Expression of COX2 and p53 in Rat Esophageal Cancer Induced by Reflux of Duodenal Contents.

Abstract

Aim. Reflux of duodenal contents can induce mucosal injury, stimulate cell proliferation, and promote tumorigenesis. We examined the expression of COX2 and p53 in rat esophageal lesions induced by duodenal content reflux. Methods. Thirty 8-week-old male Wistar rats were exposed to duodenal content esophageal reflux. All animals underwent an esophagoduodenal anastomosis (EDA) with total gastrectomy in order to produce chronic esophagitis. Ten rats were the sham. Control. They were sacrificed at the 40th week. Their esophagi were examined for HE, COX2, p53, and proliferating cell nuclear antigen (PCNA). Results. After 40 weeks of reflux, dysplasia, squamous cell carcinoma (SCC), and adenocarcinoma (ADC) were found. PCNA labeling index was higher in dysplastic and cancer tissue than that in normal. Overexpression of COX2 was shown in ADC and SCC. Wild-type p53 accumulation was found in ADC, and not in SCC. Conclusion. Reflux of duodenal contents into the esophagus led to ADC and SCC in rats. COX2 may play an important role in esophageal cancer by duodenal content reflux. Our present results suggest an association between wild-type p53 accumulation and COX2 expression in ADC, with no such relation seen in SCC.

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International Scholarly Research Network
ISRN Gastroenterology
Volume 2012, Article ID 914824, 5 pages
doi:10.5402/2012/914824
Research Article
ExpressionofCOX2andp53inRatEsophagealCancerInducedby
Refluxof DuodenalContents
Naoki Hashimoto
Department of Surgery, Kinki University, 377-2 Ohno-Higashi, Osaka Sayama, Osaka 589-8511, Japan
Correspondence should be addressed to Naoki Hashimoto, gojigen000@gmail.com
Received 2 September 2011; Accepted 3 October 2011
Academic Editor: A. Weimann
Copyright © 2012 Naoki Hashimoto. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Aim. Reflux of duodenal contents can induce mucosal injury, stimulate cell proliferation, and promote tumorigenesis. We
examined the expression of COX2 and p53 in rat esophageal lesions induced by duodenal content reflux. Methods. Thirty 8-
week-old male Wistar rats were exposed to duodenal content esophageal reflux. All animals underwent an esophagoduodenal
anastomosis (EDA) with total gastrectomy in order to produce chronic esophagitis. Ten rats were the sham. Control. They were
sacrificed at the 40th week. Their esophagi were examined for HE, COX2, p53, and proliferating cell nuclear antigen (PCNA).
Results. After 40 weeks of reflux, dysplasia, squamous cell carcinoma (SCC), and adenocarcinoma (ADC) were found. PCNA
labeling index was higher in dysplastic and cancer tissue than that in normal. Overexpression of COX2 was shown in ADC and
SCC.Wild-typep53accumulationwasfoundinADC,andnotinSCC.Conclusion.Refluxofduodenalcontentsintotheesophagus
ledtoADCandSCCinrats.COX2mayplayanimportantroleinesophagealcancerbyduodenalcontentreflux.Ourpresentresults
suggest an association between wild-type p53 accumulation and COX2 expression in ADC, with no such relation seen in SCC.
1.Introduction
Esophageal cancer ranks the 10th most common cancer
worldwide. Esophageal squamous cell carcinoma is the pre-
dominant histological subtype of esophageal cancer. Many
geneshaverecentlybeenreportedtobeoverexpressedduring
esophageal cancer development. Tumorigenesis is increas-
ingly recognized as a process that involves the coordinated
action of a group of genes, rather than a single gene [1].
These genes and the associated control mechanisms may
represent potential targets for the prognosis and therapy of
esophageal cancer.
Alteration in the p53 tumor suppressor gene seems to
be one of the most important events in human cancer. p53
is known to play a central role in sensing and signaling for
growtharrestandapoptosisincellswithDNAdamage.There
is overwhelming evidence that p53 gene alterations are early
and frequent events in esophageal cancer and that this gene
is associated with the malignant transformation of Barrett’s
esophagus [2].
Cyclooxygenase2 (COX2) affects many processes includ-
ing apoptosis, proliferation, angiogenesis, invasiveness, im-
munosuppression, and inflammation, which are important
in carcinogenesis, and is therefore an attractive therapeutic
target. Increased expression of COX2 was found in many
premalignant tissues and malignant tumors [3]. COX2 is
not only becoming a sensitive marker for high-grade squa-
mous epithelial dysplasia of the esophagus and of Barrett’s
esophagus but also a target for the therapy and prevention of
esophageal cancer.
Although many studies have been performed to assess
the possible pivotal role of p53 and COX2 in esophageal
cancer progression, there are few on the mutual relationships
between p53 and COX2 from the standpoint of a series
of mechanisms. Therefore, we conducted an immunohisto-
chemical evaluation of the expression of p53 and COX2 in
esophageal cancer induced by chronic duodenal reflux.
2.MaterialandMethods
Eight-week-old male Wistar rats with a body weight of about
300g were used for the experiments. They were allowed
to acclimate for 2 weeks prior to surgery. Solid food was

