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Introduction
Inflammatory bowel disease (IBD), which includes
ulcerative colitis and Crohn's disease, affects approximately one
million individuals in the US each
year[1]. Clinical studies have shown that patients with ulcerative colitis have a
2_8-fold relative risk of developing colorectal cancer as
compared to the general population. The risk of colorectal
malignancies in colitis patients increases with the extent of
colonic involvement, age of onset, severity and duration of
disease[2]. Recent data suggest a cumulative risk of
malignancy below 1% for the first 8_10 years, which increases in
increments of 0.5%_1.0% annually, reaching 5%_10% after
20 years and 15%_20% after 30 years[3]. It is estimated that
25%_40% of ulcerative colitis patients who have not had a
prophylactic colectomy will develop colorectal cancer after
40 years of having the disease[4]. It should be noted that
data collected prospectively from a 30-year surveillance
program and reported recently suggest that the risk of colorectal
cancer among patients undergoing surveillance may be lower
than reported previously (cumulative incidence of 10.8% at
40 years)[5].
Although significant effort has been dedicated to the
establishment of surveillance programs for individuals with
ulcerative colitis, their effectiveness in preventing
colitis-associated colorectal cancer remains
controversial[5,6]. It is estimated that only 20%_50% of colorectal neoplasias are
identified during routine
colonoscopy[7]. The significant lag (at least 7 years) in the implementation of endoscopic
screening in individuals with long-standing disease,
indecisiveness regarding the optimal frequency of colonoscopies and
the number of biopsies that should be obtained during the
procedure, and the lack of strong adherence of physicians
to existing guidelines make it extremely difficult to assess
the value of such programs[6]. These limitations in clinical
practice, when combined with the routine recommendation
of colectomy for patients with colorectal dysplasia, dictate
the critical need for early chemopreventive intervention in
patients with ulcerative colitis.
Data from studies conducted to date suggest that the
molecular targets for early chemopreventive intervention in
colitis-associated colorectal carcinogenesis may differ from
those of sporadic colorectal cancer. Although significant
overlap exists between the genetic alterations associated
with these two colorectal diseases, the timing of these events
is distinct. Mutation of adenomatous polyposis coli
(APC), the putative gatekeeper of sporadic colorectal
carcino-genesis, is a late event in ulcerative colitis, occurring
primarily in high-grade dysplasias and
cancers[8,9]. In direct contrast, mutation or loss of heterozygosity (LOH)
of p53, a hallmark of late-stage sporadic colorectal cancer, is a
frequent event in both inflamed, non-neoplastic mucosa and
dysplasias[10]. Mutational events during this early time
period have been attributed to microsatellite instability
induced by inflammation-associated oxidative
stress[11,12]. It is interesting to note that LOH of 17p
(p53 gene locus) is found more frequently in flat dysplasias as compared to polypoid
dysplasias, while the percentage of each subtype exhibiting
LOH of 5p (APC gene locus) is
comparable[13]. Based on the association of
k-ras mutations with the polypoid growth of
sporadic colorectal adenomas[14] and their presence in only
30% of colitis-associated cancers[15], it has been suggested
that flat dysplasias develop in the absence of
k-ras mutations.
Reliable, reproducible and clinically relevant animal
models of colitis are needed to identify the molecular events
associated with disease progression and to develop
efficacious strategies for tumor inhibition. It is essential that
models chosen for study closely mimic the course of human
ulcerative colitis, ultimately leading to the development of
colorectal tumors that are pathologically similar to those of
humans. The dextran sulfate sodium (DSS) model of induced
colitis is an excellent preclinical model that exhibits many
phenotypic features of relevance to human ulcerative colitis.
