Gastroenterology Section, Veterans Affairs Palo Alto Health Care System, Palo Alto 94304; and Division of Gastroenterology and Hepatology, Stanford University, Stanford, California 94305
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ABSTRACT |
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Barrett's esophagus (BE) results from acid and bile reflux and predisposes to cancer. To further understand the mechanisms of acid- and bile-induced hyperproliferation in BE, we investigated the release of PGE2 in response to acid or bile salt exposure. Biopsies of esophagus, BE, and duodenum were exposed to a bile salt mixture as a 1-h pulse and compared with exposure to pH 7.4 for up to 24 h, and PGE2 release, cyclooxygenase-2 (COX-2), and protein kinase C (PKC) expression were compared. Similar experiments were also performed with acidified media (pH 3.5) alone, in the presence or absence of bisindolylmaleimide (BIM), a selective PKC inhibitor, and NS-398, a COX-2 inhibitor. One-hour pulses of bile salts or acid significantly enhanced proliferation, COX-2 expression, and PGE2 release in BE. In contrast, the combination pulse of acid and bile salts had no such effect. Treatment with either BIM or NS-398 led to a dramatic decrease in PGE2 release in BE explants and a suppression of proliferation. The acid- or bile salt-mediated hyperproliferation is related to PGE2 release. Acid- and bile salt-induced induction of COX-2 and PKC may explain, at least in part, the tumor-promoting effects of acid and bile in BE.
acid; bile salts; gastroesophageal reflux disease; protein kinase C; cyclooxygenase-2; prostaglandins; duodenogastric reflux
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INTRODUCTION |
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BARRETT'S ESOPHAGUS (BE), or specialized intestinal metaplasia, is a complication of gastroesophageal reflux disease and predisposes to dysplasia and esophageal adenocarcinoma (34). Adenocarcinoma in BE does not arise de novo but rather follows a multistep process of low-grade dysplasia, high-grade dysplasia, early adenocarcinoma, and eventually invasive cancer (11, 27). Several studies have examined the role of duodenogastroesophageal refluxate in the pathophysiology of BE (12, 38). Patients with BE have severe, long-standing reflux of acid and bile compared with patients with esophagitis or normal controls. Because the BE epithelium is continually undergoing renewal and is exposed to these external factors, it is possible that acid and/or bile may alter its cellular signal transduction pathways involved in cell growth, thereby inducing cell proliferation and promoting malignant transformation (39).
We have recently shown (10, 24) that acid has a direct, dynamic effect on the cell proliferation and differentiation of BE both in vivo and ex vivo. Whereas long-term acid exposure results in a relatively differentiated phenotype and decreased cell proliferation, short pulses of acid exposure induce cell hyperproliferation. These changes may, in turn, increase the risk for dysplasia and adenocarcinoma. In similar experiments (17), we have also investigated the effects of bile salts on proliferation of BE epithelia in ex vivo organ culture compared with normal esophageal (squamous) and duodenal (columnar) controls and explored whether such an effect occurs in synergism with acid. In these experiments, acid or bile salt pulses significantly increased proliferating cell nuclear antigen (PCNA) expression in BE, possibly through a protein kinase C (PKC)-dependent mechanism (16). Paradoxically, when acid and bile salts were combined, partial or complete inhibition of PCNA expression was noted. Thus bile salts, independent of acid, contribute to the proliferative alterations in BE in a dynamic fashion but manifest a complex effect when they interact with acid.
Because of their known role in epithelial injury and carcinogenesis, bile acids may act as tumor promoters by increasing cell proliferation via PKC activation (7, 16). For example, dihydroxy bile acids have been shown to activate the transcription of cyclooxygenase-2 (COX-2) in a human esophageal adenocarcinoma cell line, again in part related to PKC activation (40). PKC is a key enzyme in signal transduction and mediates a number of cellular functions, including the control of growth and differentiation (2, 13, 21). Although the exact causal link(s) between the activity of COX-2 and carcinogenesis remains uncertain, there are several different mechanisms potentially involved (25). Overexpression of COX-2 inhibits apoptosis, increases the invasiveness of malignant cells, and enhances the synthesis of tissue prostaglandins (4, 15, 18, 31).
To further understand the mechanisms of acid- and bile salt-mediated cell proliferation, we have examined the possible roles of PKC and COX-2 in our ex vivo model of BE. Our experiments demonstrate an acid- and bile salt-mediated enhancement of PGE2 release that correlates well with PCNA expression as a marker of proliferation and may be suppressed by PKC or COX-2 inhibitors.