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Esophagoduodenal anastomosis with total gastrectomy (EDA)
Esophagus
Esophagus
Duodenum
Duodenum
EDA
Stomach
Figure 1: EDA model: Esophagoduodenal anastomosis with total
gastrectomy.
withdrawn 1 day before and for 1 day after surgery. In 30
rats EDA was performed under general anesthesia (pento-
barbital 50mg/kg body wt ip) through an upper midline
incision. The gastroesophageal junction was ligated and the
distal esophagus was transected 2mm above the ligature.
Moreover, the gastroduodenal junction was also ligated and
the proximal duodenum was transected 3mm distal to the
pylorus.Atotalgastrectomywasperformedwiththeremoval
of the entire stomach and end-to-end anastomosis of the
esophagus and duodenum. The abdominal incision was
closed in two layers (Figure 1). In 10 rats the sham operation
inducedamidlinelaparotomyalone(normalcontrolgroup).
Postoperatively the rats were allowed to drink water after
six hours and were fed the following day. This procedure
was approved by the Animal Care and Facilities Committee,
Kinki University.
All the rats were killed as described previously. Special
carewastakentoseparatetheesophagusfromtheduodenum
based on the suture line. For the animals killed at the 40th
week, all the esophagi were cut longitudinally and were fixed
in 10% buffered formalin. The formalin-fixed esophagus
was Swiss-rolled, processed, and embedded in paraffin. Five-
micron sections were mounted onto glass slides and used for
pathological and immunohistochemical analysis.
2.1. Immunohistochemistry
COX2: Localization of COX2 protein was determined by
immunohistochemical staining using specific antibodies.
The EnVision system (Dako, Denmark) was used with
autoclave acceleration. Deparaffinized 5µm sections of a
formalin-fixed, paraffin-embedded block were immersed in
absolutemethanolcontaining0.3%hydrogenperoxidaseand
then covered with normal goat serum (1:30). Sections were
incubated overnight at 4◦C with primary antibody to rat
COX2 protein (diluted 1:100) (Transduction Laboratory,
Lexington, KY). The sections were treated with labeled
polymer (Dako) for 2h. Immersing the sections in 3,3?-
diaminobenzil tetrahydrochloride developed the reaction
products. The slides were counterstained lightly with hema-
toxylin.
Macroscopic finding of esophagus in EDA
Duodenum Esophagus
Figure 2: Macroscopic appearance of the esophagus in rats
autopsied 40 weeks after surgery.
PCNA (proliferating cell nuclear antigen): Immunohis-
tochemical detection of the PCNA was performed by the
avidin-biotin complex method using mouse monoclonal
anti-human PCNA antibody and appropriate Histostain
Gold AEC kit. The PCNA labeling index has been widely
used for the assessment of cell proliferation. In this study,
the index was defined as the number of squamous epithelial
cellswithaPCNA-positivenucleus(ornuclei)/100squamous
epithelial cells (%).
p53: immunohistochemistry staining was performed using
the Ventana Benchmark automated immunostainer follow-
ingtheprotocolsprovidedbythemanufacture.Theantibody
used was DO-7 for wild-type p53.
2.2. Statistical Analysis. Data are expressed as mean ± SD of
eachgroup.AnalysisofStudent’st-testwasusedforstatistical
analysis. P < 0.05 was considered statistically significant.
3.Results
3.1. General Observation. A total of 37 of 40 (92.5%) rats
completed the study. In the EDA group, 27 (90%) rats
completed the study and 3 rats died of complications such
as malnutrition and pneumonia. In the control group, 10
(100%) rats completed the study.
3.2. Macroscopic Findings (Figure 2). The middle and lower
esophagus of animals in the EDA group was wide and
thickened. The animals usually had very sever inflammation
across the whole esophagus, manifested by esophageal short-
ening, enlargement of the esophageal cavity (especially the
lower and middle parts), hyperkeratinization, and large-area
epithelial sloughing and ulceration.
The mucosa showed longitudinal zone changes: a nodu-
lar but smooth and glistening surface giving it a cobble-stone
appearanceinthelowerportion,nodularandunevensurface

