Alterations of Gut Microbiota From Colorectal Adenoma to Carcinoma
2006-04-10 Thesis
1. 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, PhD
Department of Pharmacology
Faculty of Medicine
Dalhousie University
Halifax, Novas Scotia
2. ii
April 10, 2006
Table of Contents
List of Figures and Tables iii
Abstract iv
List of Abbreviations v
Acknowledgements vi
Introduction
History of NSAIDs 1
Colorectal Cancer – The Problem 1
Colorectal Cancer – Prevention 3
NSAIDs inhibit cyclooxygenase enzymes 4
CXCR4, a chemoreceptor implicated in tumour progression and metastasis 6
Hypothesis 7
Specific Objectives 7
Materials and Methods 8
Results
Validation of the assay system 9
COX1-selective NSAIDs produce significant but variable inhibition of
cell-surface CXCR4 9
COX2-selective NSAIDs produce significant but variable inhibition of
cell-surface CXCR4 12
Sulindac and its metabolites are potent inhibitors of CXCR4 expression 14
Only sulfasalazine’s activated metabolite, 5-ASA, decreases CXCR4 expression 16
Discussion
The down-regulation of CXCR4 by NSAIDs shows features of COX dependence 22
The COX-related down-regulation of CXCR4 does not seem to be through either
COX isoenzyme alone 26
Possible COX-independent pathways 27
Significance of findings 30
Conclusion 31
References 32
3. iii
List of Figures and Tables
Figure 1 Genetic model of colorectal tumorigenesis 2
Figure 2 Eicosanoid biosynthesis by cyclooxygenase enzymes 5
Figure 3 COX-1 selective NSAIDs produce significant but variable
inhibition of cell-surface CXCR4. 11
Figure 4 COX-2 selective NSAIDs produce significant but variable
inhibition of cell-surface CXCR4. 13
Figure 5 Sulindac compounds are potent inhibitors of CXCR4 expression. 15
Figure 6 Sulfasalazine and its metabolites have little effect on CXCR4 expression. 17
Figure 7 Relation between COX and CXCR4 inhibition 20
Figure 8 Relation between CXCR4 inhibition and relative COX selectivity 21
Figure 9 Proposed mechanism by which Sulindac and its two metabolites
down-regulate CXCR4 25
Figure 10 COX-independent action of NSAIDs 28
Table 1 Potency of NSAIDs in inhibiting cell-surface CXCR4 18
Table 2 Comparison of CXCR4 inhibition to COX potency and selectivity 23
5. 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.
6. 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.
7. vii
Introduction
History of NSAIDs
One 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
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).
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8. viii
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).
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9. 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 – Prevention
Given 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
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).
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10. x
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
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).
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11. xi
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).
5
12. xii
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 metastasis
CXCR4 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.,
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
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13. xiii
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.
Hypothesis
NSAIDs 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.
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.
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14. xiv
Materials and Methods
Materials
The 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). 125
I-labeled sheep anti-mouse
IgG, F(ab')2 fragment was obtained from PerkinElmer Life Sciences (NEN, Boston, MA).
Cell culture
Cells 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.
Assay for cell-surface CXCR4
An 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 125
I-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
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15. xv
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 Analysis
Each 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 Values
Cell 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 system
Adenosine, a purine nucleotide found in high concentrations within the tumour
microenvironment (Blay et al., 1997), was used to show that CXCR4 could be positively
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
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high potency was further reflected in its IC25 value, which was eight times lower than that of
piroxicam (Fig. 1. Panel D).
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17. xvii
Figure 3. COX-1 selective NSAIDs produce significant but variable inhibition of cell-
surface CXCR4.
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).
(a) (b)
(c) (d)
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18. xviii
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).
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Figure 4. COX-2 selective NSAIDs produce significant but variable inhibition of cell-
surface CXCR4.
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).
(a) (b)
(c) (d)
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20. xx
Sulindac and its metabolites are potent inhibitors of CXCR4 expression
Sulindac, 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).
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Figure 5. Sulindac compounds are potent inhibitors of CXCR4 expression.
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).
(a) (b)
(c) (d)
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22. xxii
Only sulfasalazine’s activated metabolite, 5-ASA, decreases CXCR4 expression
Sulfasalazine 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.
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23. xxiii
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).
(a) (b)
(c) (d)
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24. xxiv
Compound
Potency
CXCR4
IC25
(µM)
COX-1
Selective
Piroxicam 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
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.
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25. xxv
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.
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26. 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)
R
2
= 0.1376 R
2
= 0.273
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27. 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.
R
2
= 0.166
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28. 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
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.
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29. xxix
Compound
Potency Selectivity
CXCR4 COX-1 COX-2 IC50 ratio
IC25
(µM)
IC50
(µM)
IC50
(µM)
log
[COX-1/COX-2]*
COX-1
Selective
Piroxicam 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
colon
Sulfasalazine >100 3242 2507 0.111
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.
23
30. xxx
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,
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.
24
31. Oxidase
Reductase
- -
Oxidase
xxxi
Sulindac sulfide Sulindac Sulindac sulfone
COX CXCR4
Figure 9. Proposed mechanism by which Sulindac and its two metabolites down-regulate
CXCR4
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.
-
?
25
32. xxxii
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
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
26
33. xxxiii
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 pathways
If 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.
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.
27
34. xxxiv
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
35. xxxv
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
36. xxxvi
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 findings
The 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
37. xxxvii
Conclusion
The 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
38. xxxviii
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