The DSS model was originally described by
Ohkusa[16] as a hamster model and was adapted to mice subsequently by
Okayasu et al[17] and this
group[18]. In general, acute colitis is induced in mice by administering the resin DSS in the
drinking water at a concentration ranging from
1%_5%[18_20] for several days. Exposure to DSS for 1_4 cycles (each cycle
= 3_7 d of DSS followed by untreated water; total 21 d) mimics
the active and inactive disease experienced by humans,
leading to ulceration of the colonic mucosa and the
establishment of chronic colitis (inflammation). The resulting
pathological features of murine DSS-induced colitis-associated
neoplasia have been characterized extensively by this group
and others on different genetic backgrounds, in the
presence of known colon carcinogens/promoters and in
genetically modified mouse strains.
DSS alone
Based on differences in the sensitivity of mouse strains
to DSS exposure, it is essential to optimize the DSS regimen
for the production of both inflammation and colorectal
tumors in each mouse strain of interest.
The protocol routinely employed by this group for
outbred female Swiss Webster mice is 4 cycles of treatment,
with each cycle consisting of 7 d of DSS followed by 14 d of
untreated water (Table 1). The histological alterations are
characteristic of those observed in patients with ulcerative
colitis[18,20]. First, after 7 d of DSS, there is loss of crypts and
ulceration. Following one cycle (and subsequent cycles),
the mucosa shows regenerative changes, distinctive
glandular disarray, separation, shortening of crypts and crypt
branching as seen in human chronic ulcerative colitis. In
some mice, changes associated with activity are still
present. A subset of mice exhibit the characteristic histopathologic
features of ulcerative colitis many months after the
discontinuation of DSS. Second, 4 cycles of DSS results in the
development of colitis-associated dyplasias and
adenocarcinomas in approximately 15%_20% of
mice[20]. This percentage is comparable to the risk of ulcerative colitis patients
developing dysplasia and/or cancer over time, providing a
model system in which to investigate the molecular basis for
the susceptibility of only certain individuals to tumor
formation. Also, as in humans, longevity of disease is
associated with a higher incidence of colitis-associated neoplasia.
If Swiss Webster mice are allowed to live an
additional 120 d after 4 cycles of DSS, the incidence of dysplasia and/or
cancer increases from 15%_20% to 37.5%, and the incidence of
cancer increases from 9.3% to 25% compared to mice
sacrificed after 4 cycles of DSS[20]. Third, as in humans, mice
develop flat neoplastic lesions as well as polypoid neoplasias
superimposed on chronic colitis (Figure 1). The potential
relevance of the observation that invasive cancers arise more
frequently from flat lesions is of great concern, considering
our current inability to accurately detect these lesions in
humans using standard endoscopic surveillance protocols.
Finally, the contribution of inflammation to the
dysplasia/cancer sequence is significant. DSS-treated Swiss Webster
mice with dysplasias/cancers have significantly higher mean
colonic inflammation scores than those without dysplasias
or cancers[20]. Interestingly, inflammation scores are
significantly higher in cancers vs dysplasias.
Several observations in the Swiss Webster model of
DSS-induced colitis suggest that the genetic profile of flat and
polypoid lesions may differ. First, flat lesions exhibit
significantly higher inflammation scores than polypoid lesions.
Second, cancers arise more frequently from flat
mucosa[20]. Third, nuclear translocation
of β-catenin is observed in the majority of DSS-induced polypoid lesions, while
-catenin remains localized to the cell membrane of flat
dysplasias and cancers. Fourth, Nosho and
colleagues[21] demonstrated recently that hierarchical clustering of the gene expression
profiles of human colon adenomas results in the
independent segregation of flat and protruding lesions. While
lesions from IBD patients were not evaluated in this
study, a similar result is anticipated based on
our observations.
A DSS regimen, similar to that described above, has been
used to induce colitis in inbred female C57BL/6J mice. A total
of 12.5% (1 of 8) of wild-type female C57BL/6J mice exposed
to 4% DSS for 4 cycles (4 d of DSS plus 17 d of water)
developed dysplasias. In contrast to DSS-treated Swiss Webster
mice, no cancers were observed[22] (Table 1).