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MATERIALS AND METHODS |
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Patients and tissue collection. Endoscopic mucosal samples from normal esophagus (squamous controls), BE, and duodenum (columnar epithelial controls) were obtained from individuals with BE undergoing endoscopic surveillance at the Veterans Affairs Palo Alto Health Care System and Stanford University Hospital as part of an endoscopic surveillance program. Biopsies were collected under a protocol that was approved by the Administrative Panel on Human Subjects in Medical Research, Stanford University.
Endoscopy and biopsy. Endoscopy was performed by using a video image endoscope, with patients receiving intravenous sedation and continuous cardiac-respiratory monitoring. BE was endoscopically defined as the presence of red columnar islands within squamous esophageal mucosa or by the presence of lighter squamous islands within a circumferential columnar mucosa present above the endoscopically identified gastroesophageal junction, as previously described (24). For the diagnosis of BE, both endoscopic and histological evidence for such an epithelium were required. Endoscopic biopsies were from each quadrant (4 biopsies) every 1-2 cm of the length of the BE (29). In addition, mucosal biopsies were obtained from the duodenum and from the proximal (squamous) esophagus and were used as controls. Endoscopic mucosal samples were immediately divided into two parts. One part was formalin fixed for histopathological assessment; the other part was maintained in tissue culture medium (see Organ culture) for subsequent experimental use.
Histopathology. All specimens were analyzed independently by a staff pathologist to categorize normal esophageal mucosa, esophageal inflammation, metaplasia, and/or dysplasia. To confirm the presence of specialized intestinal metaplasia, Alcian blue staining was performed on all BE samples (10). Only BE biopsy samples with incomplete intestinal metaplasia, as defined by a specialized (intestinalized) surface and pit epithelium with goblet cells on hematoxylin and eosin (H&E) and Alcian blue stains, were included in the organ culture studies. Since all patients had established BE and were undergoing surveillance while receiving proton pump inhibitor therapy, there was no evidence of inflammation in the tissues other than the usual degree of scattered chronic inflammatory cells in the lamina propria. Furthermore, since dysplasia and COX-2 expression have been linearly related, we did not include any dysplastic samples in our ex vivo experiments to minimize variability of results. Morphological assessment by H&E stain was performed to ensure histological integrity of organ culture tissues for each time point up to 24 h.
Reagents.
All bile acid/salts (sodium glycocholate and taurocholate, glycocholic
acid, and taurochenodeoxycholate) and anti-PCNA monoclonal antibodies
(MAbs) were obtained from Sigma (St. Louis, MO). Anti-PKC MAbs were
obtained from Oncogene Research Products (Cambridge, MA). The specific
PKC inhibitor bisindolylmaleimide (BIM) was purchased from
Boehringer-Mannheim (Indianapolis, IN). COX-2 antibodies were obtained
from Cayman Laboratories (Ann Arbor, MI). Anti-PKC- MAbs were
obtained from Oncogene.
Organ culture. Organ culture was performed essentially as previously described (8, 9). Briefly, multiple mucosal biopsies were cut into two fragments by using an aseptic technique and were randomly assigned to acid, bile salts, acid plus bile salts, or control groups to avoid sampling bias. Patient samples were not pooled together, and a single patient's specimens were used for each individual experiment. Four to six biopsy fragments were placed on a sterilized stainless steel grid within 12-well plates so that culture medium (1 ml) covered the surface of the biopsy. Plates were placed on racks in a sterile sealed jar (Torsion Balance, Clifton, NJ), perfused with 95% O2-5% CO2, and then cultured at 37°C. For all experiments, we used medium 199 supplemented with 10% heat-inactivated fetal calf serum, 1 µg/ml insulin, 500 U/ml streptomycin, and 250 U/ml penicillin. Tissues were exposed to a bile salt mixture (sodium glycocholate and taurocholate, glycocholic acid, and taurochenodeoxycholate; total final concentration 1 mM; pH 7.4) as a 1-h pulse, followed by incubation in normal media (pH 7.4). This bile salt mixture concentration was selected because its effects have been previously described on patients with BE, and hence it represents near-physiological constituents and concentrations (7). The concentration of 1 mM was chosen because it was not toxic to our experimental and control epithelia in dose-response experiments and because it exhibits uniformity of proliferative response in BE tissues in our ex vivo model. For acidic culture conditions, the medium was acidified for 1-h pulse with 0.1 M HCl (~20% vol/vol, pH 3.5). In all experiments, a volume of distilled water (~20% vol/vol) was added to the control nonacidified media to achieve an osmolality equal to that of the acid-treated media. Tissues were exposed to acid alone (in 1-h pulse, pH 3.5), bile salts alone (in 1-h pulse, pH 7.4), or control media (pH 7.4). Experiments were conducted for 24 h, with collection time points of 1 h and 24 h. For the PKC experiments, a 1-h pulse of bile salts was administered either in the absence or presence of the PKC inhibitor BIM (17). Prior experiments (not shown) have demonstrated that the BIM dose chosen (10 µM) gives the best reproducibility of response in our ex vivo system. To confirm tissue viability after organ culture, lactate dehydrogenase (LDH) assays were performed by using an aliquot of tissue culture medium taken at the end of the culture period and an LDH assay kit (Sigma) (8). Explants that were homogenized in media with Triton X-100 and then sonicated (before measuring the LDH of the media) were used as positive controls. Control tissues were processed at the same pH as the experimental samples to adjust for pH sensitivity of the assay. The absorbance values (at 490 nm) were expressed as a percentage of the positive control and adjusted for protein content of the explants.
