Expression and Molecular Regulation of the Cox2 Gene in Gastroenteropancreatic Neuroendocrine Tumors and Antiproliferation of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs).

BACKGROUND Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has had a significant increase over the past 4 decades. The pathophysiological role of the cyclooxygenase-2 (cox-2) gene and factors responsible for the expression in GEP-NETs is of clinical value. Current study determined the expression of cox-2 gene in human GEP-NET tissues and corresponding cell lines, investigated the molecular mechanisms underlying the regulation of cox-2 gene expression and assessed the effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on both anchorage-dependent and independent growth of GEP-NET cells. MATERIAL AND METHODS GEP-NET tissues and QGP-1, BON, and LCC-18 GEP-NET cell lines were used. The expression of cox-2 gene was analyzed by immunohistochemistry, western blot, RT-PCR, and enzyme immunoassay. Transient transfection and luciferase assays along with electrophoretic mobility shift assays were conducted to explore the regulation of cox-2 gene expression. The effect of COX-inhibitors on GEP-NET cell growth was determined by proliferation assays and colony growth assessment. RESULTS We found 87.8% of GEP-NET tissues stained positive for COX-2. QGP-1 and LCC-18 cells expressed cox-2 gene. PGE2 (prostaglandin E2) amounts quantified in the supernatants of NET cells matched to cox-2 expression level. The CRE-E-box element (-56 to -48 bp) and binding of USF1, USF2, and CREB transcription factors to this proximal promoter element were essential for cox-2 promoter activity in GEP-NET cells. COX-2-specific inhibitor NS-398 potently and dose-dependently inhibited PGE2 release from QGP-1 cells. Interestingly, both NS-398 and acetylic salicylic acid effectively suppressed proliferation of QGP-1 and BON cells in a dose-dependent manner. CONCLUSIONS The majority of GEP-NETs over express cox-2 gene. The binding of CREB and USF-1/-2 transcription factors to a proximal, overlapping CRE-Ebox element is the underlying mechanism for cox-2 gene expression. NSAIDs potently suppressed the proliferations and may offer a novel approach for chemoprevention and therapy of GEP-NETs.

Gastrin transactivates the chromogranin A gene through MEK-1/ERK- and PKC-dependent phosphorylation of Sp1 and CREB.

Our previous work revealed that gastrin regulates chromogranin A (CgA) transcription through enhanced binding of Sp1, CREB and Egr-1 to a proximal gastrin-responsive promoter element (Gas-RE). Here, we provide a detailed characterization of the signalling pathways transmitting the effect of gastrin on the CgA promoter. Gastrin treatment of gastric AGS-B cells potently stimulated MEK-1 as well as MAP kinases ERK-1/-2, JNK and p38 in a time-dependent manner. Interruption of ERK-1/-2/MEK-1 pathways abolished the transactivating effect of gastrin, whereas blockade of JNK or p38 activity was without effect. Functional promoter analysis revealed that the minimal element CgA-85/-64 was sufficient and necessary to confer MEK-1/ERK responsiveness. Analysis of proximal signalling pathways showed that activation of the MEK-1/ERK-1/2 module by gastrin does not require Ras, PI3-kinase or intracellular calcium signals, but depends on activation of kinases of the PKC family. This report demonstrates that a pathway comprising PKCs>Raf-1>MEK-1>ERK-1/-2 mediates the effect of gastrin on the CgA promoter, and strongly suggests that enhanced phosphorylation of Sp1 and CREB is crucial for CgA transactivation through the G protein-coupled CCK-B/gastrin receptor.

An upstream CRE-E-box element is essential for gastrin-dependent activation of the cyclooxygenase-2 gene in human colon cancer cells.

Cyclooxygenase-2, the inducible enzyme of arachidonic acid metabolism and prostaglandin synthesis, is over expressed in colorectal cancer. Inhibition of COX-1/-2 by non-steroidal anti-inflammatory drugs is associated with a decreased risk for these malignancies, whereas high serum gastrin levels elevate this risk. As gastrin exhibits trophical effects on colonic epithelium we sought to explore whether it is capable to induce COX-2 expression in a human colon cancer cell line. The aim of this study is the description of the gastrin evoked effects on the transcriptional activity of the COX-2 gene in colorectal cancer cells and the identification of regulatory promoter elements. Reporter gene assays were performed with the gastrin-stimulated human colorectal cancer cell-line Colo-320, which was stable transfected with the human cholecystokinin-B/gastrin receptor cDNA and COX-2-promoter-luciferase constructs containing different segments of the 5'-region of the COX-2 gene or with mutated promoter constructs. Transcription factors were characterized with electrophoretic mobility shift assays. Gastrin-dependent induction of COX-2 mRNA was shown using "real-time" PCR. Resulting elevated Prostaglandin E2-levels were measured using ELISA. Gastrin stimulated the PGE2-generation and COX-2-mRNA expression in human Colo-320-B cells potently, obviously by transactivating the COX-2-promoter using a region between - 68 bp and + 70 bp. Further examinations identified a CRE-E-box element between - 56 bp and - 48 bp mediating the gastrin-effects on the COX-2 gene. Transcription factors binding to this promoter element were USF-1 und -2. These results show the necessity to perform succeeding studies, which could describe possible mechanisms in which gastrin and COX-2 contribute to the induction of colorectal carcinomas.