DSS in combination with a colon carcinogen/promoter
Although the pathological features of the Swiss Webster
mice with DSS-induced colitis mimic those of humans with
ulcerative colitis, intervention studies in this model are costly
due to the need for large numbers of animals to achieve
statistical significance and the length of time required for
tumor formation (a minimum of 84 d). Carcinogens, such as
azoxymethane (AOM) and heterocyclic amines (HCAs), and
other promoters (ie, iron) have been added to the DSS
regimen to enhance tumor incidence, multiplicity, and/or lesion
progression.
AOM Based on the experience of this group, use of
DSS in combination with AOM, a classic chemical
carcinogen that induces colorectal cancer in rodents, results in
100% incidence of colonic tumors[23] as compared to
15%_20% when DSS is administered alone (Table 1). Although
the length of study can be shortened from 4 cycles to 3
cycles (63 d) of DSS due to the accelerated development of
colorectal lesions, no invasive carcinomas are observed. It
should be noted that no dysplasias are observed in mice
receiving only AOM, suggesting that the dose of
carcinogen used (7.4 mg/kg) is insufficient to induce colorectal
tumors in the absence of inflammation. Tanaka and
colleagues[24,25] also observed enhanced development of colitis-associated
neoplasia when ICR mice were injected with AOM prior to
DSS exposure. Use of a lower percentage of DSS (1%_2%
for 4_7 d) in combination with AOM was sufficient to induce
colitis-associated neoplasia as long as the animals were
allowed to live an additional 16_18 weeks following DSS
exposure.
The morphology and representation of flat and polypoid
lesions generated by AOM/DSS treatment is consistent with
that observed following treatment with DSS alone (Table 1).
However, in the presence of AOM and DSS, nuclear
translocation of β-catenin is observed in both flat and polypoid
lesions induced by AOM and DSS due to mutation of
β-catenin. This finding is consistent with the presence of
β-catenin mutations in AOM-induced colon lesions in the
rat[26] and mouse[27] and the altered distribution of
β-catenin in human colitis-associated colorectal
cancer[28]. Because of the small number of studies carried out on human samples to
date, the role of β-catenin mutations in human
colitis-associated neoplasia remains
unclear[29].
Strain differences in susceptibility to AOM/DSS have
been reported[25]. Treatment of 4 different strains of mice
(balb/c, C3H/HeN, C57BL/6N and DBA/2N) with an identical
regimen of AOM/DSS by Suzuki et
al[25] resulted in distinct differences in tumor incidence, multiplicity and inflammation
among the strains. In our hands, C57BL/6J mice were unable
to tolerate the AOM/DSS regimen used for Swiss Webster
mice (7 d of DSS plus 14 d of untreated water, over 3 cycles).
The optimal protocol that we have established for this strain
includes 3 cycles of abbreviated exposure to DSS (4 d of
DSS and 17 d of untreated water). The survival rate of
animals receiving AOM (control) or AOM/DSS (100% and 60%,
respectively) is comparable to that of Swiss Webster mice
receiving identical treatment (100% and 56%, respectively).
These data suggest that genetic background may play an
important role in dictating the risk of colitis-associated
neoplasia.
Comparison of AOM/DSS-induced colitis-associated
neoplasia in Swiss Webster (outbred) and C57BL/6J (inbred)
mice reveals many similar features. First, all (100%) female
Swiss Webster and C57BL/6J mice developed colitis
following exposure to AOM and DSS and with a similar degree of
inflammation. Second, the incidence of colorectal
dysplasias in C57BL/6J mice receiving AOM alone or AOM/DSS is
0% and 100%, respectively; identical to that of treated Swiss
Webster mice. Third, as in Swiss Webster mice, invasive
cancers are not observed in AOM/DSS-treated C57BL/6J
mice. Fourth, the mean multiplicity of colonic dysplasias
observed in C57BL/6J mice exposed to AOM and DSS
(15.4±2.3, mean±SEM) is comparable to that observed in
AOM/DSS-treated Swiss Webster mice (16.0±2.2). Finally, similar
percentages of flat and polypoid dysplasias (Table 1) are
observed in AOM/DSS-treated C57BL/6J and Swiss Webster
mice. These data demonstrate that, although C57BL/6J mice
are more sensitive to AOM/DSS exposure than outbred Swiss
Webster mice, administration of AOM/DSS to inbred
C57BL/6J mice induces colitis-associated colonic dysplasias that
are identical to those of AOM/DSS-treated Swiss Webster
mice with respect to tumor incidence, multiplicity and
morphology.