Protein extraction and immunoblot analysis.
This was performed to assess PCNA, PKC-, or COX-2 expression in BE
epithelia treated with 1-h pulse of bile salts or acid (10,
17). Samples from 1 and 24 h of organ culture were
assessed. For each time point, tissues were homogenized (4°C) in 1%
deoxycholate, 1% Nonidet P-40, and 0.1% SDS in PBS (pH 7.4)
containing 5 mM EDTA, 15 µg/ml aprotinin, 10 µM leupeptin, 10 µM
pepstatin, and 0.1 mM phenylmethylsulfonyl fluoride. The protease
inhibitors were added just before solubilization, and homogenization
was performed by using Kontes glass tissue grinders. Lysates were centrifuged at 16,000 g (20 min, 4°C), and protein
concentrations were measured by the bicinchoninic acid protein assay as
recommended by the manufacturer (Pierce, Rockford, IL). Proteins were
separated by using 12% SDS-PAGE and then transferred to polyvinylidene
difluoride membranes (Schleicher & Schuell, Keene, NH) in transfer
buffer containing 12.5 mM Tris · HCl (pH 8.3), 100 mM glycine,
and 20% methanol (40 V, 12 h, 4°C). After transfer, membranes
were blocked for 3 h in MT buffer (5% dry nonfat milk in PBS
containing 0.1% Tween 20), then incubated for 2 h (22°C) in
1:1,000 dilution of the anti-PCNA antibody, 1:1,000 dilution of the
anti-COX-2 antibody, or 1:2,000 dilution of the anti-PKC antibody,
respectively. Membranes were washed in PBS buffer (containing 0.1%
Tween 20) and then incubated with peroxidase-conjugated secondary
antibody (1:3,000 dilution) for 1 h at 22°C. After being washed
once in PBS buffer (containing 0.1% Tween 20) for 1 h, bands were
visualized by using an enhanced chemiluminescence system (NEN,
Wilmington, DE).
PGE2 immunoassays. PGE2 release was measured by using the Biotrak EIA assay (Amersham Pharmacia Biotech). PGE2 was extracted from the organ culture medium by using the Biotrak assay kit protocol and evaporated to dryness under nitrogen. Samples were reconstituted in the assay buffer provided in the kit. Twenty-five microliters of each sample were assayed in duplicates and triplicates for accuracy. A standard curve was run in duplicates and under similar conditions as the samples. Assay data were calculated from the standard curve by using Excel software. PGE2 release was measured in separate experiments with biopsy tissues from four different patients. Since pilot experiments (not shown) revealed an excellent correlation among tissue weight, PGE2 release, and protein content, we report all of the PGE2 data for milligrams of tissue placed in each dish.
To examine the synthesis of PGE2 in more detail, we also measured the effects of acid and bile salts on the production of PGE2 when an excess of arachidonic acid (500 µM) was added to the system. These experiments were done because PGE2 production can be affected by changes in the activity of phospholipase A2, which provides the substrate for COX-catalyzed reactions. Adding excess arachidonate minimizes any contribution of phospholipase A2 activity to the rate of production of PGE2. In these experiments, arachidonic acid was added to the media along with other treatments or alone, in the case of controls. The treatment lasted 1 h in each case.COX-2 and PKC- inhibition.
