ceruletide and Pancreatic-Neoplasms

Gastrointestinal hormones (GI hormones) have received growing interest in endocrinology, gastroenterology and neuroendocrinology. Because of new methodological techniques, they can be measured in plasma and therefore be related to different pathophysiological conditions. In childhood, our present knowledge is as yet limited to the physiological rôle of gastrin at different ages and in some diseases (gastrinoma; Verner-Morrison syndrome) caused by humoral dysfunction. The present review relates the clinical important GI hormones to chemically classified families. The diagnostic value of determining endogenous hormone concentration in plasma and the validity of function tests carried out by administration of exogenous hormones are pointed out. Particular emphasis is given to the trophic action of GI hormones in the development and function of the gastrointestinal tract during childhood. More speculatively, GI hormones are involved in the complex function of the central nervous system, thus making food intake a trophotropic action in a broader sense.

Topics: Adenoma, Islet Cell; Bombesin; Ceruletide; Child; Cholecystokinin; Gastric Inhibitory Polypeptide; Gastrins; Gastrointestinal Hormones; Glucagon-Like Peptides; Humans; Motilin; Neurotensin; Pancreatic Neoplasms; Pancreatic Polypeptide; Secretin; Somatostatin; Substance P; Vasoactive Intestinal Peptide

Develop a novel murine models of malignant pancreatitis.. Although patients with chronic pancreatitis are at a greater risk of developing pancreatic cancer, there is no definitive mouse model that currently develops chronic pancreatitis-induced pancreatic cancer.. Characterization of eosinophilic inflammation-mediated malignant pancreatitis in novel murine model.. We developed a murine model of chronic eosinophilic inflammation associated with pancreatitis that also shows characteristic features of pancreatic malignancy. The mouse received cerulein and azoxymethane via intraperitoneal administration developed pathological malignant phenotype, as well as concomitant lung inflammation.. We discovered pathological alterations in the pancreas that were associated with chronic pancreatitis, including a buildup of eosinophilic inflammation. Eosinophil degranulation was reported nearby in the pancreas tissue sections that show acinar-to-ductal metaplasia and acinar cell atrophy, both of which are characteristic of pancreatic malignancies. Additionally, we also observed the formation of PanIN lesions after three initial doses of AOM and eight weeks of cerulein with the AOM treatment regimen. We discovered that persistent pancreatic eosinophilic inflammation linked with a pancreatic malignant phenotype contributes to pulmonary damage. The RNA seq analysis also confirmed the induction of fibro-inflammatory and oncogenic proteins in pancreas and lung tissues. Further, in the current manuscript, we now report the stepwise kinetically time-dependent cellular inflammation, genes and proteins involved in the development of pancreatitis malignancy and associated acute lung injury by analyzing the mice of 3 AOM with 3, 8, and 12 weeks of the cerulein challenged protocol regime.. We first show that sustained long-term eosinophilic inflammation induces time-dependent proinflammatory, profibrotic and malignancy-associated genes that promote pancreatic malignancy and acute lung injury in mice.

Topics: Animals; Ceruletide; Disease Models, Animal; Inflammation; Mice; Pancreatic Neoplasms; Pancreatitis, Chronic

Ubiquitin-binding associated protein 2 (UBAP2) is reported to promote macropinocytosis and pancreatic adenocarcinoma (PDAC) growth, however, its role in normal pancreatic function remains unknown. We addressed this knowledge gap by generating UBAP2 knockout (U2KO) mice under a pancreas-specific Cre recombinase (Pdx1-Cre). Pancreatic architecture remained intact in U2KO animals, but they demonstrated slight glucose intolerance compared to controls. Upon cerulein challenge to induce pancreatitis, U2KO animals had reduced levels of several pancreatitis-relevant cytokines, amylase and lipase in the serum, reduced tissue damage, and lessened neutrophil infiltration into the pancreatic tissue. Mechanistically, cerulein-challenged U2KO animals revealed reduced NF-κB activation compared to controls. In vitro promoter binding studies confirmed the reduction of NF-κB binding to its target molecules supporting UBAP2 as a new regulator of inflammation in pancreatitis and may be exploited as a therapeutic target in future to inhibit pancreatitis.