HCAs A variety of heterocyclic amines (HCAs),
important food-derived carcinogens generated during heating
amino acids and proteins[30_32], have been evaluated for their
ability to promote the formation of DSS-induced colitis-associated neoplasia. Of the HCAs examined to date,
2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) elevated the
incidence and multiplicity of total lesions and
adenocarcinomas to the greatest extent as compared to either
2amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or
2-amino-3-methylimidazo[4,5-f]quinoxaline
(IQ)[27,33]. Both polypoid and flat colorectal neoplasias were observed in mice exposed to
HCAs and DSS[27]. β-catenin mutations (codons 32 or 34)
were found in all PhIP/DSS-induced adenocarcinomas and
accompanied by strong nuclear and cytoplasmic staining of
β-catenin[27]. These lesions also exhibited overexpression of
cyclooxygenase2 (COX2) and iNOS protein.
Iron Oral iron supplements are routinely prescribed to
patients with chronic ulcerative colitis to ameliorate any iron
deficiency associated with colitis-induced anemia. Because
iron is poorly absorbed in the upper gastrointestinal tract,
the majority of oral iron reaches the colonic lumen where it
reacts with superoxides and hydrogen peroxide to form
hydroxyl radicals and other reactive oxygen
species[34].
Results from animal studies suggest that iron may
exacerbate colitis and enhance the formation of colitis-associated
neoplasia[34,35]. Both disease activity and severity of colitis
increased in a dose-dependent manner when DSS-treated
animals were fed diets supplemented with
iron[35]. Long-term exposure to an iron-enriched diet (2fold) in combination with
cyclic administration of 0.7% DSS caused a significant
increase in the incidence of colorectal adenocarcinomas as
compared to that of controls maintained on a standard diet
and administered DSS (94% and 38%,
respectively)[35].
DSS in genetically defined mouse models
Apc+/Min mouse model Mutation of the
APC gene, the putative gatekeeper of colorectal tumorigenesis, occurs in
more than 80% of sporadic colorectal cancer. In contrast,
only 4%_27% of human colitis-associated colorectal
cancers harbor mutant APC[2]. In order to determine the
contribution of mutant APC to colitis-associated neoplasia, colitis
was induced in Apc+/Min mice carrying a germline mutation in
Apc, and tumor incidence and multiplicity were
evaluated[19,22]. Our study was carried out in a unique strain of
Apc+/Min mice, which, unlike the conventional strain, develop multiple
adenomas in the large intestine[36]. Female C57BL/6J
Apc+/Min-FCCC mice were treated with 2 cycles of DSS. Cycle 1 consisted
of 4 d of 4% DSS plus 17 d of untreated water, and Cycle 2
consisted of 3 d of DSS plus 18 d of untreated water.
Wild-type female C57BL/6J mice were treated with 4 cycles of
DSS, with each cycle consisting of 4 d of 4% DSS plus 17 d
of untreated water. DSS-treated animals exhibited a 2-fold
increase in tumor incidence (50% vs 100%) and a dramatic
increase in tumor multiplicity (mean 1.9±0.7 and 29.3±4.1 for
untreated and DSS-treated Apc+/Min-FCCC mice, respectively)
(Table 1). No invasive colorectal cancers were observed in
untreated Apc+/Min-FCCC mice, while 40% of DSS-treated mice
had colorectal cancer. Tumor incidence in wild-type mice
exposed to DSS was 12.5%, and the mean number of tumors
per tumor-bearing mouse was 1.0. Interestingly, all of the
dysplasias in DSS-treated wild-type mice were polypoid; no
flat lesions were found. The majority of the neoplastic
lesions observed in both untreated and DSS-treated
Apc+/Min-FCCC mice were also polypoid (86.7% and 52.7%,
respectively). Dysplastic colorectal lesions from both
untreated and DSS-treated ApcMin mice showed LOH of
Apc (100% and 90.6%, respectively). These findings indicate that
both mutation of Apc and inflammation accelerate the
formation of colitis-associated dysplasias and their progression
to invasive cancers. Even when colorectal carcinogenesis is
driven by inflammation, LOH of Apc remains a critical event
in the formation of colorectal dysplasias.