NS-398, a specific COX-2 inhibitor, was used at 0.1-, 0.5-, 1.0-, 5.0-, 10.0-, and 20.0-µM concentrations to determine the ideal
concentration for our experiments with biopsies in the ex vivo organ
culture setting (17). Final concentration of 10.0 µM
NS-398 was then used in all of our experiments. To inhibit PKC-
, BIM
at 10 µM was used in all experiments. However, BIM is not a specific
inhibitor of PKC-
; rather, it inhibits all isoforms of PKC.
PKC- activity.
Acid- and bile salt-treated BE samples were homogenized, and the
soluble fraction of total protein was collected. Protein (50-100
µg) was used to immunoprecipitate PKC-
with specific PKC-
antibody. Immunoprecipitates (equal amounts) were used for PKC
activity assay. Immunoprecipitates were incubated for 10 min at 30°C
with 2 µg myelin basic protein (MBP/whole protein) in the presence of
5 µCi of [
-32P]ATP and phospholipids. Kinase
reaction mixture was subjected to SDS-PAGE to resolve the
phosphorylated MBP and then to autoradiography.
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RESULTS |
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PGE2 release in BE.
We first measured PGE2 release at 1 and 24 h in the
media of mucosal biopsy explants from normal esophagus, BE, and
duodenum using a specific immunoassay. Figure
1 shows the amount of PGE2 (pg/ml) released in the medium of such explants at 1 and 24 h of
organ culture in standard media (see MATERIALS AND
METHODS). The amount of PGE2 released was
significantly higher in BE tissues compared with either normal
esophageal or duodenal control tissues at both 1 and 24 h
(P < 0.001). These changes could not be attributed to
inflammation, since the number of chronic inflammatory cells in the
lamina propria of BE epithelia was similar to that of normal duodenum.
At 24 h, there was no significant LDH release compared with the
Triton X-extracted, positive controls; light microscopy of H&E-stained
sections revealed that tissue architecture was well preserved (data not
shown).
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PGE2 release in BE is enhanced in response to either
acid or bile salt pulses.
We then determined the pattern of PGE2 release from BE
explants in response to either acid or bile salt 1-h pulses in the presence or absence of excess arachidonic acid (500 µM). All
experiments were performed in the presence and absence of arachidonic
acid (not all shown). Figure 2 depicts
the PGE2 release from mucosal biopsy BE explants after
1 h of acid or bile salt exposure, followed by culture in regular
media for 24 h. Organ culture media were retrieved after 1 and
24 h of organ culture and then analyzed by immunoassay as
described in MATERIALS AND METHODS. An increase in
PGE2 was noted in response to 1-h pulse of acid (Fig.
2A) or bile salts (Fig. 2B) compared with
arachidonic acid-containing control media. This effect was noted as
early as 1 h after exposure and was more pronounced at 24 h
and in the presence of arachidonic acid (P < 0.005).
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Modulation of acid- or bile salt-induced PGE2 release
in BE.
We then studied the effects of acid (pH 3.5, Fig.
3A) and bile salt (1 mM, Fig.
3B) pulses (1 and 24 h) in the presence of the COX-2
inhibitor NS-398, the PKC inhibitor BIM, and their combination on
PGE2 release under similar experimental conditions. For
both acid and bile salt-treated tissues, there was a significant degree (>80%) of inhibition of PGE2 release by the nonselective
PKC inhibitor BIM and the selective COX-2 inhibitor NS-398 at both time
points. A near-complete inhibition of PGE2 release was also
accomplished by the combination of inhibitors.
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Effect of acid or bile salts on PCNA expression.
We also examined the effect of acid or bile salts on cell proliferation
by assessing PCNA expression. For this, we performed organ
culture experiments on BE exposed to 1-h pulses of media alone (pH
7.4), acid (pH 3.5), and bile salts (pH 7.4), followed by continuous
culture in regular media (pH 7.4). Figure
5 depicts PCNA immunoblot results of a
representative of three experiments. Baseline PCNA expression (36 kDa,
Fig. 5, top) was markedly increased after a pulse of acid or
bile salts (Fig. 5, middle). Both baseline and acid or bile
salt-stimulated PCNA expression were significantly decreased by either
of the two inhibitors.
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Effects of COX-2 and PKC inhibitors.