Topics: Acute Disease; Adenocarcinoma; Animals; Ceruletide; Glucose; Inflammation; Mice; NF-kappa B; Pancreas; Pancreatic Neoplasms; Pancreatitis

Chronic inflammation is known to be associated with pancreatic cancer, however a complete picture regarding how these pathologies intersect is still being characterized. In vivo model systems are critical for the study of mechanisms underlying how inflammation accelerates neoplasia. Repeat injection of cerulein, a cholecystokinin (CCK) analog, is widely used to experimentally induce acute and chronic pancreatitis in vivo. Chronic cerulein administration into genetically engineered mouse models (GEMMs) with predisposition to pancreatic cancer can induce a pro-inflammatory immune response, pancreatic acinar cell damage, pancreatic stellate cell activation, and accelerate the onset of neoplasia. Here we provide a detailed protocol and insights into using cerulein to induce pancreatitis in GEMMs, and methods to experimentally assess inflammation and pancreatic neoplasia.

Topics: Acinar Cells; Animals; Ceruletide; Mice; Pancreas; Pancreatic Neoplasms; Pancreatitis

Polo-like kinase 1 (Plk1) plays an important role in cell-cycle regulation. Recent work has suggested that Plk1 could be a biomarker of gemcitabine response in pancreatic ductal adenocarcinoma (PDAC). Although targeting Plk1 to treat PDAC has been attempted in clinical trials, the results were not promising, and the mechanisms of resistance to Plk1 inhibition is poorly understood. In addition, the role of Plk1 in PDAC progression requires further elucidation. Here, we showed that Plk1 was associated with poor outcomes in patients with PDAC. In an inducible transgenic mouse line with specific expression of Plk1 in the pancreas, Plk1 overexpression significantly inhibited caerulein-induced acute pancreatitis and delayed development of acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Bioinformatics analyses identified the regulatory networks in which Plk1 is involved in PDAC disease progression, including multiple inflammation-related pathways. Unexpectedly, inhibition or depletion of Plk1 resulted in upregulation of PD-L1 via activation of the NF-κB pathway. Mechanistically, Plk1-mediated phosphorylation of RB at S758 inhibited the translocation of NF-κB to nucleus, inactivating the pathway. Inhibition of Plk1 sensitized PDAC to immune checkpoint blockade therapy through activation of an antitumor immune response. Together, Plk1 suppresses PDAC progression and inhibits NF-κB activity, and targeting Plk1 can potentiate the efficacy of immunotherapy in PDAC.. Inhibition of Plk1 induces upregulation of PD-L1 expression in pancreatic ductal adenocarcinoma, stimulating antitumor immunity and sensitizing tumors to immunotherapy.

Topics: Acute Disease; Animals; B7-H1 Antigen; Carcinoma, Pancreatic Ductal; Cell Cycle Proteins; Ceruletide; Humans; Immune Checkpoint Inhibitors; Mice; NF-kappa B; Pancreatic Neoplasms; Pancreatitis; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins

Pancreatic ductal adenocarcinoma (PDAC) is highly metastatic and represents one of the deadliest forms of human cancers. Previous studies showed that activation of Yes-associated protein 1 (YAP1) plays a key role in malignant transformation in the pancreas. In this study, we found that YAP1 regulates the expression of epithelial cell transforming 2 (ECT2), a guanine nucleotide exchange factor for Rho-like GTPases. By immunohistochemistry analysis of human tissues, we show that ECT2 is highly expressed in primary PDAC and liver metastasis but not in normal pancreas. These correlations were also observed in a mouse model of PDAC, where pancreatic transformation is driven by mutants of

Topics: Adaptor Proteins, Signal Transducing; Cell Cycle Proteins; Cell Line; Cell Movement; Cell Proliferation; Cell Survival; Ceruletide; Humans; Neoplasms, Experimental; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins; Transcription Factors; Up-Regulation; YAP-Signaling Proteins

The unfolded protein response (UPR) is activated in pancreatic pathologies and suggested as a target for therapeutic intervention. In this study, we examined activating transcription factor 3 (ATF3), a mediator of the UPR that promotes acinar-to-ductal metaplasia (ADM) in response to pancreatic injury. Since ADM is an initial step in the progression to pancreatic ductal adenocarcinoma (PDAC), we hypothesized that ATF3 is required for initiation and progression of PDAC. We generated mice carrying a germline mutation of Atf3 (Atf3

Topics: Acinar Cells; Activating Transcription Factor 3; Animals; Ceruletide; Humans; Mice; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras)