The role of DSS-induced colitis as a promoter of
neoplasia in ApcMin mice has also been studied by Tanaka
et al[19]. In their experiment, mice were exposed to 2% DSS in the
drinking water for 7 d, followed by 4 weeks of untreated
water (total of 35 d). One hundred percent of the mice
developed gross lesions with a multiplicity of 8.3±5 (3.3±3
adenomas and 5.0±2 adenocarcinomas). In addition, all mice
developed microscopic dysplastic lesions (13.3±3.4 per
mouse). One explanation for the higher incidence of
adenocarcinomas in this study as compared to that of Cooper
et al[22] is the difference in the definition of adenocarcinoma. In the
latter study, lesions were considered adenocarcinomas only
when they had invaded through the muscularis mucosae
and into the submucosa. In the study carried out by Tanaka
et al[19], 100% of adenocarcinomas showed LOH of
Apc. There was strong nuclear expression of p53 and
β-catenin; however, no β-catenin mutations were detected. COX2 and
iNOS were strongly expressed in the cytoplasm of adenomas,
adenocarcinomas, mononuclear cells and endothelial cells.
p53-deficient mouse model Results from many studies
have suggested that loss of p53 function is an early and
critical event in colitis-associated colorectal
cancer[10,37]; one that is perhaps comparable to the gatekeeper function of
APC in sporadic colorectal carcinogenesis. In order to
further investigate the function of p53 in colitis-associated
colorectal neoplasia, colitis was induced in
p53_/_, p53+/_, and
p53+/+ mice (C57BL/6J background) using 3 or 4 cycles of
DSS followed by 120 d of water[38]. Each cycle of DSS
consisted of 4 d of 4% DSS in the drinking water followed by 17
d of untreated water. No colorectal lesions were found in
untreated p53_/_, p53+/_, and
p53+/+ mice. The incidence of neoplastic lesions in DSS-treated
p53_/_, p53+/_, and
p53+/+ mice was 57%, 20%, and 20%, respectively (Table 2). DSS-treated
p53_/_ mice had a significantly greater number of total lesions,
cancers, and dysplasias per mouse than either DSS-treated
p53+/_ or p53+/+ mice. Two important pathological features
were observed in these animals. First, cancers were found in
DSS-treated p53+/_ and
p53_/_ mice but not in
p53+/+ mice. Second, the predominant morphologic subtype of colorectal
neoplasia varied depending on the number of copies of
wild-type p53. The representation of lesion subtypes in
DSS-treated p53_/_ mice was 38.5% flat cancers, 46.1% flat
dysplasias, 15.4% polypoid cancers, and 0% polypoid
dysplasias. In DSS-treated p53+/_ mice, the representation of
lesion subtypes was 16.7% flat cancers, 0% flat dysplasias,
5.6% polypoid cancers, and 77.8% polypoid dysplasias. In
DSS-treated p53+/+ mice, all lesions were polypoid dysplasias.
These data indicate that flat lesions are associated with the
p53_/_ genotype, while polypoid lesions are associated with
the p53+/_ and p53+/+ genotypes. Irrespective of the
p53 genotype, nuclear translocation and mutation of
β-catenin were observed only in polypoid lesions (91% and 43.7%
respectively). These data indicate that loss of p53 enhances
the induction of colitis-associated colorectal neoplasia, in
particular flat lesions, and dysregulation of β-catenin
signaling plays an important role in the formation of polypoid
lesions in the p53 model of colitis-associated dysplasia.
