Secretory Phospholipase A2 IIa Mediates Expression of Growth Factor Receptors in Esophageal Adenocarcinoma.

BACKGROUND: Receptor tyrosine kinases of the epidermal growth factor receptor (EGFR) family such as human epidermal receptor-2 (HER2) are involved in the development and progression of esophageal adenocarcinoma (EAC). Prior studies have demonstrated that group IIa secretory phospholipase A2 (sPLA2 IIa) can function as a ligand for the EGFR family of receptors and lead to an increase in receptor signaling. AIMS: We hypothesized that sPLA2 IIa inhibition downregulates the expression of EGFR and HER-2 in EAC and through this mechanism decreases proliferation in EAC. METHODS: Normal human esophageal epithelium, Barrett's esophagus (BE), and EAC tissue samples were assayed for baseline expression of EGFR, HER-2, and sPLA2 IIa. sPLA2 IIa was attenuated via inhibitor or lentiviral knockdown in esophageal cell lines, and cells were assayed for EGFR and HER2 expression as well as proliferation. FLO1 EAC cells were injected into the flank of nude mice. After randomization, mice received daily group IIA sPLA2 inhibitor or a control solution, and tumor volume was measured with calipers. RESULTS: sPLA2 IIa, EGFR, and HER2 expression increased across the spectrum of normal esophageal epithelium to EAC. sPLA2 IIa inhibition and knockdown decreased the expression of HER-2 and EGFR and proliferation. Mice treated with sPLA2 IIa inhibitor had smaller tumors than controls. CONCLUSIONS: sPLA2 IIa inhibition decreases EGFR and HER2 expression and lowers proliferation of human EAC. The discovery of sPLA2 IIa inhibition's ability to attenuate growth factor receptor signaling underscores the exciting potential of sPLA2 IIa inhibitors as therapeutics in the treatment of EAC.

In Barrett's epithelial cells, weakly acidic bile salt solutions cause oxidative DNA damage with response and repair mediated by p38.

The frequency of esophageal adenocarcinoma is rising despite widespread use of proton pump inhibitors (PPIs), which heal reflux esophagitis but do not prevent reflux of weakly acidic gastric juice and bile in Barrett's esophagus patients. We aimed to determine if weakly acidic (pH 5.5) bile salt medium (WABM) causes DNA damage in Barrett's cells. Because p53 is inactivated frequently in Barrett's esophagus and p38 can assume p53 functions, we explored p38's role in DNA damage response and repair. We exposed Barrett's cells with or without p53 knockdown to WABM, and evaluated DNA damage, its response and repair, and whether these effects are p38 dependent. We also measured phospho-p38 in biopsies of Barrett's metaplasia exposed to deoxycholic acid (DCA). WABM caused phospho-H2AX increases that were blocked by a reactive oxygen species (ROS) scavenger. WABM increased phospho-p38 and reduced bromodeoxyuridine incorporation (an index of S phase entry). Repair of WABM-induced DNA damage proceeded through p38-mediated base excision repair (BER) associated with reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease I (Ref-1/APE1). Cells treated with WABM supplemented with ursodeoxycholic acid (UDCA) exhibited enhanced p38-mediated responses to DNA damage. All of these effects were observed in p53-intact and p53-deficient Barrett's cells. In patients, esophageal DCA perfusion significantly increased phospho-p38 in Barrett's metaplasia. WABM exposure generates ROS, causing oxidative DNA damage in Barrett's cells, a mechanism possibly underlying the rising frequency of esophageal adenocarcinoma despite PPI usage. p38 plays a central role in oxidative DNA damage response and Ref-1/APE1-associated BER, suggesting potential chemopreventive roles for agents like UDCA that increase p38 activity in Barrett's esophagus.NEW & NOTEWORTHY We found that weakly acidic bile salt solutions, with compositions similar to the refluxed gastric juice of gastroesophageal reflux disease patients on proton pump inhibitors, cause oxidative DNA damage in Barrett's metaplasia that could contribute to the development of esophageal adenocarcinoma. We also have elucidated a critical role for p38 in Barrett's metaplasia in its response to and repair of oxidative DNA damage, suggesting a potential chemopreventive role for agents like ursodeoxycholic acid that increase p38 activity in Barrett's esophagus.

Constitutively Higher Level of GSTT2 in Esophageal Tissues From African Americans Protects Cells Against DNA Damage.

