in mRNA expression of cell cycle- and differentiation-related molecules in human colon carcinoma cells. Int. J. Oncol. 28:1193-1199.

Tachibana, K., S. Hirota, H. Iizasa, H. Yoshida, K. Kawabata, Y. Kataoka, Y. Kitamura, K. Matsushima, N. Yoshida, S. Nishikawa, T. Kishimoto, and T. Nagasawa. 1998. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 393:591-594.

Tan, E. Y., M. Mujoomdar, and J. Blay. 2004. Adenosine down-regulates the surface expression of dipeptidyl peptidase IV on HT-29 human colorectal carcinoma cells: implications for cancer cell behavior. Am. J. Pathol. 165:319-330.

Terry, M. B., M. D. Gammon, F. F. Zhang, H. Tawfik, S. L. Teitelbaum, J. A. Britton, K. Subbaramaiah, A. J. Dannenberg, and A. I. Neugut. 2004. Association of frequency and duration of aspirin use and hormone receptor status with breast cancer risk. JAMA 291:2433-2440.

38

xlvi

Ulrich, C. M., J. Bigler, and J. D. Potter. 2006. Non-steroidal anti-inflammatory drugs for cancer prevention: promise, perils and pharmacogenetics. Nat. Rev. Cancer. 6:130-140.

van Staa, T. P., T. Card, R. F. Logan, and H. G. Leufkens. 2005. 5-Aminosalicylate use and colorectal cancer risk in inflammatory bowel disease: a large epidemiological study. Gut 54:1573-1578.

van Stolk, R., G. Stoner, W. L. Hayton, K. Chan, B. DeYoung, L. Kresty, B. H. Kemmenoe, P. Elson, L. Rybicki, J. Church, K. Provencher, D. McLain, E. Hawk, B. Fryer, G. Kelloff, R. Ganapathi, and G. T. Budd. 2000. Phase I trial of exisulind (sulindac sulfone, FGN-1) as a chemopreventive agent in patients with familial adenomatous polyposis. Clin. Cancer Res. 6:78-89.

Vogelstein, B. and K. W. Kinzler. 2004. Cancer genes and the pathways they control. Nat. Med. 10:789-799.

Wallace, K., J. A. Baron, B. F. Cole, R. S. Sandler, M. R. Karagas, M. A. Beach, R. W. Haile, C. A. Burke, L. H. Pearson, J. S. Mandel, R. Rothstein, and D. C. Snover. 2004. Effect of calcium supplementation on the risk of large bowel polyps. J. Natl. Cancer Inst. 96:921-925.

Wang, W. H., J. Q. Huang, G. F. Zheng, S. K. Lam, J. Karlberg, and B. C. Wong. 2003. Non-steroidal anti-inflammatory drug use and the risk of gastric cancer: a systematic review and meta-analysis. J. Natl. Cancer Inst. 95:1784-1791.

Warner, T. D., F. Giuliano, I. Vojnovic, A. Bukasa, J. A. Mitchell, and J. R. Vane. 1999. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc. Natl. Acad. Sci. U. S. A. 96:7563-7568.

Winawer, S. J. 2005. Screening of colorectal cancer. Surg. Oncol. Clin. N. Am. 14:699-722.

Witta, S. E., D. L. Gustafson, A. S. Pierson, A. Menter, S. N. Holden, M. Basche, M. Persky, C. L. O'Bryant, C. Zeng, A. Baron, M. E. Long, A. Gibbs, K. Kelly, P. A. Bunn Jr, D. C. Chan, P. Pallansch, and S. G. Eckhardt. 2004. A phase I and pharmacokinetic study of exisulind and docetaxel in patients with advanced solid tumors. Clin. Cancer Res. 10:7229-7237.

Wolfe, M. M., D. R. Lichtenstein, and G. Singh. 1999. Gastrointestinal toxicity of nonsteroidal antiinflammatory drugs. N. Engl. J. Med. 340:1888-1899.

Yamamoto, Y., M. J. Yin, K. M. Lin, and R. B. Gaynor. 1999. Sulindac inhibits activation of the NF-kappaB pathway. J. Biol. Chem. 274:27307-27314.

Yasumoto, K., K. Koizumi, A. Kawashima, Y. Saitoh, Y. Arita, K. Shinohara, T. Minami, T. Nakayama, H. Sakurai, Y. Takahashi, O. Yoshie, and I. Saiki. 2006. Role of the CXCL12/CXCR4 axis in peritoneal carcinomatosis of gastric cancer. Cancer Res. 66:2181-2187.

39

xlvii

Yin, M. J., Y. Yamamoto, and R. B. Gaynor. 1998. The anti-inflammatory agents aspirin and salicylate inhibit the activity of I(kappa)B kinase-beta. Nature 396:77-80.

Zeelenberg, I. S., L. Ruuls-Van Stalle, and E. Roos. 2003. The chemokine receptor CXCR4 is required for outgrowth of colon carcinoma micrometastases. Cancer Res. 63:3833-3839.

Zou, Y. R., A. H. Kottmann, M. Kuroda, I. Taniuchi, and D. R. Littman. 1998. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393:595-599.

40