Richard T. Ethridge

Selective inhibition of NF-κB attenuates the severity of cerulein-induced acute pancreatitis☆

BACKGROUND Acute pancreatitis (AP) is associated with increased cytokine production, which can ultimately produce deleterious local and systemic effects. The transcription factor NF-kappaB is activated by degradation of its inhibitory factor, IkappaB, and can stimulate various cytokines. The purpose of this study was to determine whether the inhibition of NF-kappaB binding activity with a novel peptide that binds to the NF-kappaB essential modifier binding domain (NBD) could attenuate the severity of AP. STUDY DESIGN AP was induced in Swiss Webster mice by hourly injections of the cholecystokinin analogue cerulein (50 microg/kg). Mice were injected with either the wild-type or control (mutated) NBD peptide at the time of the first cerulein injection; they were then sacrificed over a time course, and pancreata and lungs were harvested for histologic analysis and scoring. Myeloperoxidase activity was measured to assess neutrophil sequestration as an indicator of inflammation. NF-kappaB binding activity and steady-state levels of IkappaB and NF-kappaB subunits were determined by gel shift and Western blot, respectively. RESULTS AP resulted in increased NF-kappaB DNA-binding activity and decreased steady-state levels of IkappaB. Treatment with NBD peptide decreased inflammation in the pancreas, decreased hemorrhage in the lungs, and decreased myeloperoxidase activity in both pancreas and lung. CONCLUSIONS The marked induction of NF-kappaB binding activity suggests a role for this transcription factor in the early inflammatory changes associated with AP. Treatment with the NBD peptide attenuated the severity of injury associated with AP. Novel compounds that selectively target NF-kappaB may prove to be useful treatment of AP and AP-associated lung injury.

Role of cyclooxygenase 2 in protein kinase C βII-mediated colon carcinogenesis

Elevated expression of protein kinase C βII (PKCβII) is an early promotive event in colon carcinogenesis (Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica, Z., and Fields, A. P. (2001) Cancer Res. 61, 1375–1381). Expression of PKCβII in the colon of transgenic mice leads to hyperproliferation and increased susceptibility to colon carcinogenesis due, at least in part, to repression of transforming growth factor beta type II receptor (TGF-βRII) expression (Murray, N. R., Davidson, L. A., Chapkin, R. S., Gustafson, W. C., Schattenberg, D. G., and Fields, A. P. (1999)J. Cell Biol., 145, 699–711). Here we report that PKCβII induces the expression of cyclooxygenase type 2 (Cox-2) in rat intestinal epithelial (RIE) cells in vitro and in transgenic PKCβII mice in vivo. Cox-2 mRNA increases more than 10-fold with corresponding increases in Cox-2 protein and PGE2 production in RIE/PKCβII cells. PKCβII activates the Cox-2 promoter by 2- to 3-fold and stabilizes Cox-2 mRNA by at least 4-fold. The selective Cox-2 inhibitor Celecoxib restores expression of TGF-βRII both in vitro and in vivo and restores TGFβ-mediated transcription in RIE/PKCβII cells. Likewise, the ω-3 fatty acid eicosapentaenoic acid (EPA), which inhibits PKCβII activity and colon carcinogenesis, causes inhibition of Cox-2 protein expression, re-expression of TGF-βRII, and restoration of TGF-β1-mediated transcription in RIE/PKCβII cells. Our data demonstrate that PKCβII promotes colon cancer, at least in part, through induction of Cox-2, suppression of TGF-β signaling, and establishment of a TGF-β-resistant, hyperproliferative state in the colonic epithelium. Our data define a procarcinogenic PKCβII → Cox-2 → TGF-β signaling axis within the colonic epithelium, and provide a molecular mechanism by which dietary ω-3 fatty acids and nonsteroidal antiinflammatory agents such as Celecoxib suppress colon carcinogenesis.