Figure 6, top, depicts Western
blots showing the effects of NS-398, BIM, and their combination on the
expression of COX-2 in BE using a specific MAb. Both inhibitors caused
a significant decrease in COX-2 expression that paralleled the decrease
in PGE2 release (see Fig. 3). Figure 6, bottom,
depicts Western blots showing the effects of NS-398, BIM, and their
combination on the expression of the 93-kDa protein (PKC-) in BE by
using a specific MAb. Both inhibitors cause a significant decrease in
PKC-
expression that parallels the decrease in COX-2 expression. We
could not show any significant differences when the combination of
inhibitors was used, but there was a trend suggesting that the
combination was more inhibitory, raising the possibility that other
factors may be involved. Similar findings were noted with acid exposure (not shown).
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PKC- activity in the presence and absence of PKC inhibition.
Figure 7 depicts acid- and bile
salt-induced phosphorylation of MBP both at 1 and 24 h, whereas a
complete inhibition is observed in the presence of BIM (see
MATERIALS AND METHODS).
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DISCUSSION |
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In contrast to normal esophagus or duodenum, the metaplastic,
hyperproliferative, and premalignant epithelium of BE releases PGE2 that may be modulated ex vivo by pulse exposure of
acid or bile salts. Such PGE2 release is COX-2- and
PKC--dependent and leads to increased PCNA expression.
The pathogenesis of BE and its propensity to dysplasia and esophageal adenocarcinoma remain poorly understood (28). A growing number of abnormalities in cell proliferation, oncogene activation, tumor suppressor gene inactivation, and growth factor effects have been described that suggest a multifactorial and multistage disease. To understand the malignant progression of BE to adenocarcinoma, an understanding of the spectrum and the relationship of environmental factors involved is critical. Understanding the role of environmental factors coming in contact with the BE, such as acid and/or bile, will allow their modification by medical or surgical therapy and may in turn alter the natural history of this condition (9). For example, one could imagine a scenario wherein an environmental trigger (i.e., acid, bile salts) for an increase in cell proliferation predisposes cells to genetic mutations. As a result of these genetic alterations, cell proliferation is deregulated further and more genetic abnormalities accumulate. A vicious cycle is thus set up, and the accumulation of critical genetic errors (e.g., p53 mutations) may eventually lead to a clone of malignant cells capable of invasion (30).
In this study, we examined the relationship between BE and
environmental factors to which this metaplastic tissue is exposed. Specifically, because BE usually develops in the context of chronic reflux of stomach acid and intestinal juices into the esophagus, we
studied how acid or bile salts affect its proliferation. In the
experiments described, we used tissue samples from patients with BE
undergoing surveillance endoscopy and monitored their cellular behavior
in the presence or absence of acid and bile in the laboratory setting
of organ culture. We hypothesized that, if components of the
duodenogastric refluxate increase PGE2 release and cell
proliferation, effective inhibition of PGE2 release through COX-2 or PKC- inhibition or prevention of acid or bile reflux with
medical or surgical therapy would substantially decrease the risk for
esophageal adenocarcinoma.
Work from our laboratory (33) and others (22, 23,
36) provides evidence that COX-2 is important for the genesis of cancer. Hence there is evidence to link the activity of PKC to carcinogenesis, in part via the induction of COX-2 and overproduction of prostaglandins. This study adds to this evidence by showing that
both acid and bile salts upregulate PGE2 release,
suggesting a plausible mechanism underlying esophageal adenocarcinoma
formation: PKC and COX-2 induction. The main emphasis of our work was
to link COX-2 (expression and activity) to proliferation (PCNA) in the
context of environmental stimulation by acid or bile salts. The
nonspecific PKC inhibitor BIM was used to point out the involvement of
PKC in these processes. Using specific antibodies to the PKC- isoform that is specific for BE, we demonstrated that PKC-
is the
dominant isoform involved. However, more work will be needed to address
the specificity and regulation of PKC involvement in these processes.
The activator protein-1 and -2 recognition and nuclear factor-B
binding sites are several possible elements that mediate the effects of
PKC (14, 19, 37). Nevertheless, irrespective of the
precise mechanism by which acid and bile salts induce COX-2, our
results suggest that PKC inhibitors could be useful for downregulating COX-2 and thereby preventing and or treating BE and adenocarcinoma. Further studies will be needed to identify the responsible COX-2 promoter elements that lead to PGE2 release by acid or bile
salts and to identify the precise role of the specific PKC-
isoform that mediates such effects. In the present study, although it appears
that PKC-
is the predominant isoenzyme involved, the experiments
with BIM are not isoform specific. Nevertheless, preliminary observations from our laboratory suggest that PKC-
(Fig. 5,
bottom) is specific to BE and contributes to enhanced COX-2
expression and proliferation (16).