Helicobacter pylori stimulates host vascular endothelial growth factor-A (vegf-A) gene expression via MEK/ERK-dependent activation of Sp1 and Sp3.

VEGF-A is a key regulator of inflammatory and tumor-associated angiogenesis. H. pylori plays a critical role in the pathogenesis of benign and malignant gastric diseases. It has been suggested that H. pylori infection is associated with activation of host angiogenesis, however, underlying mechanisms as well as angiogenic growth factors activated by the bacterium have not yet been identified. Therefore, we investigated the influence of the bacterium on VEGF-A as a candidate host target gene in vivo and in vitro. We show that H. pylori potently up-regulates production and release of VEGF-A protein as well as vegf-A mRNA levels, and we provide strong evidence that enhanced recruitment of Sp1 and Sp3 transcription factors to two proximal GC-rich vegf-A promoter elements mediates H. pylori-triggered vegf-A gene expression. In addition, H. pylori infection increased the transactivating capacity of both Sp1 and Sp3, which suggests additional mechanism(s) of vegf-A gene regulation by the bacterium. Signaling studies identified the MEK>ERK1/-2 kinase cascade as principal host signaling pathway mediating H. pylori-stimulated vegf-A transcription. By identifying H. pylori as potent activator of vegf-A gene expression and characterization of underlying molecular mechanisms, our results provide novel insights into pathways linking the bacterium to host angiogenesis and may help to develop strategies to influence vegf-A gene expression in the setting of H. pylori infection.

Small-molecule insulin mimetic reduces hyperglycemia and obesity in a nongenetic mouse model of type 2 diabetes.

Adiposity positively correlates with insulin resistance and is a major risk factor of type 2 diabetes. Administration of exogenous insulin, which acts as an anabolic factor, facilitates adipogenesis. Recently nonpeptidal insulin receptor (IR) activators have been discovered. Here we evaluate the effects of the orally bioavailable small-molecule IR activator (Compound-2) on metabolic abnormalities associated with type 2 diabetes using a nongenetic mouse model in comparison with the effects of a novel non-thiazolidinedione (nTZD) peroxisome proliferator-activated receptor-gamma agonist. Both Compound-2 and nTZD alleviated fasting and postprandial hyperglycemia; accelerated glucose clearance rate; and normalized plasma levels of nonesterified fatty acids, triglycerides, and leptin. Unlike nTZD, which increased body weight gain, and total fat mass, which is a common feature for PPARgamma agonists, Compound-2 prevented body weight gain and hypertrophy of brown, and white adipose tissue depots and the development of hepatic steatosis in the mouse model of type 2 diabetes. The effect of the two compounds on proximal steps in insulin signal transduction pathway was analyzed in tissues. Compound-2 enhanced insulin-stimulated phosphorylation of IR tyrosine and/or Akt in the liver, skeletal muscle, and white adipose tissue, whereas nTZD potentiated the phosphorylation of IR and Akt in the adipose tissue only. In conclusion, small-molecule IR activators have unique features as insulin sensitizers and hold potential utility in the treatment of type 2 diabetes and obesity.

Vascular endothelial growth factor-D and its receptor VEGFR-3: two novel independent prognostic markers in gastric adenocarcinoma.