Reports indicate that accumulated macrophages in the pancreas are responsible for promoting the pathogenesis of chronic pancreatitis (CP). Recently, macrophage-secreted cytokines have been implicated in promoting pancreatic acinar-to-ductal metaplasia (ADM). This study aims to establish the role of accumulated macrophage-activated NLRP3-IL-18-eosinophil mechanistic pathway in promoting several characteristics of pancreatic malignancy in CP. We report that in a murine model of pancreatic cancer (PC), accumulated macrophages are the source of NLRP3-regulated IL-18, which promotes eosinophilic inflammation-mediated accumulation to periductal mucin and collagen, including the formation of ADM, pancreatic intraepithelial neoplasia (PanINs), and intraductal papillary mucinous neoplasm. Most importantly, we show improved malignant characteristics with reduced levels of oncogenes in an anti-IL-18 neutralized and IL-18 gene deficient murine model of CP. Last, human biopsies validated that NLRP3-IL-18-induced eosinophils accumulate near the ducts, showing PanINs formation in PC. Taken together, we present the evidence on the role of IL-18-induced eosinophilia in the development of PC phenotype like ADM, PanINs, and ductal cell differentiation in inflammation-induced CP.

Topics: Animals; Azoxymethane; Ceruletide; Eosinophils; Humans; Interleukin-18; Macrophages; Male; Mice; Mucins; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreatic Neoplasms; Phenotype; Proteomics; Signal Transduction

Topics: Alcohol Drinking; Animals; Carcinogens; Carcinoma, Pancreatic Ductal; Central Nervous System Depressants; Ceruletide; Cytokines; Diet, Western; Ethanol; Hepatocytes; Homeodomain Proteins; Liver Neoplasms; Mice; Mutation; Nicotine; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras); Trans-Activators

Topics: Amylases; Animals; Ceruletide; Disease Models, Animal; Endothelin-1; Endothelin-Converting Enzymes; Gene Expression Regulation; Humans; Inflammation; Mice; Oncogenes; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins p21(ras); Receptor, Endothelin A; Receptor, Endothelin B

Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis.. We performed studies with LSL-Kras. Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D. In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Energy Metabolism; Fibrosis; Humans; Interleukins; Intramolecular Oxidoreductases; Mice, Transgenic; Mutation; Octamer Transcription Factors; Pancreas; Pancreatic Neoplasms; Pancreatitis; Prostaglandin D2; Proto-Oncogene Proteins p21(ras); Time Factors; Transcription Factors

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.

Topics: Acinar Cells; Adenocarcinoma; Animals; Antineoplastic Agents; Calcium; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Dedifferentiation; Cell Transformation, Neoplastic; Cells, Cultured; Ceruletide; Deoxycytidine; Disease Progression; Gemcitabine; GTP-Binding Proteins; Histone Deacetylase Inhibitors; Mice; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins p21(ras); RGS Proteins; Signal Transduction

Pancreatitis is a major risk factor for the development of pancreatic cancer. In genetically engineered mouse models, induction of pancreatic inflammation dramatically accelerates oncogenic KRas-induced fibrosis, precancerous PanIN formation, and tumorigenesis. Here we describe simple methods of secretagogue-induced experimental acute and chronic pancreatitis, the most commonly used pancreatitis models, and their applications in pancreatic cancer research. Additionally, the preparation of primary pancreatic acinar cells is introduced. Primary acinar cells can be used to study the early events of pancreatic inflammation and pancreatic acinar-to-ductal (ADM) metaplasia.

Topics: Acinar Cells; Animals; Carcinogenesis; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Humans; Metaplasia; Mice; Mice, Transgenic; Mutation; Pancreas; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Primary Cell Culture; Proto-Oncogene Proteins p21(ras); Tumor Cells, Cultured

Acinar-to-ductal metaplasia (ADM) of the pancreas is a process that pancreatic acinar cells differentiate into ductal-like cells with ductal cell traits. The metaplasia of pancreatic acinar cells manifests their ability to adapt to the genetic and environmental pressure they encounter. However, with oncogenic genetic insults and/or sustained environmental stress, ADM may lead to pancreatic intraepithelial neoplasia (PanIN), which is a common precancerous lesion that precedes pancreatic cancer. Understanding the intermediate states of ADM and important molecules that regulate ADM formation may help the development of novel preventive strategies that could be translated to the clinic to benefit the people with high risk of pancreatic cancer. Mouse model is widely used in both in vivo and ex vivo studies of ADM. In this chapter, we describe detailed protocols of injury models of the adult mouse pancreas that can function as a tool to study mechanisms of ADM formation.