Acute Pancreatitis Results in Induction of Heat Shock Proteins 70 and 27 and Heat Shock Factor-1

Heat shock proteins (HSPs) 70 and 27 are stress-responsive proteins that are important for cell survival after injury; the expression of these HSPs is regulated primarily by the transcription factor heat shock factor-1 (HSF-1). The purpose of this study was to determine the effect of acute pancreatitis on pancreatic HSPs (70, 27, 60, and 90) expression and to assess potential mechanisms for HSP induction using a murine model of cerulein-induced pancreatitis. We found an increase of both HSP70 and HSP27 levels with expression noted throughout the pancreas after induction of pancreatitis. HSP60 and HSP90 levels were constitutively expressed in the pancreas and did not significantly change with acute pancreatitis. HSF-1 DNA binding activity increased in accordance with increased HSP expression. We conclude that acute pancreatitis results in a marked increase in the expression of HSP70 and HSP27. Furthermore, the induction of HSP70 and HSP27 expression was associated with a concomitant increase in HSF-1 binding activity. The increased expression of both HSP70 and HSP27 noted with pancreatic inflammation may confer a protective effect for the remaining acini after acute pancreatitis.

Molecular Mechanisms Contributing to Necrotizing Enterocolitis

ObjectiveTo examine the cellular mechanisms involved in the pathogenesis of necrotizing enterocolitis (NEC). Summary Background DataNecrotizing enterocolitis is a major cause of death and complications in neonates; the cellular mechanisms responsible for NEC are unknown. The inducible form of cyclooxygenase (i.e., COX-2) is activated by the transcription factor nuclear factor (NF)-&kgr;B and is thought to play a role in inflammation. MethodsSegments of perforated and adjacent uninvolved small intestine from neonates with NEC were analyzed for COX-2 expression by immunohistochemistry. NEC was induced in weanling (18 days old) rats by occlusion of superior mesenteric vessels for 1 hour and intraluminal injection of platelet activating factor (50 &mgr;g/kg). Small intestine was harvested for protein extraction. Western immunoblot was performed to determine expression of COX-2. Gel shift assays were performed to assess NF-&kgr;B binding activity. ResultsImmunohistochemical analysis showed increased COX-2 protein expression in the perforated intestinal sections of all 36 neonates but not in adjacent normal intestine. Increased expression of COX-2 protein and NF-&kgr;B binding activity was noted in the small intestine of weanling rats at 0 and 3 hours after induction of NEC. ConclusionsIncreased COX-2 expression was identified in all neonatal intestinal segments resected for perforated NEC. In addition, a coordinate induction of COX-2 expression and NF-&kgr;B binding was noted in a rodent model of NEC. These findings suggest that the COX-2/NF-&kgr;B pathway may play a role in the pathogenesis of NEC. Therapeutic agents that target this pathway may prove useful in the treatment or possible prevention of NEC.

Gut peptide receptor expression in human pancreatic cancers.

OBJECTIVE To determine the prevalence of gastrointestinal (GI) peptide receptor expression in pancreatic cancers, and to further assess signaling mechanisms regulating neurotensin (NT)-mediated pancreatic cancer growth. SUMMARY BACKGROUND DATA Pancreatic cancer remains one of the leading causes of GI cancer death; novel strategies for the early detection and treatment of these cancers is required. Previously, the authors have shown that NT, an important GI hormone, stimulates the proliferation of an NT receptor (NTR)-positive pancreatic cancer. METHODS A total of 26 human pancreatic adenocarcinomas, obtained after resection, and 5 pancreatic cancer xenografts were analyzed for expression of NTR, vasoactive intestinal peptide receptor (VIPR), substance P receptor (SPR), and gastrin-releasing peptide receptor (GRPR). In addition, NTR expression, [Ca2+]i mobilization, and growth in response to NT was determined in L3.6, a metastatic pancreatic cancer cell line. RESULTS Neurotensin receptor was expressed in 88% of the surgical specimens examined and all five of the pancreatic cancer xenografts. In contrast, VIPR, SPR, and GRPR expression was detected in 31%, 27%, and 8% of pancreatic cancers examined, respectively. Expression of NTR, functionally coupled to the Ca2+ signaling pathway, was identified in L3.6 cells; treatment with NT (10 micromol/L) stimulated proliferation of these cells. CONCLUSIONS The authors demonstrated NTR expression in most of the pancreatic adenocarcinomas examined. In contrast, VIPR, SPR, and GRPR expression was detected in fewer of the pancreatic cancers. The expression of NTR and other peptide receptors suggests the potential role of endocrine manipulation in the treatment of these cancers. Further, the presence of GI receptors may provide for targeted chemotherapy or radiation therapy or in vivo scintigraphy for early detection.

