Mohamed M.M. Abdel-Latif

NF-kappaB Activation in Esophageal Adenocarcinoma: Relationship to Barrett's Metaplasia, Survival, and Response to Neoadjuvant Chemoradiotherapy

Objective:To examine the expression of the transcription factor nuclear factor kappa B (NF-&kgr;B) in Barretts epithelium and adenocarcinoma and the impact of NF-&kgr;B expression on tumor stage and response to neoadjuvant chemotherapy and radiation therapy. Summary Background Data:Progression of Barretts esophagus to adenocarcinoma is associated with a wide range of cellular and molecular abnormalities. Nuclear factor-kappa B (NF-&kgr;B) regulates several genes involved in inflammatory, immune and apoptotic responses, but its role in esophageal inflammation and tumorigenesis has not been reported. Methods:Mobility shift assay was used to measure NF-&kgr;B activity in nuclear extracts of fresh-frozen biopsies from tumor and uninvolved tissues (n = 30) and esophageal cell lines OE33, SKGT-4, and OE21. RelA expression was assessed by immunohistochemical staining (n = 97). The NF-&kgr;B/RelA and I&kgr;B protein expressions were also examined by Western blotting. Results:NF-&kgr;B was not expressed in normal esophageal squamous epithelium, in contrast to increased expression in 40% of patients with Barretts epithelium. Sixty-one percent of resected tumors (n = 97) displayed NF-&kgr;B immunoreactivity, and 87.5% of the NF-&kgr;B- positive tumors were Stage IIb and III compared with only 12.5% of patients with Stage I and IIa disease (P < 0.05). The expression of NF-&kgr;B inversely correlated with major or complete pathologic responses to neoadjuvant chemotherapy and radiation therapy, with 15/20 (75%) responders in the NF-&kgr;B-negative group compared with 7/38 (18%) in the NF-&kgr;B-positive group (P < 0.00001). Moreover, incubation of esophageal cell lines OE33, SKGT-4, and OE21 with deoxycholic acid or low pH induced NF-&kgr;B expression. Conclusions:Bile acids and low pH induce NF-&kgr;B expression in esophageal cell lines. NF-&kgr;B activation is common in esophageal adenocarcinoma. In patients with Barretts epithelium and an associated esophageal adenocarcinoma, there is a progressive expression of NF-&kgr;B through Barretts tumorigenesis. The absence of NF-&kgr;B expression in esophageal adenocarcinoma correlates with response to neoadjuvant chemoradiotherapy and may be of value in predicting response to neoadjuvant therapy.

Proinflammatory Cytokine and Nuclear Factor Kappa-B Expression along the Inflammation–Metaplasia–Dysplasia–Adenocarcinoma Sequence in the Esophagus

BACKGROUND:The incidence of esophageal adenocarcinoma has increased significantly in the western world over the last 20 yr. Most cases arise in a background of chronic gastroesophageal reflux, and specialized intestinal metaplasia in Barretts esophagus is frequently an antecedent phenotype or evident in association with adenocarcinoma. The molecular events that characterize the pathway from inflammation to metaplasia to dysplasia and adenocarcinoma are poorly understood.AIMS:To examine the expression of the proinflammatory cytokines IL-8 and IL-1β along the esophagitis, metaplasia, dysplasia, and adenocarcinoma pathway, and to correlate this with histological changes and expression of the transcription factor NF-κB.PATIENTS AND  METHODS:Fresh biopsy specimens were collected from patients with reflux esophagitis (n= 15), Barretts esophagus (n = 35), Barretts adjacent to adenocarcinoma (n = 8), and esophageal adenocarcinoma (n = 35). IL-8 and IL-1β expression were measured using enzyme-linked immunosorbent assay. NF-κB expression was measured by electrophoretic mobility shift assay.RESULTS:Elevated expression of NF-κB was found in 2 (13%) out of 15 patients with reflux esophagitis, 21 (60%) out of 35 patients with Barretts esophagus, and 28 (80%) out of 35 patients with esophageal adenocarcinoma. All 5 patients with Barretts esophagus and high-grade dysplasia showed elevated expression of NF-κB. IL-8 and IL-1β were significantly increased in esophagitis, Barretts, and adenocarcinoma compared with squamous epithelium, and in adenocarcinoma compared with all other groups. There was a stepwise increase in the expression of IL-8, IL-1β, and NF-κB from normal through Barretts epithelium to adenocarcinoma in eight cases of esophageal adenocarcinoma. The levels of both IL-8 and IL-1β in adenocarcinoma patients correlated with stage of disease. Patients with adenocarcinoma who were NF-κB positive had significantly higher levels of both IL-8 (p= 0.04) and IL-1β (p= 0.03) compared to adenocarcinoma patients who were NF-κB negative.CONCLUSIONS:The proinflammatory cytokines IL-8 and IL-1β are elevated in esophagitis and Barretts epithelium, and markedly elevated in adenocarcinoma. NF-κB activation is infrequent in esophagitis, but is increased in Barretts epithelium and adenocarcinoma. The association of NF-κB activation with cytokine upregulation was only evident in patients with adenocarcinoma. These patterns may play an important role in Barretts inflammation and tumourigenesis, and inhibition of the NF-κB/proinflammatory cytokine pathway may be an important target for future chemoprevention strategies.