Topics: Acinar Cells; Animals; Carcinoma, Pancreatic Ductal; Cell Transdifferentiation; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Humans; Metaplasia; Mice; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Primary Cell Culture; Tumor Cells, Cultured

Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy.. We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-μm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2. Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen.. In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

Topics: Acinar Cells; Animals; Autophagy; Cell Membrane; Ceruletide; Exocytosis; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas; Pancreatic Neoplasms; Pancreatitis; Secretory Vesicles; Syntaxin 1; Trypsinogen

Topics: Animals; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Disease Progression; Gene Knockdown Techniques; Heterografts; Humans; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Nude; Mice, Transgenic; Naphthoquinones; Pancreas; Pancreatic Neoplasms; Precancerous Conditions; Snail Family Transcription Factors; Tumor Cells, Cultured

Chronic or repeated episodes of acute pancreatic inflammation, or pancreatitis, are risk factors for the development of pancreatic cancer. Pancreatic cancer is characterized by a strong fibro-inflammatory tumor microenvironment. In pancreatitis, the same fibro-inflammatory reaction is observed concurrently with a loss of normal pancreatic cells. Mouse models are commonly employed to study the progression of pancreatitis and pancreatic cancer, with genetic and pharmacological tools used to elucidate cellular and acellular interactions within pancreatic tumors. Described in this article is a protocol for using Kras

Topics: Adenocarcinoma; Animals; Ceruletide; Disease Models, Animal; Pancreas; Pancreatic Neoplasms; Pancreatitis; Point Mutation; Proto-Oncogene Proteins p21(ras)

Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in Kras

Topics: Acinar Cells; Animals; Carcinoma in Situ; Cell Proliferation; Cell Transdifferentiation; Cell Transformation, Neoplastic; Cells, Cultured; Ceruletide; Disease Models, Animal; Disease Progression; Genetic Predisposition to Disease; Glycogen Synthase Kinase 3 beta; Homeodomain Proteins; Male; Metaplasia; Mice, Knockout; Pancreas, Exocrine; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Phenotype; Proto-Oncogene Proteins p21(ras); Ribosomal Protein S6 Kinases; Signal Transduction; Time Factors; TOR Serine-Threonine Kinases; Trans-Activators; Tumor Necrosis Factor-alpha

Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result,

Topics: Acinar Cells; Activating Transcription Factor 3; Animals; Carcinoma, Pancreatic Ductal; Cell Differentiation; Ceruletide; Down-Regulation; Male; Mice; Mice, Knockout; Pancreatic Neoplasms; Pancreatitis; Phenotype

The ability of exocrine pancreatic cells to change the cellular phenotype is required for tissue regeneration upon injury, but also contributes to their malignant transformation and tumor progression. We investigated context-dependent signaling and transcription mechanisms that determine pancreatic cell fate decisions toward regeneration and malignancy. In particular, we studied the function and regulation of the inflammatory transcription factor nuclear factor of activated T cells 1 (NFATC1) in pancreatic cell plasticity and tissue adaptation.. We analyzed cell plasticity during pancreatic regeneration and transformation in mice with pancreas-specific expression of a constitutively active form of NFATC1, or depletion of enhancer of zeste 2 homologue 2 (EZH2), in the context of wild-type or constitutively activate Kras, respectively. Acute and chronic pancreatitis were induced by intraperitoneal injection of caerulein. EZH2-dependent regulation of NFATC1 expression was studied in mouse in human pancreatic tissue and cells by immunohistochemistry, immunoblotting, and quantitative reverse transcription polymerase chain reaction. We used genetic and pharmacologic approaches of EZH2 and NFATC1 inhibition to study the consequences of pathway disruption on pancreatic morphology and function. Epigenetic modifications on the NFATC1 gene were investigated by chromatin immunoprecipitation assays.. NFATC1 was rapidly and transiently induced in early adaptation to acinar cell injury in human samples and in mice, where it promoted acinar cell transdifferentiation and blocked proliferation of metaplastic pancreatic cells. However, in late stages of regeneration, Nfatc1 was epigenetically silenced by EZH2-dependent histone methylation, to enable acinar cell redifferentiation and prevent organ atrophy and exocrine insufficiency. In contrast, oncogenic activation of KRAS signaling in pancreatic ductal adenocarcinoma cells reversed the EZH2-dependent effects on the NFATC1 gene and was required for EZH2-mediated transcriptional activation of NFATC1.. In studies of human and mouse pancreatic cells and tissue, we identified context-specific epigenetic regulation of NFATc1 activity as an important mechanism of pancreatic cell plasticity. Inhibitors of EZH2 might therefore interfere with oncogenic activity of NFATC1 and be used in treatment of pancreatic ductal adenocarcinoma.