Fujii et al[39] have also studied the development of
colonic neoplasia in p53-deficient mice with DSS-induced
colitis. In this study, p53+/+,
p53+/_, and p53_/_ mice were on a
C57BL/6 × CBA background. Mice were exposed to DSS for
2 cycles (7 d of 4% DSS plus 14 d of untreated water per
cycle) followed by 84 d of untreated water (126 d total).
Neoplastic lesions were found in 100%, 46.2%, and 13.3% of
p53_/_, p53+/_, and
p53+/+ mice, respectively. The mean number of lesions per mouse was 5.0, 0.62, and 0.2 in
p53_/_, p53+/_, and
p53+/+ mice, respectively. Invasive adenocarcinomas
were seen in 5% (2 of 40) of mice. Similar to the study by this
group[38], the majority of lesions in
p53_/_ mice were flat
(91.7%), while the majority of lesions in
p53+/+ mice were polypoid (66.7%). Unlike our study, in which nuclear
localization was confined to polypoid lesions, Fujii
et al[39]
observed nuclear staining of β-catenin in both flat and
polypoid neoplasias (82.6%).
iNOS An overproduction of reactive oxygen and
nitrogen species in chronic ulcerative colitis leads to colonic
nitrosative and oxidative stress and depletion of antioxidant
molecules. Activation of iNOS causes prolonged
production of NO in cytotoxic concentrations. iNOS has been shown
to be overexpressed in the colons of patients with ulcerative
colitis and may contribute to the pathogenesis of
colitis-associated neoplasia[40,41]. Recently, Seril
et al[42] studied the role of iNOS in the development of DSS colitis-associated
neoplasia. iNOS_/_ (C57BL/6 background) and
iNOS+/+ (C57BL/6 background) mice were treated with 15 cycles of
1% DSS (1 cycle = 7 d of DSS plus 10 d of water) and fed a
high-iron AIN76A diet. Mice were sacrificed on day 255. A
total of 65.2% of iNOS+/+ mice developed tumors as
compared to 68.4% of iNOS_/_ mice. Tumor multiplicity was 1.5±
0.2 and 2.0±0.2 in iNOS+/+ and
iNOS_/_ mice, respectively. There was no difference in staining intensity for nitrotyrosine
between iNOS_/_ and iNOS+/+ mice. These results suggest
that in the absence of iNOS, other factors such as eNos may
play a role in nitrosative stress and colitis-associated cancer.
MSH2 Mutation of MSH2, one of the mismatch repair
genes, in humans is associated with the hereditary colorectal
cancer syndrome HNPCC (hereditary nonpolyposis colon
cancer). The role of loss of function of mismatch repair is
unclear in the development of colitis-associated neoplasia
in humans[43,44]. Colitis was induced in
Msh2_/_, Msh2+/_, and
Msh2+/+ mice (on a 129/OLA × C57BL/6 background) using
3_8 cycles of DSS. There was no difference in the severity of
colitis between genotypes. After 5 cycles of DSS, 12.5%,
8.0%, and 46.7% of Msh2_/_,
Msh2+/_, and Msh2+/+ mice developed high-grade dysplasia, and 16.7%, 8.0%, and
13.3% of Msh2+/+, Msh2+/_, and
Msh2_/_ mice developed adenocarcinomas. The majority of adenocarcinomas were of
the mucinous type. In Msh2_/_ mice, 77.8% of tumors were
microsatellite instability-high as compared to 0% in
Msh2+/_ and Msh2+/+ mice. This model provides an avenue to study
the role of DNA mismatch repair in colitis-associated
neoplasia in the human[45].
Chemoprevention of DSS-induced colitis-associated neoplasia
Although the number of chemoprevention studies
reported to date in the DSS model is quite limited, emerging
data show great promise that colitis-associated colorectal
carcinogenesis is indeed a preventable disease. The
following section summarizes the ability of select classes of both
synthetic and dietary agents to inhibit the formation of
DSS-induced neoplasms.