The concentrations of acid and bile salts required to induce COX-2 and PGE2 release in our study were similar to those found in bile and gastric secretions (5) and similar to those used in other studies (40). There is no physiological level of bile salts in the esophagus. Bile acids may enter the mucosal cells in the nonionized form through the lipophilic cell membrane and then accumulate, because intracellular ionization results in entrapment (32). Furthermore, conjugated bile acids may be precipitated out of solution at higher pH (3). In our experiments, however, we did not observe any precipitation of bile salts, nor did we observe any morphological damage by either histology or LDH assays of the explants and surrounding media. Recently, observations with a rat model of esophageal carcinogenesis have shown bile-induced COX-2 overexpression in vivo and suppression of such expression with selective and nonselective COX-2 inhibitors (6). Furthermore, PKC inhibitors block the transcription of COX-2 in Barrett's adenocarcinoma cell lines (40). In other cell systems, inhibition of PKC inhibits COX-2 induction and transcription (26, 35), whereas PKC activation stimulates PGE2 release (20). All of these efforts should help our understanding of the precise mechanisms by which components of the gastroduodenal refluxate induce proliferation and facilitate the development of chemopreventive strategies to diminish the risk of adenocarcinoma in BE. To what degree such strategies will regress the BE surface also remains unclear.
In conclusion, we have demonstrated by using a nondysplastic, ex vivo
BE culture model that acid or bile salts, administered as 1-h pulses,
induce PGE2 release and cell proliferation in BE through
PKC-- and COX-2-dependent mechanisms. Indeed, our results suggest
that the sequence of events is that of an early PKC-
activation,
followed by upregulation of COX-2 expression, enhanced PGE2
production, and, thus, enhanced cell proliferation (Fig. 8). However, such a sequence should be
seen only as a working model, before further experiments clarify the
precise events involved. Additional studies will be needed to address
other COX-2-modulating factors and to assess whether such altered
proliferation leads to the development of dysplasia and adenocarcinoma.
One possible therapeutic implication of our proposed model is that the
recently available COX-2 inhibitory therapy may suppress cell
proliferation in BE, particularly if it is associated with effective
inhibition of acid and bile reflux. Therefore, the current clinical
practice of acid suppressive therapy using proton pump inhibitors and
aiming at completely abolishing any acid pulses affecting the BE
epithelia may be enhanced by COX-2 inhibitors to prevent dysplasia and
adenocarcinoma. Indeed, all clinical studies to date have failed to
show a reduction of dysplasia risk in patients with BE treated with
acid-suppressive therapy alone (1). Because of the
disturbing increase in the incidence of BE and adenocarcinoma in the
western world, understanding of the mechanisms by which the various
components of the gastroduodenal refluxate affect the phenotype and
behavior of BE epithelia will, in turn, allow more effective disease
prevention and treatment.
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ACKNOWLEDGEMENTS |
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We thank Kris Morrow for medical illustration and the faculty, fellows, and staff of the Endoscopy Units of the Veterans Affairs Palo Alto Health Care System and Stanford University Hospital for their assistance in retrieval of mucosal specimens.
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FOOTNOTES |
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This research was funded in part by the Cancer Research Foundation of America (to B. S. Kaur).
Address for reprint requests and other correspondence: G. Triadafilopoulos, Gastroenterology Section (111-GI), Veterans Affairs Palo Alto Health Care System, 3801 Miranda Ave., Palo Alto, CA 94304 (E-mail: vagt{at}stanford.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 20, 2002;10.1152/ajpgi.00543.2001
Received 26 December 2001; accepted in final form 17 March 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bammer, T,
Hinder RA,
Klaus A,
Trastek VF,
and
Achem SR.
Rationale for surgical therapy in Barrett's esophagus.
Mayo Clin Proc
76:
335-342,
2001[ISI][Medline].
2.
Barry, OP,
Kazanietz MG,
Pratico D,
and
Fitzgerald GA.
Arachidonic acid in platelet microparticles up-regulates cyclooxygenase-2-dependent prostaglandin formation via a protein kinase C/mitogen-activated protein kinase-dependent pathway.
J Biol Chem
274:
7545-7556,
1999
3.
Batzri, S,
Harmon JW,
Schweitzer EJ,
and
Toles R.
Bile acid accumulation in gastric mucosal cells.
Proc Soc Exp Biol Med
197:
393-399,
1991[Abstract].
4.