Peroxisome proliferator-activated receptor γ ligand inhibits cell growth and invasion of human pancreatic cancer cells

SummaryBackground. Peroxisome proliferator-activated receptor γ (PPARγ) is expressed in certain human cancers; ligand-induced PPARγ activation can result in growth inhibition and differentiation in these cells. However, the precise mechanism for the antiproliferative effect of PPARγ ligands is not entirely known.Aim of Study. The purpose of this study was to examine the effect of PPARγ ligands on pancreatic cancer cell growth and invasiveness.Methods. The effect of two PPARγligands, 15 deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) and ciglitazone, on the growth of four human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Panc-1, and L3.6) was assessed. Expression of cell-cycle and apoptotic-related proteins was measured. Finally, the effect of 15d-PGJ2 on pancreatic cancer cell invasiveness and matrix metalloproteinase expression was determined.Results. Both 15d-PGJ2 and ciglitazone inhibited the growth of all four pancreatic cancer cell lines in a dose- and time-dependent fashion. Treatment of BxPC-3 cells with 15d-PGJ2 resulted in a time-dependent decrease in cyclin D1 expression associated with a concomitant induction of p21waf1 and p27kip1. In addition, 15d-PGJ2 treatment induced apoptosis through activation of caspase-8, -9, and -3. Moreover, pancreatic cancer cell invasiveness was significantly suppressed after treatment with a nontoxic dose of 15d-PGJ2, which was associated with a reduction of MMP-2 and MMP-9 protein levels and activity.Conclusion. These results demonstrate that PPARγ ligands have the dual advantage of inhibiting pancreatic cancer cell growth while reducing the invasiveness of the tumor cells, suggesting a potential role for these agents in the adjuvant treatment of pancreatic cancer.

The PPARγ Ligand, 15d-PGJ2, Attenuates the Severity of Cerulein-Induced Acute Pancreatitis

Introduction The prostaglandin D2 metabolite, 15d-PGJ2, a potent natural ligand for peroxisome proliferator–activated receptor &ggr; (PPAR&ggr;), exerts antiinflammatory effects by inhibiting the induction of inflammatory response genes and NF-&kgr;B-dependent transcription. Aim To determine whether 15d-PGJ2 decreases the severity of secretagogue-induced acute pancreatitis (AP) and to assess cellular mechanisms contributing to these effects. Methodology Swiss Webster mice were injected with either saline or cerulein (50 &mgr;g/kg) hourly for 8 hours and received either 15d-PGJ2 (2 mg/kg) or vehicle 1 hour before and 4 hours after induction of AP. Results Treatment with 15d-PGJ2 significantly attenuated AP, as determined by histologic assessment of edema, vacuolization, inflammation, and necrosis. This attenuation was associated with decreased cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression and decreased serum and pancreatic IL-6 levels. Treatment with 15d-PGJ2 markedly inhibited NF-&kgr;B DNA-binding activity, and, moreover, this decreased activity was associated with a concomitant inhibition of I&kgr;B protein degradation. Conclusion Our findings demonstrate that 15d-PGJ2 attenuates the severity of AP most likely through the inhibition of COX-2 expression, IL-6 production, and NF-&kgr;B activation. Ligands specific for PPAR&ggr; may represent novel and effective means of clinical therapy for AP.

Inhibition of heat-shock protein 70 induction in intestinal cells overexpressing cyclooxygenase 2