BACKGROUND & AIMS: African American and European American individuals have a similar prevalence of gastroesophageal reflux disease (GERD), yet esophageal adenocarcinoma (EAC) disproportionately affects European American individuals. We investigated whether the esophageal squamous mucosa of African American individuals has features that protect against GERD-induced damage, compared with European American individuals. METHODS: We performed transcriptional profile analysis of esophageal squamous mucosa tissues from 20 African American and 20 European American individuals (24 with no disease and 16 with Barrett's esophagus and/or EAC). We confirmed our findings in a cohort of 56 patients and analyzed DNA samples from patients to identify associated variants. Observations were validated using matched genomic sequence and expression data from lymphoblasts from the 1000 Genomes Project. A panel of esophageal samples from African American and European American subjects was used to confirm allele-related differences in protein levels. The esophageal squamous-derived cell line Het-1A and a rat esophagogastroduodenal anastomosis model for reflux-generated esophageal damage were used to investigate the effects of the DNA-damaging agent cumene-hydroperoxide (cum-OOH) and a chemopreventive cranberry proanthocyanidin (C-PAC) extract, respectively, on levels of protein and messenger RNA (mRNA). RESULTS: We found significantly higher levels of glutathione S-transferase theta 2 (GSTT2) mRNA in squamous mucosa from African American compared with European American individuals and associated these with variants within the GSTT2 locus in African American individuals. We confirmed that 2 previously identified genomic variants at the GSTT2 locus, a 37-kb deletion and a 17-bp promoter duplication, reduce expression of GSTT2 in tissues from European American individuals. The nonduplicated 17-bp promoter was more common in tissue samples from populations of African descendant. GSTT2 protected Het-1A esophageal squamous cells from cum-OOH-induced DNA damage. Addition of C-PAC increased GSTT2 expression in Het-1A cells incubated with cum-OOH and in rats with reflux-induced esophageal damage. C-PAC also reduced levels of DNA damage in reflux-exposed rat esophagi, as observed by reduced levels of phospho-H2A histone family member X. CONCLUSIONS: We found GSTT2 to protect esophageal squamous cells against DNA damage from genotoxic stress and that GSTT2 expression can be induced by C-PAC. Increased levels of GSTT2 in esophageal tissues of African American individuals might protect them from GERD-induced damage and contribute to the low incidence of EAC in this population.

Expression analysis of cyclooxygenase-2 in patients suffering from esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the aggressive malignancies and mechanisms underlying its pathogenesis remain unclear. Cyclooxygenase-2 (COX-2) enzyme system plays a crucial role in many gastrointestinal malignancies and is an important regulator of cell growth, proliferation, apoptosis, differentiation and transformation. More precise outcome of COX-2 in ESCC is less investigated. In this study we investigated the risk factors of ESCC and expression of COX-2 in Carcinoma in situ (CIS) and ESCC compared to normal esophageal mucosa. ESCC relationship to clinico-pathological parameters using immunohistochemistry was also part of this investigation. Current study was conducted in the Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan. A total of 69 diagnosed patients of ESCC, both Pakistanis and Afghans were enrolled. Various risk factors associated with ESCC were recorded. Mean age at the time of diagnosis was 55 years. Out of 69 patients of ESCC 46 (67%) were users of dipping tobacco (Naswar). Expression of COX-2 was determined in normal esophageal mucosa, CIS and invasive ESCC using Immunohistochemistry (IHC). Differences of mean were computed using ANOVA followed by applying Post Hoc test. Patients were categorized as positive with high expression or negative with low to nil expression. ANOVA showed large differences in expression of COX-2 in normal healthy mucosa compared with CIS and ESCC with the mean difference of -9.529 and -7.370 respectively, p-value being <.05 at 95% confidence interval (CI). No significant difference was noticed in the expression of COX-2 in CIS compared with ESCC with p-value >.05 at 95% CI. Our complete cohort (23-85 years) showed statistically significant difference in the expression of COX-2 gene in ESCC and CIS tissue samples compared with normal healthy mucosa. Results of this study indicate that over-expression of COX-2 is positively associated with ESCC.

Measurement of Inflammation in Eosinophilic Esophagitis Using an Eosinophil Peroxidase Assay.

OBJECTIVES: We describe a simple, quick method to measure an eosinophil granule protein, eosinophil peroxidase (EPO), as a marker of eosinophil activity, in eosinophilic esophagitis (EoE). METHODS: Esophageal mucosal brushings initially were collected from 36 patients with active EoE (n=13), resolved EoE (n=13), and controls (n=10) before endoscopic biopsy collection; the brushes were frozen at -80 ºC until assayed. EPO on the brush was measured in a colorimetric assay visually and by spectrophotometric absorbance measurements (at 492 nm), and was compared with peak eosinophil counts in esophageal biopsy specimens. The assay was calibrated with known EPO concentrations; as EPO increased in the assay, the color changed from light yellow to dark brown. RESULTS: Mucosal brush specimens from active EoE yielded orange to dark brown colors with absorbance measurements > 1.1 U; in contrast, control and resolved EoE brush specimens yielded a light to dark yellow color with absorbance measurements < 1.1. We then corroborated the results at the bedside (real time) in 16 additional patients. EPO on the brush was measured directly in < 1 h in the assay visually and by absorbance at 492 nm. Absorbance units strongly correlated with peak eosinophil counts both with the frozen brush (rs=0.79, P<0.0001) and with the bedside (rs=0.86, P<0.00017) approaches. CONCLUSIONS: The results support the use of this rapid method to detect and monitor EoE disease activity. Moreover, because eosinophils infiltrate and degranulate in the esophagus in EoE in a patchy manner, this method may be more accurate than current practice by testing for an eosinophil constituent from both intact and degranulated cells, and by sampling large portions of the esophageal lumen rather than small biopsy specimens that may not be representative of eosinophil involvement.