Bennett, A,
Civier A,
Hensby CN,
Melhuish PB,
and
Stamford IF.
Measurement of arachidonate and its metabolites extracted from human normal and malignant gastrointestinal tissues.
Gut
28:
315-318,
1987[Abstract].
5.
Bremmer, CG,
and
Mason RJ.
Bile in the oesophagus.
Br J Surg
80:
1374-1376,
1993[ISI][Medline].
6.
Buttar, NS,
Wang KK,
Krishnadath KK,
Lutzke LS,
Anderson MA,
and
Burgart LJ.
Can cyclo-oxygenase-2 inhibitors prevent cancer in an animal model of Barrett's esophagus? A randomized, placebo-controlled chemoprevention trial (Abstract).
Gastroenterology
120, Suppl1:
A79,
2001.
7.
Craven, PA,
Pfanstiel J,
and
DeRubertis FR.
Role of activation of protein kinase C in the stimulation of colonic epithelial proliferation and reactive oxygen formation by bile acids.
J Clin Invest
79:
532-541,
1987[ISI][Medline].
8.
Decker, T,
and
Lohmann-Matthes ML.
A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumour necrosis factor (TNF) activity.
J Immunol Methods
15:
61-69,
1988.
9.
DeMeester, SR,
and
DeMeester TR.
The diagnosis and management of Barrett's esophagus.
Adv Surg
33:
29-68,
1999[Medline].
10.
Fitzgerald, RC,
Omary MB,
and
Triadafilopoulos G.
Dynamic effects of acid on Barrett's esophagus: an ex vivo proliferation and differentiation model.
J Clin Invest
98:
2120-2128,
1996
11.
Fitzgerald, RC,
and
Triadafilopoulos G.
Recent developments in the molecular characterization of Barrett's esophagus.
Dig Dis
16:
63-80,
1998[ISI][Medline].
12.
Gillen, P,
Keeling P,
Byrne PJ,
Healy M,
O'Moore RR,
and
Hennessy TPJ
Implication of duodenogastric reflux in the pathogenesis of Barrett's esophagus.
Br J Surg
75:
540-543,
1988[ISI][Medline].
13.
Guan, Z,
Buckman SY,
Pentland AP,
Templeton DJ,
and
Morrison AR.
Induction of cyclooxygenase-2 by the activated MEKK1 to SEK1/MKK4 to p38 mitogen-activated protein kinase pathway.
J Biol Chem
273:
12901-12908,
1998
14.
Imagawa, M,
Chiu R,
and
Karin M.
Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP.
Cell
51:
251-260,
1987[ISI][Medline].
15.
Kargman, SL,
O'Neill GP,
Vickers PJ,
Evans JF,
Mancini JA,
and
Jothy S.
Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer.
Cancer Res
55:
2556-2559,
1995[Abstract].
16.
Kaur, BS,
Omary MB,
and
Triadafilopoulos G.
Bile salt-induced cell proliferation in an ex-vivo model of Barrett's esophagus is associated with specific PKC isoform modulation (Abstract).
Gastroenterology
116:
A338,
1999.
17.
Kaur, BS,
Ouatu-Lascar R,
Omary MB,
and
Triadafilopoulos G.
Bile salts induce or blunt cell proliferation in Barrett's esophagus in an acid-dependent fashion.
Am J Physiol Gastrointest Liver Physiol
278:
G1000-G1009,
2000
18.
Kutchera, W,
Jones DA,
Matsunami N,
Groden J,
McIntyre TM,
Zimmerman GA,
White RL,
and
Prescott SM.
Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.
Proc Natl Acad Sci USA
93:
4816-4820,
1996
19.
Lenardo, M,
and
Baltimore D.
NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control.
Cell
58:
227-229,
1989[ISI][Medline].
20.
Liu, PS,
Sun L,
and
Hayashi J.
Stimulation of prostaglandin production in rat thymic epithelial cells by protein kinase C-mediated activation of phospholipase A2.
Biochem Int
27:
931-938,
1992[ISI][Medline].
21.
Mellor, H,
and
Parker PJ.
The extended protein kinase C superfamily.
Biochem J
332:
281-292,
1998[ISI][Medline].
22.
Miller, BW,
Baier LD,
and
Morrison AR.
Over-expression of protein kinase C-zeta isoform increases cyclooxygenase-2 and inducible nitric oxide synthase.
Am J Physiol Cell Physiol
273:
C130-C136,
1997
23.