PURPOSE: Vascular endothelial growth factor (VEGF)-D and its homolog VEGF-C influence lymphangiogenesis through activation of VEGF receptor 3 (VEGFR-3), and have been implicated in lymphatic tumor spread. Nodal dissemination of gastric adenocarcinomas critically determines clinical outcome and therapeutic options of affected patients. Therefore, we analyzed expression and prognostic significance of VEGF-D along with VEGF-C, and VEGFR-3 in gastric adenocarcinomas. MATERIALS AND METHODS: VEGF-C, VEGF-D, and VEGFR-3 were analyzed in 91 R(0)-resected primary gastric adenocarcinomas, corresponding noncancerous gastric mucosa, and lymph node metastases employing immunohistochemistry and/or in situ hybridization. Blood and lymph vessel densities were assessed after staining with CD31 and LYVE-1-specific antibodies. RESULTS: VEGF-D and VEGF-C were detected in 67.0% and 50.5% of gastric cancers, respectively. Healthy gastric mucosa was negative for VEGF-C and in 12.5% positive for VEGF-D. Presence of VEGF-D (P = .005) or VEGF-C (P = .006) was correlated with lymphatic metastases and decreased survival (VEGF-D, P < .05; VEGF-C, P < .05). VEGFR-3 was correlated with reduced carcinoma-specific survival (P < .05), and Cox multivariate regression analysis qualified VEGF-D and VEGFR-3, but not VEGF-C, as independent prognostic parameters. In lymph node-positive gastric cancers, presence of VEGF-D/VEGFR-3 was associated with poor survival, whereas absence of VEGF-D/VEGFR-3 defined a subgroup of patients with clearly favorable prognosis. CONCLUSION: VEGF-D and VEGFR-3 are novel independent prognostic marker molecules aiding to identify patients with poor prognosis after curative resection of gastric adenocarcinomas. Combined analysis of the VEGF-C/VEGF-D/VEGFR-3 system can be useful to identify patients with unfavorable clinical outcome and thereby may help to refine therapeutic decisions in gastric cancer.

Cyclosporin A-mediated killing of Leishmania major by macrophages is independent of reactive nitrogen and endogenous TNF-alpha and is not inhibited by IL-10 and 13.

Murine macrophages were treated with various doses of cyclosporin A (CsA) to enhance the killing of Leishmania major parasites. CsA reduced the rate of infected cells from 75% in non-treated controls to less than 15% with 1 micro g CsA/ml in a dose-dependent manner. The leishmanicidal effect was also observed when CsA was added 48 h after the infection of macrophages. In contrast, FK506, another structural non-related immunosuppressive drug with antiparasitic activities, showed no effect on the ability of macrophages to kill intracellular Leishmania parasites. Since nitric oxide has been identified as a key molecule for the leishmanicidal function of macrophages, we analyzed the role of this molecule. There was no influence on the leishmanicidal effect of CsA when L- N-(1-iminoethyl)lysine, a potent and selective inhibitor of mouse inducible nitric oxide synthase, was added. Furthermore, the presence of the macrophage-inhibiting cytokines interleukin (IL)-10 and IL-13 simultaneously or prior to CsA did not inhibit leishmania killing, while both cytokines completely prevented parasite killing by macrophages activated with gamma interferon and tumor necrosis factor (TNF). CsA was fully active on macrophages from TNF-receptor p55 knockout mice arguing against autocrine activation by TNF. We therefore conclude that the antileishmanial effect of CsA is independent of effector mechanisms employed by macrophage-activating cytokines.

Reliable detection of macrolide-resistant Helicobacter pylori via fluorescence in situ hybridization in formalin-fixed tissue.

Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem. Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test. A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy. In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy. In such biopsies macrolide-resistant H. pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH). Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue. Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H. pylori gastritis. To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available. Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts. In all 104 patients, H. pylori was detected by FISH and could thus be investigated for macrolide resistance. Overall, macrolide-resistant bacteria were found in 71 patients (68.3%). In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test). Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H. pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy.

Helicobacter pylori stimulates host cyclooxygenase-2 gene transcription: critical importance of MEK/ERK-dependent activation of USF1/-2 and CREB transcription factors.

Cyclooxygenase-2 (COX-2) represents the inducible key enzyme of arachidonic acid metabolism and contributes to the pathogenesis of gastroduodenal ulcers and gastric cancer. Helicobacter pylori infection is associated with elevated gastric COX-2 levels, but the mechanisms underlying H. pylori-dependent cox-2 gene expression are unclear. H. pylori stimulated cox-2 mRNA and protein abundance in gastric epithelial cells in vitro and in vivo, and functional analysis of the cox-2 gene promoter mapped its H. pylori-responsive region to a proximal CRE/Ebox element at -56 to -48. Moreover, USF1/-2 and CREB transcription factors binding to this site were identified to transmit H. pylori-dependent cox-2 transcription. Activation of MEK/ERK1/-2 signalling by bacterial virulence factors located outside the H. pylori cag pathogenicity island (cagPAI) was found to mediate bacterial effects on the cox-2 promoter. Our study provides a detailed description of the molecular pathways underlying H. pylori-dependent cox-2 gene expression in gastric epithelial cells, and may thus contribute to a better understanding of mechanisms underlying H. pylori pathogenicity.