COX-2 inhibitors The significant contribution of
COX-2 to the development of sporadic colorectal tumors has been well
documented in both preclinical[46] and
clinical[47] studies. Based on chronic inflammation as the hallmark of ulcerative
colitis, it is anticipated that COX-2 inhibitors will also be
highly effective in this disease setting. A small retrospective
analysis of celecoxib and rofecoxib use indicates that
COX-2 inhibitors are well tolerated by the majority of patients with
inflammatory bowel disease[48]. Administration of celecoxib
(300 ppm in the diet) to Swiss Webster mice with
AOM/DSS-induced colitis decreased the multiplicity of colorectal
dysplasias by 50%[23]. Interestingly, this inhibitory response
appeared to be specific to polypoid (vs flat) lesions and
unrelated to its anti-inflammatory activity. The degree of
inflammation in mice receiving AOM, DSS and celecoxib was
significantly more severe than that of control mice
administered only AOM and DSS.
Nimesulide is a selective COX-2 inhibitor of the
sulfonamide class, which is less ulcerogenic than other
nonsteroidal anti-inflammatory
drugs[49]. Long-term feeding of diets supplemented with nimesulide (0.04%,
w/w) to ICR mice with AOM/DSS-induced colitis caused a significant reduction in
the incidence of both adenomas and adenocarcinomas (40%
and 60%, respectively)[50]. Drug treatment also decreased
total tumor multiplicity 3-fold. Associated reductions in
staining for proliferation cell nuclear antigen (PCNA), COX-2,
iNOS, b-catenin and nitrotyrosine, a marker of nitrosative
damage, were observed.
PPAR ligands Peroxisome proliferator-activated
receptors (PPARs) are nuclear hormone receptors that
function as ligand-activated transcription factors. The ability of
PPAR ligands to inhibit colorectal adenomas in
Apc+/Min mice remains
controversial[51_54]. Administration of 0.05%
troglitazone (w/w), a ligand of PPARg, to ICR mice with
AOM/DSS-induced colitis significantly inhibited both the incidence
and multiplicity of colonic adenocarcinomas by 60%, while
bezafibrate, a PPARα ligand, reduced only tumor incidence
significantly[50]. These data confirm previous findings by
this same group that demonstrate that troglitazone and
bezafibrate, as well as another PPARg ligand, pioglitazone,
can suppress the formation of aberrant crypt foci in rats with
AOM/DSS-induced colitis[55].
Aminosalicylates Aminosalicylates such as
5-amino-salicylate (5-ASA) have evolved over the past 60 years as
the gold standard for the treatment of mild to moderate
ulcerative colitis and maintenance of therapeutically induced
remissions. However, the ability of this class of agents to
prevent colitis-associated neoplasia in patients with
ulcerative colitis remains unclear. The effect of 5ASA on colorectal
carcinogenesis has been evaluated in the AOM/DSS mouse
model[56,57]. An inverse trend between dose of 5-ASA and
multiplicity of colorectal dysplasias was observed when Swiss
Webster mice with AOM/DSS-induced colitis were
administered 5-ASA at 75, 150, or
225 mg/kg[56]. Mice receiving the lowest dose of 5-ASA (75 mg/kg, a clinically relevant dose)
exhibited the fewest tumors per animal (7.6±1.4 and 13.6±2.7
for drug-treated and AOM/DSS controls, respectively).
Grimm et al[57] studied the chemopreventive effects of low
(100 mg/kg) and high (300 mg/kg) dose 5ASA in the
AOM/DSS model. Mice underwent 2 cycles of DSS treatment after
a single ip injection of AOM (8 mg/kg). High-dose 5-ASA
given for 1 week immediately after the second treatment of
DSS significantly reduced the incidence of low- and
high-grade dysplasia by 35% and 100%, respectively, as well as
inflammation scores. High-dose 5-ASA given 1 week after
the end of the second treatment of DSS blocked the
progression of established dysplasia.