Oshima, M,
Dinchuk JE,
Kargman SL,
Oshima H,
Hancock B,
Kwong E,
Trzaskos JM,
Evans JF,
and
Taketo MM.
Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase-2 (COX-2).
Cell
87:
803-809,
1996[ISI][Medline].
24.
Ouatu-Lascar, R,
Fitzgerald RC,
and
Triadafilopoulos G.
Differentiation and proliferation in Barrett's esophagus and the effects of acid suppression.
Gastroenterology
117:
327-335,
1999[ISI][Medline].
25.
Prescott, SM,
and
White RL.
Self-promotion? Intimate connections between APC and prostaglandin H synthase-2.
Cell
87:
783-786,
1996[ISI][Medline].
26.
Rao, YP,
Stravitz RI,
Vlahcevic ZR,
Gurley EC,
Sando JJ,
and
Hylemon PB.
Activation of protein kinase C-alpha and delta by bile acids: correlation with bile acid structure and diacylglycerol formation.
J Lipid Res
38:
2446-2454,
1997[Abstract].
27.
Reid, B.
Barrett's esophagus and esophageal adenocarcinoma.
Gastroenterol Clin North Am
20:
817-834,
1991[ISI][Medline].
28.
Reid, BJ,
Levine DS,
Longton G,
Blount PL,
and
Rabinovitch PS.
Predictors of progression to cancer in Barrett's esophagus: baseline histology and flow-cytometry identify low- and high-risk patients subsets.
Am J Gastroenterol
95:
1669-1676,
2000[ISI][Medline].
29.
Reid, BJ,
Weinstein WM,
Lewin KJ,
Haggitt RC,
VanDeventer G,
DenBesten L,
and
Rubin CE.
Endoscopic biopsies diagnose high-grade dysplasia or early operable adenocarcinoma without grossly recognizable neoplastic lesions.
Gastroenterology
94:
81-90,
1988[ISI][Medline].
30.
Rice, TW,
Goldblum JR,
Falk GW,
Tubbs RR,
Kirby TJ,
and
Casey G.
p53 immunoreactivity in Barrett's metaplasia, dysplasia, and carcinoma.
J Thorac Cardiovasc Surg
108:
1132-1137,
1994
31.
Ristimaki, A,
Honkanen N,
Jankala H,
Sipponen P,
and
Harkonen M.
Expression of cyclooxygenase-2 in human gastric carcinoma.
Cancer Res
57:
1276-1280,
1997[Abstract].
32.
Salo, J,
and
Kivilaasko E.
Role of luminal H+ in the pathogenesis of experimental esophagitis.
Surgery
97:
662-667,
1982.
33.
Shirvani, VN,
Ouatu-Lascar R,
Kaur BS,
Omary MB,
and
Triadafilopoulos G.
Cyclooxygenase-2 expression in Barrett's esophagus and esophageal adenocarcinoma: ex-vivo induction by bile salts and acid exposure.
Gastroenterology
118:
487-496,
2000[ISI][Medline].
34.
Spechler, S,
and
Goyal R.
The columnar-lined esophagus, intestinal metaplasia, and Norman Barrett.
Gastroenterology
110:
614-621,
1996[ISI][Medline].
35.
Subbaramaiah, K,
Jing W,
and
Dannenberg AJ.
Ceramide regulates the transcription of cyclo-oxygenase-2.
J Biol Chem
273:
32943-32949,
1998
36.
Tsujii, M,
and
DuBois RN.
Alterations in cellular adhesion and apoptosis in epithelial cells over-expressing prostaglandin endoperoxide synthase-2.
Cell
83:
493-501,
1995[ISI][Medline].
37.
Xie, W,
and
Herschman HR.
v-src Induces prostaglandin synthase 2 gene expression by activation of the c-jun N-terminal kinase and the c-jun transcription factor.
J Biol Chem
270:
27622-27628,
1995
38.
Vaezi, MF,
and
Richter JE.
Synergism of acid and duodenogastroesophageal reflux in complicated Barrett's esophagus.
Surgery
117:
699-704,
1995[ISI][Medline].
39.
Vaezi, MF,
Singh S,
and
Richter JE.
Role of acid and duodenogastric reflux in esophageal injury: a review of animal and human studies.
Gastroenterology
108:
1897-1907,
1995[ISI][Medline].
40.
Zhang, F,
Subbaramaiah K,
Altorki N,
and
Dannenberg AJ.
Dihydroxy-bile acids activate the transcription of cyclo-oxygenase-2.
J Biol Chem
273:
2424-2428,
1998