Rab11 in dysplasia of Barrett's epithelia

Barrett's esophagus predisposes affected patients to the development of esophageal adenocarcinoma. The development of adenocarcinoma proceeds along a progression through low- and high-grade dysplasia. Surveillance of Barrett's patients requires serial endoscopic investigations and grading mucosal biopsies. Unfortunately, grading of biopsies by conventional hematoxylin and eosin staining is fraught with significant interobserver variations. We have found in both biopsy and resection specimens that immunostaining for the small GTP binding protein Rab11 is increased in low-grade dysplastic cells. This staining is lost in high-grade dysplastic cells. These results suggest that low-grade dysplastic cells undergo an apical trafficking blockade, which is released as cells progress to the less differentiated phenotype of high-grade dysplasia and adenocarcinoma. Examination of the SKGT-4 esophageal adenocarcinoma cell line demonstrated prominent mRNA and protein expression for Rab11. Rab11 immunostaining was present in SKGT-4 cells as a perinuclear nidus of punctate staining along with a more diffuse punctate pattern. Thus, Rab11 expression was present in a esophageal adenocarcinoma cells in culture. Markers of vesicle trafficking may be critical factors for grading of mucosal dysplastic transitions leading to adenocarcinoma.     

Molecular imaging using fluorescent lectins permits rapid endoscopic identification of dysplasia in Barrett's esophagus

Barrett's esophagus is an example of a pre-invasive state, for which current endoscopic surveillance methods to detect dysplasia are time consuming and inadequate. The prognosis of cancer arising in Barrett's esophagus is improved by early detection at the stage of mucosal carcinoma or high-grade dysplasia. Molecular imaging methods could revolutionize the detection of dysplasia, provided they permit a wide field of view and highlight abnormalities in real time. We show here that cell-surface glycans are altered in the progression from Barrett's esophagus to adenocarcinoma and lead to specific changes in lectin binding patterns. We chose wheat germ agglutinin as a candidate lectin with clinical potential. The binding of wheat germ agglutinin to human tissue was determined to be specific, and we validated this specific binding by successful endoscopic visualization of high-grade dysplastic lesions, which were not detectable by conventional endoscopy, with a high signal-to-background ratio of over 5.     

Association of metallothionein expression and lack of apoptosis with progression of carcinogenesis in Barrett's esophagus

Barrett's esophagus is the transformation of normal esophageal squamous epithelium to specialized intestinal metaplasia (SIM). Among the Barrett's specialized cells, those that can develop protective mechanisms against apoptosis may have potential to become malignant. Studies have shown that overexpression of metallothionein (MT), low molecular protein that protects cells from apoptotic stimuli, appears to be associated with more advanced, highly malignant tumors. We thus investigated the relationship between MT expression and apoptosis in different stages of Barrett's carcinogenesis. Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling and immunohistochemical dual-staining assay were performed in human biopsy samples of normal, SIM, dysplasia, and adenocarcinoma. Apoptotic index and MT expression were quantified by using an image system to analyze the converted digital data. A negative correlation between MT expression and apoptotic index was found. MT expression was significantly increased along with the histologic progression towards adenocarcinoma. This study thus suggests that MT may contribute to cytoprotection, thereby inhibiting apoptosis and leading to carcinogenesis of Barrett's esophageal cells.     

Endoscopic screening and surveillance for Barrett's esophagus--clinical implications

There is now a clear causal relationship between symptomatic gastroesophageal reflux and esophageal adenocarcinoma (Lagergren et al, 1999). The risk factor is now identified as Barrett's metaplasia (Solaymani et al, 2004). Chronic reflux results in Barrett's metaplastic change, and the route to carcinoma is a stepwise progression, through dysplasia to invasive carcinoma (Jankowski et al, 2000). Earlier-stage disease is found in patients undergoing surveillance and is the major predictor of survival following surgery (Fountoulakis et al, 2004). Screening and surveillance by endoscopic biopsy regimen has profound implications for the allocation of healthcare resources and the provision of clinical services. Screening a high-risk group such as men with gastroesophageal reflux disease (GERD) will result in the detection of more patients with Barrett's esophagus, many of whom are asymptomatic. Once detected, questions remain as to surveillance intervals and the current methodology for surveillance. There are profound challenges with the accurate endoscopic and pathologic detection and categorization of Barrett's metaplasia, dysplasia , and, indeed, cancer. New endoscopic detection methods are being investigated to improve the diagnosis and definition of the premalignant phenotype. The detection of dysplasia requires increased surveillance and usually intervention either endoscopically or with surgery.     

Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus

Control of the G1/S-phase transition as well as angiogenic switch are two of the most studied mechanisms in cancer. The current study examined the correlation between the immunohistochemical expression of pRb2/p130, VEGF, EZH2, p53, p16, p21waf-1, p27, and PCNA in Barrett's esophagus (BE). Overall, p53 showed a much higher expression in BE patients (up to 50%) than in controls (1-10%) (P < 0.005). Also p21 showed a downregulation in BE when compared to normal esophagus (70% of cells vs. 65%), but the difference did not show any statistical significance (P = 0.45). pRb2/p130 was detected in 80% of cells in normal controls, but showed positive in only 20% of cells in BE biopsies. Additionally, Rb2/p130 expression was inversely correlated to that of VEGF, EZH2, and PCNA (P < 0.0001, P = 0.0032, P < 0.001, respectively). p27 stained more intensely and in a widespread manner (70%) cells in normal esophageal tissues but about only 30% in BE samples (P < 0.001). Lastly, in accordance with other reports, we also found p16 expressed by immunohistochemistry at high levels in normal controls and at low levels in BE (P < 0.001). In conclusion, p16, p21, p27, and p53 staining confirmed previously published data. Interestingly, pRb2/p130 expression was found significantly decreased in metaplastic epithelium compared to normal controls and showed significant inverse correlation with the expression of other markers, such as VEGF, EZH2, and PCNA. These data, taken together, indicate that these molecular events occurring in Barrett's metaplasia (BM) may represent one of the many steps taking place during esophageal malignant progression such as impairment of cell-cycle control, altered differentiation, and unbalanced angiogenesis.     

Image analysis of p53 and cyclin D1 expression in premalignant lesions of the oral mucosa

OBJECTIVE: The expression of p53 and cyclin D1 proteins was analyzed by image analysis in oral premalignant lesions and normal oral mucosa. STUDY DESIGN: Punch biopsies from the normal oral mucosa were obtained from 20 normal donors and 41 patients with oral dysplastic leukoplakias. After controlled formaldehyde fixation and paraffin embedding, immunohistochemistry was used to detect cyclin D1 and p53. Image analysis was performed using stain intensity levels established by determining color thresholds (nuclear score) in the basal and parabasal layers. RESULTS: Analysis of sections showed a similar pattern: only two normal donors had a few intensely positive p53 cells in the basal layer of the floor of the mouth and the tongue epithelia. Similarly, only three donors had intensely positive cyclin D1 cells in the normal epithelia of the same sites. Most cells fell in the range of negative or marginal stain (lower quartiles or terciles of nuclear score). These data on normal mucosa were compared with low grade oral leukoplakias (LGD) with mild to moderate dysplasia and with high grade leukoplakias (HGD) with severe dysplasia. Both markers were differentially expressed in precursor lesions versus normal epithelia. Statistical analysis of our data shows that the intensity of the immunohistochemical stain, as reflected in the nuclear scores of p53, is a reliable parameter that can differentiate between LGD and HGD of the oral mucosa. This was especially true when higher nuclear scores were compared. In contrast, low nuclear scores are more effective in differentiating normal epithelia from dysplastic epithelia. Although cyclin D1 immunohistochemistry does not stain as intensely as p53 stain, similar conclusions can be derived from those data. CONCLUSION: Image analysis of these two markers proved useful in distinguishing normal oral epithelia from low grade and high grade leukoplakias. With further developments in this field it is hoped that image analysis procedures could be used in different types of studies in which variations of protein expression in tissue sections could have prognostic implications or could be useful to determine subtle effects of curative or preventive treatment.     

p53 and the malignant progression of Barrett's esophagus

Barrett's esophagus (BE) is a metaplastic disorder in which specialized columnar epithelium replaces healthy squamous epithelium (intestinal metaplasia). Even though its pathophysiology and the steps of its neoplastic progression are not completely understood, BE can be considered as a complication of gastroesophageal reflux disease (GERD). Given that esophageal adenocarcinoma, which is continually increasing in the Western world, still has a poor prognosis and suffers from late diagnosis, and because BE is a precancerous lesion, there is a strong need for good molecular markers of malignant progression in Barrett's metaplasia (BM). The aim of this review is to examine the published data regarding the role that assessment of p53 may play in the management of BE, trying to understand if it may be a useful marker to early diagnose BE malignant transformation.     