Antioxidants Seril et
al[58] have examined the chemo-preventive activity of
N-acetylcysteine (NAC), an antioxidant and mucolytic agent, in C57BL/6J mice treated with
0.7% DSS for 12 cycles and maintained on an iron-enriched
diet. Exposure to 0.2% NAC significantly reduced tumor
incidence from 88.5% (DSS controls) to 68% and mean tumor
multiplicity from 2.1±0.2 (DSS controls) to 1.5±0.1. Based on
an observed decrease in the number of colonic epithelial
cells in NAC-treated mice that stained positive for PCNA,
nitrotyrosine and iNOS, the authors conclude that NAC
inhibits tumor formation by decreasing both proliferation
and the cellular damage induced by nitrosative stress.
Statins Statins are coenzyme A reductase inhibitors that
suppress the production of mevalonate, a precursor of
cholesterol and geranyl-geranyl diphosphate, and ultimately the
prenylation of signal transduction
proteins[59]. Fluvastatin is a lipophilic statin that readily crosses the cell membrane
and decreases the oxidation of low density
lipoproteins[60]. Administration of fluvastatin to CBA/J mice receiving a single
injection of AOM and 3 cycles of 3% DSS dramatically
decreased the mean number of high-grade colonic
dysplasias per mouse from 27.9±2.8 (controls) to 0.8±0.5
(drug-treated animals)[61]. A drug-induced decrease in the degree
of inflammation was confirmed by the detection of fewer
cells with positive staining for oxidative damage (8-OHdG).
These data provide strong support for further analysis of
the efficacy of other statins in the chemoprevention of
colitis-associated colorectal neoplasia.
Inositol compounds Polyphosphate inositol compounds
are active constituents of high-fiber foods. Inositol
hexaphos-phate (IP6) is found in whole grains, cereals, legumes, nuts
and seeds where it serves as the primary energy source and
antioxidant for the germinating
plant[62]. Phosphoinositides are metabolized to compounds that regulate enzyme
activities and protein phosphorylation in the cell membrane,
nucleus and cytoplasm. The antitumor activity of IP6 has
been documented in several models of chemically induced
carcinogenesis (see Fox et al for
review)[63]. The efficacy of IP6 and inositol against inflammation-associated
carcinogenesis has been examined in C57BL/6J mice given DSS for 15
cycles and fed a diet enriched in iron for the duration of the
study[64]. The addition of 1% inositol to the drinking fluid of
DSS-treated mice reduced tumor incidence, multiplicity and
volume significantly to 61%, 40%, and 20% of that of
DSS-treated controls, respectively. In contrast, administration of
1% IP6 in the drinking fluid had no significant effect on the
formation of colitis-associated neoplasias. Results from
associated mechanistic studies suggest that inositol may
inhibit colitis-associated colorectal carcinogenesis by
reducing macrophage-mediated inflammation, nitrooxidative stress
and cell proliferation.
Prenyloxycoumarins Prenyloxycoumarins are
secondary metabolites found in plants of the Rutaceae (ie, orange,
lemon, lime, grapefruit) and Umbelliferae (ie, carrots, parsley,
caraway, fennel) families that possess anti-inflammatory
properties. Tanaka et al[65] reported that Citrus auraptene
inhibits chemically induced aberrant crypt foci in rats.
Extension of this observation to inflammatory bowel disease
yielded a similar result. Exposure of ICR mice with
AOM/DSS-induced colitis to diets supplemented with either
auraptene or collinin for 17 weeks significantly reduced both
tumor incidence and the number of lesions per animal at
both doses tested (0.01 and 0.05%)[66]. Enhanced apoptosis
and decreased proliferation, iNOS and COX-2 were noted.
In summary, preclinical studies conducted to date
support the use of the mouse model of DSS-induced colitis as a
highly relevant system in which to further characterize the
molecular events required for the formation of colorectal
neoplasia in a background of chronic inflammation. Results
from chemopreventive intervention studies provide evidence
that colitis-associated colorectal cancer can be prevented.
Future experimentation in this model is anticipated to
facilitate the development of an efficacious chemopreventive
regimen for patients with ulcerative colitis.
Acknowledgments
We thank Dr Tony KONG for providing us with an
opportunity to review this exciting area of research, and
Maureen CLIMALDI for her excellent assistance in
preparing this manuscript for publication.
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