Pilot translational study of dietary vitamin C supplementation in Barrett's esophagus.

The transcription factor Nuclear factor kappa B (NF-kappaB) is central to the regulation of genes encoding for mediators of inflammation and carcinogenesis. In the esophagus, NF-kappaB is progressively activated from inflammation to Barrett's metaplasia and adenocarcinoma. Vitamin C, an antioxidant, can inhibit NF-kappaB in in vitro models, and the aim of this study was to prospectively assess the effect of supplemental vitamin C on NF-kappaB and associated cytokines in patients with Barrett's esophagus. Twenty-five patients with long-segment Barrett's and specialized intestinal metaplasia received dietary vitamin C (1000 mg/day) orally for four weeks, and had pre- and post-vitamin C endoscopic biopsies. NF-kappaB activity (activated p50 and p65 subunits) of nuclear extracts was assessed using the Active Motif NF-kappaB assay, and cytokines and growth factors were measured using the Evidence Investigator biochip array. NF-kappaB and related pro-inflammatory cytokines and growth factors (IL-8, VEGF, IL-10) were activated in all Barrett's tissue pre-treatment. Down-regulation in activated NF-kappaB and cytokines was observed in 8/25 (35%) patients. Dietary vitamin C supplementation may down-regulate pro-inflammatory markers in a subset of Barrett's patients. Further studies with larger numbers of endpoints will be needed to further evaluate this effect.

Vitamin C enhances chemosensitization of esophageal cancer cells in vitro.

Chemotherapy is increasingly utilised in multimodal protocols to try and improve outcomes. Cisplatin and 5-fluorouracil (5-Fu) are the mainstay of chemotherapeutic regimens, and an understanding of sensitivity and resistance of esophageal cancer to these agents is of considerable clinical importance. Antioxidants may modulate the response to chemotherapy, and in this study we examined the effect of vitamin C on 5-Fu and cisplatin cytotoxicity and related pathways in the esophageal cancer cell lines OE33 and SKGT-4. The antiproliferative effect of antitumor agents was measured by the MTT assay, and the transcription factors NF-kappaB and AP-1 pathways were assessed by electrophoretic mobility gel shift assay. 5-Fu and cisplatin demonstrated marked morphological changes and decreased cell proliferation. A combination of vitamin C with 5-Fu or cisplatin exerted a significantly enhanced cytotoxic effect compared to both drugs individually. Treatment of esophageal cancer cells with 5-Fu and cisplatin induced NF-kappaB and AP-1 activation. Pretreatment with vitamin C inhibited 5-Fu or cisplatin induced NF-kappaB nuclear translocation and DNA binding activity, but vitamin C had no effect on IkappaB-alpha protein levels. Vitamin C also inhibited 5-Fu- and cisplatin-induced AP-1 activation. Our data demonstrate that vitamin C enhances the antitumor activity of 5-Fu and cisplatin, in part by inhibiting translocation of NF-kappaB and AP-1, and sensitizes cancer cells to drug-induced cell death. The data suggest that vitamin C supplementation may improve the efficacy of chemotherapy for esophageal cancer.

Activated nuclear factor-kappa B and cytokine profiles in the esophagus parallel tumor regression following neoadjuvant chemoradiotherapy.

Esophageal adenocarcinoma is increasing in incidence; it relates to chronic gastroesophageal reflux, it is difficult to cure, and treatment modalities increasingly use chemotherapy and radiation therapy prior to resectional surgery. Nuclear factor-kappa B (NF-kappaB) is a pleiotropic transcription factor that regulates several genes for cytokines and enzymes involved in inflammation and immunity, and we have previously described sequential expression of NF-kappaB from the normal esophagus through Barrett's metaplasia to adenocarcinoma. The aim of this exploratory study was to assess the NF-kappaB status and cytokine profiles pre- and post-chemoradiotherapy for esophageal adenocarcinoma. Fresh biopsy specimens obtained from 20 patients with esophageal adenocarcinoma and normal adjacent squamous epithelium were obtained pre-, during and post-chemoradiotherapy, and NF-kappaB expression was analyzed by electrophoretic mobility shift assay. The cytokine protein content of interleukin-1 beta (IL-1beta) and interleukin-8 (IL-8) of tissue homogenates was measured using the ELISA technique. NF-kappaB was constitutively activated in tumor tissues from esophageal adenocarcinoma but was not detected in adjacent normal esophageal mucosa. Elevated levels of IL-1beta and IL-8 were significantly (P < 0.05) higher in tumor tissues compared to control tissues. Patients with a major or complete pathological response (responders) were associated with absence of activated NF-kappaB from nuclear extracts after treatment. Moreover, IL-1beta and IL-8 levels were significantly (P < 0.05) down-regulated in tumor tissues from patients who demonstrated a complete pathological response. No differences in NF-kappaB, IL-1beta and IL-8 levels were detected pre- and post-treatment in patients who did not have a major or complete pathological response (non-responders). The study suggests that monitoring of molecular and cytokine patterns in patients undergoing this neoadjuvant regimen may help subselect the cohort that derives most benefit from the multimodal approach.

Helicobacter pylori activates the early growth response 1 protein in gastric epithelial cells.

The early growth response 1 (Egr-1) transcription factor is rapidly induced by various stimuli and is implicated in the regulation of cell growth, differentiation, and gene expression. The aim of this study was to examine the effect of Helicobacter pylori on the expression of Egr-1 and Egr-1-regulated genes in gastric epithelial AGS cells. Egr-1 expression was assayed by immunoblotting and electrophoretic mobility shift assays using H. pylori-stimulated AGS cells. Transient transfection experiments with promoter-reporter constructs of CD44, ICAM-1, and CD95L were used for expression studies. H. pylori induced the expression of Egr-1 in gastric epithelial cell lines in a dose-dependent manner, with the rapid kinetics that are typical of this class of transcription factors. Immunohistochemical studies of biopsies revealed that Egr-1 expression is more abundant in H. pylori-positive patients than in uninfected individuals. Reporter-promoter transfection studies indicated that Egr-1 binding is required for the H. pylori-induced transcriptional promoter activity of the CD44, ICAM-1, and CD95L (APO-1/Fas) constructs. The blocking of egr-1 with an antisense sequence prevented H. pylori-induced Egr-1 and CD44 protein expression. The MEK1/2 signaling cascade participates in H. pylori-mediated Egr-1 expression, but the p38 pathway does not. The data indicate that H. pylori induces Egr-1 expression in AGS cells in vitro and that the Egr-1 protein is readily detectable in biopsies from H. pylori-positive subjects. These observations suggest that H. pylori-associated Egr-1 expression may play a role, in part, in H. pylori-induced pathology.