Downregulation of p63 upon exposure to bile salts and acid in normal and cancer esophageal cells in culture

p63 is a member of the p53 protein family that regulates differentiation and morphogenesis in epithelial tissues and is required for the formation of squamous epithelia. Barrett's mucosa is a glandular metaplasia of the squamous epithelium that develops in the lower esophagus in the context of chronic, gastroesophageal reflux and is considered as a precursor for adenocarcinoma. Normal or squamous cancer esophageal cells were exposed to deoxycholic acid (DCA, 50, 100, or 200 microM) and chenodeoxycholic and taurochenodeoxycholic acid at pH 5. p63 and cyclooxygenase-2 (COX-2) expressions were studied by Western blot and RT-PCR. DCA exposure at pH 5 led to a spectacular decrease in the levels of all isoforms of the p63 proteins. This decrease was observed within minutes of exposure, with a synergistic effect between DCA and acid. Within the same time frame, levels of p63 mRNA were relatively unaffected, whereas levels of COX-2, a marker of stress responses often induced in Barrett's mucosa, were increased. Similar results were obtained with chenodeoxycholic acid but not its taurine conjugate at pH 5. Proteasome inhibition by lactacystin or MG-132 partially blocked the decrease in p63, suggesting a posttranslational degradation mechanism. These results show that combined exposure to bile salt and acid downregulates a critical regulator of squamous differentiation, providing a mechanism to explain the replacement of squamous epithelium by a glandular metaplasia upon exposure of the lower esophagus to gastric reflux.     

Failure of capsaicin-containing red pepper sauce suspension to induce esophageal motility response in patients with Barrett's esophagus.

The physiologic importance of afferent sensory pathways in the esophageal motor functions has been recently recognised. Capsaicin-sensitive sensory afferents were shown to play a role in the maintenance of mucosal integrity of the GI tract, and regulation of human esophageal motility. The aim of this study was to investigate the effect of topical application of capsaicin-containing red pepper sauce (Tabasco, 25%v/v, pH:7.0) suspension on the phasic activity of the human esophagus of healthy volunteers and patients with Barrett's esophagus. METHODS: The diagnosis of Barrett's esophagus was based on the findings of esophagoscopy and histology taken from the squamocolumnar junction of the esophagus. Esophageal motility was measured by perfusion manometry before and after application of red pepper sauce. RESULTS: Capsaicin containing red pepper sauce increases the motility response (LES tone, contraction amplitude, propagation velocity) of the human esophagus in healthy volunteers. This response failed in patients with Barrett's esophagus. CONCLUSION: Impaired esophageal sensory motor function may serve as one etiologic role in the development of Barrett's esophagus.     

Barrett's esophagus: treatment or observation of a major precursor factor of esophageal cancer?

Barrett's esophagus (BE) is a major precursor factor of esophageal cancer (EC). The appropriate management of patients with BE depends on the presence or not of dysplasia and the type of dysplasia that occurs. Due to the small proportion of BE patients that progress to cancer, the value of surveillance programs are a matter of debate. On the contrary, in high risk group of patients surveillance programs have significant impact. Large prospective trials are needed to define the optimal management strategy. Elucidation of carcinogenesis' steps and signal transduction pathways could reveal potential biomarkers in the order of early prediction for a highly malignant neoplasm with dismal prognosis. An efficacious tailored-made manner focusing to the safety profile and associated costs should be practised for less severe disease. In this review a thorough investigation of all available methods dealing with the clinical management of BE is provided.     