BACKGROUND & AIMS The cyclooxygenase (COX) enzymes catalyze the initial step of prostaglandin formation; the inducible form, COX-2, plays a role in inflammation. Heat-shock protein 70 (hsp70) is a stress-responsive gene important for cell survival; induction of hsp70 appears to be mediated, in part, by the prostaglandin pathway. We determined the effect of COX-2 overexpression on hsp70 induction in rat intestinal epithelial (RIE) cells. METHODS RIE cells transfected with COX-2 complementary DNA oriented in the sense (RIE-S) or antisense (RIE-AS) direction were subjected to a heat shock; RNA and protein were harvested and analyzed by Northern and Western blots, respectively. Gel shift assays were performed to assess DNA binding. RESULTS Both hsp70 messenger RNA and HSP70 protein levels were increased in the RIE-AS cells, whereas induction was markedly inhibited in the RIE-S cells after heat shock. Inhibition of heat-shock factor binding was noted in RIE-S cells, suggesting that heat-shock transcription factor regulation may explain the inhibition of hsp70. The COX-2 selective inhibitor, NS-398, reversed the effects of COX-2 overexpression. CONCLUSIONS The results support a functional role for the prostaglandin/COX pathway in the induction of hsp70. The findings underscore a potential regulatory mechanism involving an inverse relationship between COX-2 expression and hsp70 induction.

Mitochondrial DNA damage and altered membrane potential (ΔΨ) in pancreatic acinar cells induced by reactive oxygen species

Background: Reactive oxygen species (ROS) have been implicated in the induction of acute pancreatitis. Mitochondria possess a distinct genome (mtDNA) that is more susceptible to ROS-induced damage than nuclear DNA (nDNA). The purpose of our study was to determine the effect of ROS on mitochondrial function and membrane potential (ΔΨmt), to identify signal transduction mechanisms activated by ROS, and to quantify damage to mtDNA in an in vitro pancreatitis model. Methods: Pancreatic acinar cells, AR4-2J, were treated with saline solution (control) or hydrogen peroxide (H2O2), a representative ROS. Mitochondrial function was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; to determine ΔΨmt, rhodamine-123 uptake was measured. Intracellular calcium levels and c-Jun N-terminal kinase activity was determined; gel mobility shift assays were performed to assess induction of the transcription factor NF-κB. To quantitate DNA damage, a novel polymerase chain reaction–based procedure was performed. Results: Mitochondrial function and ΔΨmt were significantly decreased with oxidative damage. H2O2 treatment resulted in increased intracellular calcium levels, activation of c-Jun N-terminal kinase, and induction of NF-κB DNA binding. Treatment of AR4-2J cells with H2O2 resulted in selective mtDNA damage; nDNA was not affected. Conclusions: Our data demonstrate that pancreatic mtDNA is more susceptible to oxidative damage than nDNA; this damage is associated with decreases in mitochondrial function and ΔΨmt and activation of downstream signal transduction pathways. Mitochondrial damage mediated by ROS may play a central role in pancreatic cell injury associated with acute pancreatitis. (Surgery 1999:126:148-55.)

COX-2 inhibition results in alterations in nuclear factor (NF)-κB activation but not cytokine production in acute pancreatitis

Acute pancreatitis is characterized by local inflammation and cytokine production, and release is thought to contribute to this process. Nuclear factor (NF)-κB activation and cytokine production are linked and inhibition of NF-κB has been shown to decrease the severity of pancreatitis. We have shown that inhibition of COX-2 ameliorates pancreatitis; however, the mechanism by which this effect occurs is unclear. Swiss Webster mice were injected intraperitoneally with either saline (control) or caerulein (CAE; 50 mg/kg) hourly for 8 hours; mice receiving CAE were further subdivided to receive saline or the cyclooxygenase-2 (COX-2) selective inhibitor (SC-58125; 10 mg, intraperitoneally) at the time of the first injection of CAE. Pancreata were harvested, histologic sections were scored, and protein was extracted to determine cytokine (interleukin [IL]-6, IL-1β) levels and NF-κB subunits by ELISA and NF-κB activation by gel shift. In addition, serum was collected for measurement of cytokines. COX-2 inhibition resulted in decreased inflammation and a decrease in NF-κB activation. IL-6 and IL-1β levels after COX-2 inhibition, however, remained elevated to levels equivalent to those of mice with histologic inflammation after CAE alone. COX-2 inhibition decreases inflammation as well as late-phase NF-κB activation but does not diminish levels of inflammatory cytokines, thus suggesting a two-phase activator of NF-κB. The attenuation of inflammation, despite unaltered cytokine levels, suggests that cytokines may not be critical for the inflammatory phase of pancreatitis.