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Microscopic finding of esophagus in EDA
Figure 3: Microscopic findings in the distal portion of the esophagus in rats autopsied 40 weeks after surgery.
with erosion in the middle portion, and normal mucosa in
the upper portion.
Therewasasmallpolypoidtumorintheloweresophagus
in the EDA group. The tumor was squamous cell carcinoma
and adenocarcinoma. Most of the nodular lesions were also
associated with carcinomas, the others with esophagitis.
3.3. Microscopic Findings (Figure 3). The esophagus of the
control rats did not reveal any pathological findings but
various squamous cell lesions were seen in the middle and
lower esophagus in the EDA group.
As shown in Table 1, all animals from the EDA group
showed histologic features of esophagitis including marked
hyperplastic changes, increased thickness of the squamous
epithelium, hyperkeratosis and regenerative changes with
papillomatosis, and basal cell hyperplasia. These features
were not found in the control group. Columnar lined epithe-
lium (CLE) developed in distal portion of the esophagi,
that is, the squamous epithelium was replaced by colum-
nar cell lined epithelium comprising absorptive cells with
brush borders. CLE was observed in 40% at the 40th
week. Sever dysplasia in the lower esophagus occurred in
100%, squamous cell carcinoma was observed in 40%, and
adenocarcinoma was observed in 30% at the 40th week.
To assess the biological behavior of various squamous
lesions, we performed immunohistochemical staining for
PCNA because the proliferative index is often increased
in dysplastic and cancer tissues. PCNA labeling index of
dysplasia and cancer (75 ± 5) was higher than that of control
(30 ± 5).
3.4. Immunohistochemistry of COX2 (Figure 4). Every ani-
mal that suffered from reflux demonstrated COX2 protein
expression in the lower esophagus. COX2 immunoreactivity
wasmainlyobservedininfiltratingcellsandfibroblastsinthe
stroma. There were some epithelial cells of SCC and ADC
which strongly expressed COX2 protein.
3.5. Immunohistochemistry of Wild-Type p53 (Figure 5).
Wild-type p53 protein accumulation was observed as a pos-
itive nuclear staining in ADC, while it was negative in SCC.
4.Discussion
The present investigation demonstrates that it is duodenal
contents, and not gastric contents, that induce esophageal
carcinogenesis through reflux. Since this carcinogenesis
required no administration of carcinogens and since spon-
taneous esophageal carcinoma is rare in animals, duodenal
contents are probably carcinogenic in the development of
esophageal carcinoma.
The histological pattern of esophageal carcinoma
induced in the present study was classified into 2 types:
adenocarcinoma and squamous cell carcinoma.
The adenocarcinoma always occurr near the esophago-
duodenostomy and always with the columnar lined epithe-
lium. Human esophageal adenocarcinoma mostly arises
in the lower third of the esophagus, and when it does,
occur it is usually associated with Barrett’s esophagus. The
majority of Barrett’s esophagus cases result from chronic
gastroesophageal reflux. SCC was observed distant from the
site of anastomosis and surrounded by chronic squamous
esophagitis with features of basal-cell hyperplasia and regen-
erative thickening.
It is widely accepted in humans that duodenal content
regurgitation is closely linked to Barrett’s esophagus and
to the development of esophageal ADC; esophageal SCC
is not reported to be related to reflux [4] but is strongly
associated with tobacco smoking and alcohol consumption.
Gastroesophageal reflux does not appear to be an inde-
pendent risk factor for esophageal SCC but may enhance
the acknowledged risk factors such as tobacco smoking
and alcohol consumption. In contrast, results of several
studies using rat duodenal content reflux models have
shown that development of esophageal carcinomas includes
squamous cell carcinoma [5]. In this study, the incidence of
pure adenocarcinoma is lower than that of squamous cell
carcinoma. It is unclear what factors lead to the formation of
carcinomas of specified histology. Miwa et al. [6] suggested
that SCC developed in places distant from the anastomosis
compared to ADC. This means that histological features may
depend on the volume of reflux contents; small amounts
of reflux cause SCC, and a large volume of reflux causes