Deoxycholic acid causes DNA damage while inducing apoptotic resistance through NF-κB activation in benign Barrett's epithelial cells

Gastroesophageal reflux is associated with adenocarcinoma in Barrett's esophagus, but the incidence of this tumor is rising, despite widespread use of acid-suppressing medications. This suggests that refluxed material other than acid might contribute to carcinogenesis. We looked for potentially carcinogenetic effects of two bile acids, deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA), on Barrett's epithelial cells in vitro and in vivo. We exposed Barrett's (BAR-T) cells to DCA or UDCA and studied the generation of reactive oxygen/nitrogen species (ROS/RNS); expression of phosphorylated H2AX (a marker of DNA damage), phosphorylated IkBα, and phosphorylated p65 (activated NF-κB pathway proteins); and apoptosis. During endoscopy in patients, we took biopsy specimens of Barrett's mucosa before and after esophageal perfusion with DCA or UDCA and assessed DNA damage and NF-κB activation. Exposure to DCA, but not UDCA, resulted in ROS/RNS production, DNA damage, and NF-κB activation but did not increase the rate of apoptosis in BAR-T cells. Pretreatment with N-acetyl-l-cysteine (a ROS scavenger) prevented DNA damage after DCA exposure, and DCA did induce apoptosis in cells treated with NF-κB inhibitors (BAY 11-7085 or AdIκB superrepressor). DNA damage and NF-κB activation were detected in biopsy specimens of Barrett's mucosa taken after esophageal perfusion with DCA, but not UDCA. These data show that, in Barrett's epithelial cells, DCA induces ROS/RNS production, which causes genotoxic injury, and simultaneously induces activation of the NF-κB pathway, which enables cells with DNA damage to resist apoptosis. We have demonstrated molecular mechanisms whereby bile reflux might contribute to carcinogenesis in Barrett's esophagus.     

Flow cytometric DNA analyses of epithelial dysplasia of the esophagus

OBJECTIVE: To investigate, with flow cytometry, DNA aneuploidy as a marker of early carcinogenesis in dysplastic esophageal lesions. STUDY DESIGN: DNA content of exfoliated cells from 789 cases of esophageal dysplasia (including mild dysplasia, 195 cases; moderate dysplasia, 383 cases; and severe dysplasia, 211 cases) was determined with a FACS 420 flow cytometer. RESULTS: Cellular DNA content was closely related to the severity of dysplasia. The carcinogenesis rate in patients with dysplasia showed that DNA aneuploidy was significantly higher than in patients showing DNA diploidy. CONCLUSION: DNA aneuploidy in dysplastic lesions is a very important early signal of carcinogenesis. Patients with dysplastic lesions showing DNA aneuploidy should be treated and closely followed.     

Deoxycholic acid impairs glycosylation and fucosylation processes in esophageal epithelial cells

It is generally accepted that esophageal adenocarcinoma arises from a Barrett's metaplastic lesion. Altered glycoprotein expression has been demonstrated in tissue from patients with Barrett's esophagus and esophageal cancer but the mechanisms regarding such changes are unknown. The bile acid deoxycholic acid (DCA) alters many cell signaling pathways and is implicated in esophageal cancer progression. We have demonstrated that DCA disrupts Golgi structure and affects protein secretion and glycosylation processes in cell lines derived from normal squamous epithelium (HET-1A) and Barrett's metaplastic epithelium (QH). Cell surface expression of glycans was identified using carbohydrate-specific probes (wheat germ agglutinate, conconavalin A, peanut agglutinin, lithocholic acid and Ulex europaeus agglutinin) that monitored N-glycosylation, O-glycosylation and core fucosylation in resting and DCA-treated cells. DCA altered intracellular localization and reduced cell surface expression of N-acetyl-D-glucosamine, α-methyl-mannopyranoside (Man/Glc) and fucose in both cell lines. Furthermore, DCA reduced the expression of epithelial growth factor receptor and E-cadherin in a manner analogous to treatment of cells with the N-glycan biosynthesis inhibitor tunicamycin. This is the first study to identify an altered Golgi structure and glycomic profile in response to DCA in esophageal epithelial cells, a process which could potentially contribute to metaplasia, dysplasia and cancer of the esophagus.     