Inflammation and esophageal carcinogenesis.

The incidence of esophageal adenocarcinoma is increasing largely in Western populations, and patients diagnosed with this cancer continue to have a poor prognosis. The major risk factors are gastroesophageal reflux disease and Barretts esophagus, both of which are associated with inflammation of the esophageal squamous epithelium, a condition called reflux esophagitis. The cellular mechanisms contributing to cancer development in the esophagus are poorly understood. The chronic inflammation that is present in Barretts esophagus creates an environment suitable for DNA damage and altered expression of genes involved in cellular proliferation and inhibition of apoptosis. Key players in the inflammatory cascade include generation of free radicals, activation of kinases pathways and transcription factors, and production of cytokines and inflammatory enzymes. The current review highlights the link between reflux-induced inflammation and esophageal carcinogenesis. Understanding the molecular pathways involved in inflammation-associated esophageal tumorigenesis could enable the development of targeted therapies and offer a better therapeutic treatment in esophageal cancer.

Caffeic acid phenethyl ester modulates Helicobacter pylori‐induced nuclear factor‐kappa B and activator protein‐1 expression in gastric epithelial cells

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives (honeybee resin), has anti‐inflammatory, anti‐carcinogenic and anti‐bacterial properties. This study was designed to investigate the anti‐inflammatory effects of CAPE on Helicobacter pylori‐induced NF‐κB and AP‐1 in the gastric epithelial cell line AGS. Electrophoretic mobility shift assay was used to measure NF‐κB‐ and AP‐1‐DNA binding activity. Western blotting was used to detect IκB‐α and COX‐2 expression in AGS cells cocultured with H. pylori. The antiproliferative effect of CAPE was measured by MTT assay. Our results showed that caffeic phenethyl ester inhibits H. pylori‐induced NF‐κB and AP‐1 DNA‐binding activity in a dose (0.1–25 μg ml−1∼0.35–88 μM) and time‐ (15–240 min) dependent manner in AGS cells. Maximum inhibition by CAPE was observed at concentrations of 25 μg ml−1 (∼88 μM) CAPE prevented H. pylori‐ and cytokine‐induced degradation of IκB‐α protein. Pretreatment of AGS cells with CAPE also blocked cytokine‐ and mitogen‐induced NF‐κB and AP‐1 expression. Furthermore, CAPE suppressed H. pylori‐induced cell proliferation and production of the cytokines TNF‐α and IL‐8. In addition, CAPE blocked H. pylori‐induced COX‐2 expression. The inhibition of such transcription by CAPE could result in suppression of many genes during H. pylori‐induced inflammation, and also provide new insights into the anti‐cancer and anti‐inflammatory properties of CAPE.

Ursodeoxycholic acid inhibits interleukin beta 1 and deoxycholic acid-induced activation of NF-κB and AP-1 in human colon cancer cells

Deoxycholic acid (DCA) has been implicated in colorectal carcinogenesis in humans with effects on proliferation and apoptosis, mediated at least in part by activation of transcription factors nuclear factor kappa B (NF‐κB), activator protein 1 (AP‐1) and protein kinase C (PKC) enzymes. Ursodeoxycholic acid (UDCA) is reported to reduce the frequency of colonic carcinogenesis in ulcerative colitis patients. Hence, we postulated that it might differ from DCA in its regulation of these transcription factors. The aim of the study was to determine effects of DCA and UDCA on NF‐κB and AP‐1 activation and explore its relationship to PKC. Human colonic tumour cell lines HCT116 were treated with DCA, UDCA, alone or pretreated with UDCA followed by DCA or IL‐1β. In other experiments, cells were pretreated with PKC inhibitors and then stimulated with DCA and IL‐1β or PMA. Gel shift assays were performed on nuclear extracts of the cells for NF‐κB and AP‐1 analysis. Western blot analyses and immunofluorescence were performed for Rel A (p65) and IκB‐α levels on the treated cells. DCA increased NF‐κB and AP‐1 DNA binding. UDCA did not increase DNA binding of NF‐κB and AP‐1 and UDCA pretreatment inhibited DCA‐induced NF‐κB and AP‐1 DNA binding. PKC inhibitors blocked DCA‐induced NF‐κB and AP‐1 activation. These results were validated by Western blot analysis for RelA and IκB‐α. In conclusion, UDCA did not induce NF‐κB and AP‐1 DNA binding but also blocked DCA‐induced NF‐κB and AP‐1 activation. These findings suggest a possible mechanistic role for UDCA in blocking pathways thought to be involved in colon carcinogenesis.

Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells

BackgroundThe progression from Barretts metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells.MethodsFollowing exposure of SKGT-4 cells to DCA, protein levels of COX-2, MAPK and PARP were examined by immunoblotting. AP-1 activity was assessed by mobility shift assay. DCA-induced toxicity was assessed by DNA fragmentation and MTT assay.ResultsDCA induced persistent activation of the AP-1 transcription factor with Fra-1 and JunB identified as the predominant components of the DCA-induced AP-1 complex. DCA activated Fra-1 via the Erk1/2- and p38 MAPK while Erk1/2 is upstream of JunB. Moreover, DCA stimulation mediated inhibition of proliferation with concomitant low levels of caspase-3-dependent PARP cleavage and DNA fragmentation. Induction of the anti-apoptotic protein COX-2 by DCA, via MAPK/AP-1 pathway appeared to balance the DCA mediated activation of pro-apoptotic markers such as PARP cleavage and DNA fragmentation. Both of these markers were increased upon COX-2 suppression by aspirin pretreatment prior to DCA exposure.ConclusionDCA regulates both apoptosis and COX-2-regulated cell survival in esophageal cells suggesting that the balance between these two opposing signals may determine the transformation potential of DCA as a component of the refluxate.

INTERCOSTAL NERVE TRANSFER IN INFANTS WITH OBSTETRIC BRACHIAL PLEXUS PALSY

The use of intercostal nerve (ICN) transfer to repair brachial plexus lesions associated with root avulsions is a well known procedure in adults. However, there is a paucity of reports on the use of ICN in infants with obstetrical brachial plexus palsy (OBPP). This study included 46 infants with obstetric brachial plexus palsy who underwent 62 neurotization procedures. Clinically, 2 cases had upper trunk injury, 19 had upper‐middle trunk injury, 3 had lower trunk injury, and 22 had total palsy. The average age at surgery was 14 months. Twelve patients underwent surgery younger than 6 months of age, 11 patients at 6 to <9 months, 9 patients at 9–12 months, and 14 patients at >12 months. The average follow‐up period was 49 months. ICN transfer resulted in 76% satisfactory (good and excellent) outcome, and was best for restoration of elbow flexion (93.5%). Functional results were best when the operation was done before the age of 9 months; however, the difference between age groups was statistically insignificant. Functional results were also independent of the extent of the original injury. Nine children had preoperative and postoperative CT chest scans. All the nine children developed basal pulmonary atelectasis postoperatively. Pulmonary atelectasis was mostly ipsilateral and was not correlated to the patient age (months), or the duration of anesthesia (in minutes). We conclude that, intercostals nerve transfer is an effective procedure for restoration of function in infants with OBPP and root avulsions. The procedure is associated with variable degree of ipsilateral pulmonary atelectasis.

Vitamin C enhances chemosensitization of esophageal cancer cells in vitro.

Abstract 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-κB 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-κB and AP-1 activation. Pretreatment with vitamin C inhibited 5-Fu or cisplatin induced NF-κB nuclear translocation and DNA binding activity, but vitamin C had no effect on IκB-α 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-κB 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.

Potential Role of NF-κB in Esophageal Adenocarcinoma: As an Emerging Molecular Target

Esophageal adenocarcinoma is increasing in incidence and arises in a background of reflux induced inflammation, metaplasia, and dysplasia. The proinflammatory transcription factor nuclear factor-kappa B (NF-kappaB) has a central role in inflammation and tumorigenesis. Because a role for NF-kappaB has been implicated in the pathogenesis of esophageal cancer, this transcription factor has been the focus of the current research of this devastating disease. NF-kappaB blocks apoptosis, mediates tumor cell proliferation, and induces resistance to chemotherapeutic drugs. Research efforts to improve the effect of chemotherapy have led to an improvement in patient survival but there is still a need for improvement, and NF-kappaB is a potential target for cancer drug development. In this review, we have attempted to highlight the possible role of NF-kappaB in esophageal adenocarcinoma and discuss the anticancer strategy with NF-kappaB as a promising molecular target in esophageal cancer therapy.

Helicobacter pylori extract induces nuclear factor-kappa B, activator protein-1, and cyclooxygenase-2 in esophageal epithelial cells.

Helicobacter pylori infection is recognized as the major cause of gastritis and gastric cancer; however, its role in the development of gastroesophageal reflux disease and Barrett’s adenocarcinoma is unclear. The expression of NF-кB, AP-1, and COX-2 may be important in inflammation and tumorigenesis in the esophagus. The aim of this study was to examine the effect of live H pylori or H pylori extract (HPE) on these factors in the esophageal epithelial cell lines SKGT-4 and OE33. NF-кB and AP-1 activity were assessed by gel shift assay and COX-2 by Western blotting. Coculture of SKGT-4 and OE33 with live H pylori and HPE induced NF-кB and AP-1 DNA-binding activity, and also decreased IкB-α levels. Treatment with the specific MEK1/2 MAPK inhibitor PD98059, but not the p38 MAPK inhibitor SB203580, inhibited NF-кB and AP-1 activity. The antioxidant vitamin C inhibited H pylori-induced NF-кB activation, but increased AP-1 expression. Moreover, HPE induced COX-2 expression and IL-8 production, and PD98059 inhibited COX-2 expression, ERK1/2 phosphorylation, and IL-8 production. These data demonstrate that both live H pylori and HPE induce NF-к B and AP-1 expression in esophageal epithelial cells. The induction of such transcription factors may play a role in the specific immune response within Barrett’s mucosa and may indirectly cause inflammation of the gastric cardia and the distal esophagus.