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Table 1: Incidence of histological lesions of esophagus.
Postoperative weekGroup
EDA
(n = 27)
ErosionRegenerative hyperplasia CLEDysplasia SCCADC
40w
100%100% 40%100% 40%30%
EDA: esophagoduodenostomy, CLE: Columnar line epithelium, SCC: squamous cell carcinoma, ADC: Adenocarcinoma.
COX2 in ADC
(a)
COX2 in SCC
(b)
Figure 4: Immunohistochemical staining for COX2 in SCC and ADC.
Wild-type p53 in ADC
Figure 5: Immunohistochemical staining for wild type p53 in SCC and ADC.
ADC. In Mukaisho’s modified model [7], they added a
serosal suture between the esophagus and the jejunum after
esophagojejunostomy. This addition of a serosal suture may
decrease the reflux of duodenal contents compared with
other models, so that the incidence of SCC was higher than
that of ADC in their study. They conclude that duodenal
content reflux has a great potential for malignant initiation
and plays a role in developing not only ADC but also
SCC.
Long-term use of NSAIDS in rheumatic patients is
related to a reduced risk of various human cancers, including
esophageal cancer [8]. A large body of genetic and biochem-
ical evidence supports a role for COX2 in human and rodent
tumors [9].
COX2 is well established as playing an important role
in the tumorigenesis of a variety of human carcinomas and
their precursor lesions. The present study demonstrated that
the persistent inflammation because of duodenal reflux may
promote the process from DYS to SCC or CLE to ADC. At
week 40, we encountered DYS in 100%, SCC in 33%, and
ADC in 35% of ED models.
In this sequence COX2 was upregulated, and cell prolif-
eration was accelerated in the esophageal epithelium. The
role of COX2 in carcinogenesis has been investigated in
various carcinomas. With regard to esophageal carcinogen-
esis, increased COX2 expression in BE, SCC, and ADC has
been reported [10]. Zimmermann et al. observed that COX2
expression was revealed immunohistochemically in 91% of
172 squamous cell carcinomas and in 78% of 27 adenocar-
cinomas and suggested that COX2-derived prostaglandins
might play an important role in the regulation of prolifer-
ation of esophageal tumor cells [11].

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In the present study, we observed a significant elevated
COX2 expression in SCC and ADC. These results are in
accordance with the increased COX2 expression reported for
other human cancers, such as lung cancer [12], colon cancer
[13], pancreatic cancer, and stomach cancer [14].
Interactions between COX2 and p53 have been shown
in vitro and in vivo. It has been demonstrated that p53 can
upregulateCOX2[15]orsuppressthetranscriptionofCOX2
[16]. Additionally, COX2 has been observed to exhibit strong
inhibitory effects on p53 transcriptional activity [17]. Benoit
et al. [18] found a correlation between COX2 expression
and TP53 wild-type status in esophageal adenocarcinoma
with Barrett’s esophagus as a precursor lesion, but not
in SCC, providing evidence that the participation of p53
in the regulation of COX2 expression in cancer may be
dependent on tumor histology. Esophageal cancer occurs
in 2 major histopathological forms, ADC, that develops
from a precursor, inflammatory metaplastic lesion, Bar-
rett’s esophagus, and SCC, that develops from the normal
mucosa through a classical hyperplasia-dysplasia-carcinoma
sequence. As compared with ADC, SCC is less frequently
inflammatory.Theyproposedalso,interestingly,thatchronic
inflammation could represent a physiopathological context
in which p53 and the transcription factor NF-kappaB could
cooperate to activate COX2. Our present results suggest an
association between p53 accumulationand COX2 expression
in ADC, with no such relation seen in SCC. On the basis of
the mechanisms envisaged for the interplay between COX2
and p53 [19], it seems likely that partially different and
partially shared conditions and regulatory events of COX2
and p53 expressions prevail in ADC and SCC histologies of
esophageal cancer.
These results suggest that wild-type p53 participates in
the upregulation of COX2 in ADC, but not in SCC.
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