Proteomic screening of a cell line model of esophageal carcinogenesis identifies cathepsin D and aldo-keto reductase 1C2 and 1B10 dysregulation in Barrett's esophagus and esophageal adenocarcinoma

Esophageal adenocarcinoma (EA) incidence is increasing rapidly and is associated with a poor prognosis. Identifying biomarkers of disease development and progression would be invaluable tools to inform clinical practice. Two-dimensional polyacrylamide gel electrophoresis was used to screen 10 esophageal cell lines representing distinct stages in the development of esophageal cancer. Thirty-three proteins were identified by MALDI-TOF-MS which demonstrated differences in expression across the cell lines. Western blotting and qRT-PCR confirmed increased cathepsin D and aldo-keto reductases 1C2 and 1B10 expression in metaplastic and dysplastic cell lines. Expression of these proteins was further assessed in esophageal epithelium from patients with nonerosive (NERD) and erosive gastro-esophageal reflux disease, Barrett's esophagus (BE) and EA. When compared with normal epithelium of NERD patients, (i) cathepsin D mRNA levels demonstrated a stepwise increase in expression (p<0.05) in erosive, metaplastic and EA tissue; (ii) AKR1B10 expression increased (p<0.05) 3- and 9-fold in erosive and Barrett's epithelium, respectively; and (iii) AKR1C2 levels increased (p<0.05) in erosive and Barrett's epithelium, but were reduced (p<0.05) in EA. These proteins may contribute to disease development via effects on apoptosis, transport of bile acids and retinoid metabolism and should be considered as candidates for further mechanistic and clinical investigations.     

Production of prostaglandinE2 via bile acid is enhanced by trypsin and acid in normal human esophageal epithelial cells

Several reports suggest that duodenogastroesophageal reflux may produce esophagitis, Barrett's esophagus and esophageal carcinoma. And it is well known that the incidence of adenocarcinoma arising from Barrett's esophagus has been increasing during the past decade. On the other hand, cyclooxygenase-2 and prostaglandins, produced by the catalytic reaction of cyclooxygenase-2, are considered to relate to carcinogenesis of the digestive tract and other malignant tumors. Recent reports suggest that cyclooxygenase-2 is induced in Barrett's esophagus and esophageal carcinoma. The purpose of this study is to investigate the reaction of cyclooxygenase-2 expression and prostaglandinE2 production on normal human esophageal epithelial cells cultured with gastroduodenal components. Normal human esophageal epithelial cells were cultured with chenodeoxycholic acid, trypsin and in acidic condition, individually and with different combinations of these three factors. After culturing, cyclooxygenase-2 expression in the cells and amount of prostglandinE2 in culture media was evaluated by immunoblotting and enzyme-immunoassay, respectively after culturing the cells. Cyclooxygenase-2 expression was up-regulated by bile acid and prostaglandinE2 production was enhanced by bile acid with trypsin, acidic condition or both of these components, without a synergistic effect on cyclooxygenase-2 expression. Production of prostaglandinE2 via these factors was suppressed by the cyclooxygenase-2 selective inhibitor JTE-522.The results suggest that duodenogastroesophageal reflux may induce cyclooxygenase-2 expression and prostaglandinE2 production in esophageal epithelial cells, cyclooxygenase-2 specific inhibitors may have a chemopreventive effect on esophageal carcinoma.     

Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus

We hypothesized that, in esophageal squamous epithelial cells, there are differences among individuals in the signal transduction pathways activated by acid reflux that might underlie the development of Barrett's esophagus. To explore that hypothesis, we immortalized nonneoplastic, esophageal squamous cells from patients with gastroesophageal reflux disease (GERD) with (NES-B3T) and without (NES-G2T) Barrett's esophagus and used those cells to study acid effects on MAPK proteins. During endoscopy in patients with GERD with and without Barrett's esophagus, we took biopsy specimens from the distal squamous esophagus to study MAPK proteins before and after esophageal perfusion with 0.1 N HCl. We used immunoblotting and Western blotting to study MEK1/2 phosphorylation at two activating sites (serines 217/221), MEK1 phosphorylation at an inhibitory site (threonine 286), and MEK1/2 activity. After acid exposure, both cell lines exhibited increased MEK1/2 phosphorylation at the activating sites; the NES-B3T cells had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the NES-G2T cells showed an acid-induced increase in MEK1/2 activity. Similarly, in the squamous epithelium of patients with GERD with and without Barrett's esophagus, acid perfusion increased MEK1/2 phosphorylation at the activating sites in both patient groups; the Barrett's patients had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the patients without Barrett's demonstrated an acid-induced increase in ERK1/2 phosphorylation. In esophageal squamous cell lines and biopsies from patients with GERD with and without Barrett's esophagus, we have found differences in MAPK pathways activated by acid exposure. We speculate that these differences might underlie the development of Barrett's metaplasia.