Topics: Acinar Cells; Animals; Carcinoma, Pancreatic Ductal; Cell Plasticity; Cell Proliferation; Cell Transdifferentiation; Cell Transformation, Neoplastic; Ceruletide; Cyclin-Dependent Kinase Inhibitor p16; Enhancer of Zeste Homolog 2 Protein; Gene Expression Regulation; Gene Silencing; Histones; Humans; Methylation; Mice; NFATC Transcription Factors; Pancreas; Pancreatic Neoplasms; Pancreatitis, Chronic; Promoter Regions, Genetic; Proto-Oncogene Proteins p21(ras); Regeneration; Signal Transduction; Transcription, Genetic

Bromodomain inhibitors (JQ1 and I-BET 762) are a new generation of selective, small molecule inhibitors that target BET (bromodomain and extra terminal) proteins. By impairing their ability to bind to acetylated lysines on histones, bromodomain inhibitors interfere with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell cycle, apoptosis and inflammation. In this study, JQ1 and I-BET 762 decreased c-Myc and p-Erk 1/2 protein levels and inhibited proliferation in pancreatic cancer cells. The tumor microenvironment is known to play an important role in pancreatic cancer, and these drugs suppressed the production of nitric oxide and a variety of inflammatory cytokines, including IL-6, CCL2, and GM-CSF, in both immune and pancreatic cancer cells in vitro. Notably, the bromodomain inhibitors also reduced protein levels of p-Erk 1/2 and p-STAT3 in mouse models of pancreatic cancer. All of these proteins are essential for tumor promotion, progression and metastasis. In conclusion, the bromodomain inhibitors JQ1 and I-BET 762 targeted and suppressed multiple pathways in pancreatic cancer. I-BET 762 and a number of other bromodomain inhibitors are currently being tested in several clinical trials, making them potentially promising drugs for the treatment of pancreatic cancer, an often-fatal disease.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Azepines; Benzodiazepines; Cell Line, Tumor; Cell Proliferation; Ceruletide; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Humans; Inflammation Mediators; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide; Pancreatic Neoplasms; Pancreatitis; Phosphorylation; Proto-Oncogene Proteins c-myc; RAW 264.7 Cells; Signal Transduction; Triazoles

Toxicants can perturb cellular homeostasis by modifying phosphorylation-based signaling. In the present study, we examined the effects of cerulein, an inducer of acute pancreatitis, on keratin 8 (K8) phosphorylation. We found that cerulein dose-dependently induced K8 phosphorylation and perinuclear reorganization in PANC-1 cells, thus leading to migration and invasion. The extracellular signal-regulated kinases (ERK) inhibitor U0126 suppressed cerulein-induced phosphorylation of serine 431 and reorganization of K8. Cerulein reduced the expressions of protein phosphatase 2A (PP2A) via ubiqutination and alpha4. PP2A's involvement in K8 phosphorylation of PANC-1 cells was also confirmed by the observation that PP2A gene silencing resulted in K8 phosphorylation and migration of PANC-1 cells. Overall, these results suggest that cerulein induced phosphorylation and reorganization through ERK activation by downregulating PP2A and alpha4, leading to increased migration and invasion of PANC-1 cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2090-2098, 2016.

Topics: Butadienes; Cell Line, Tumor; Cell Movement; Ceruletide; Down-Regulation; Extracellular Signal-Regulated MAP Kinases; Humans; Keratin-8; Nitriles; Pancreatic Neoplasms; Phosphorylation; Protein Phosphatase 2; Serine; Signal Transduction

Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFβ-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFβ-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFβ-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.

Topics: Acinar Cells; Amylases; Animals; Carcinoma, Pancreatic Ductal; Cell Cycle Checkpoints; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Ceruletide; Epithelial Cells; Fibrosis; Irritants; Lipase; Male; Metaplasia; Mice, Knockout; Mice, Transgenic; Pancreas; Pancreatic Neoplasms; Pancreatitis; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta

Mutation of Kirsten rat sarcoma viral oncogene homolog (KRAS) and chronic pancreatitis are the most common pathogenic events involved in human pancreatic carcinogenesis. In the process of long-standing chronic inflammation, aberrant metabolites of arachidonic acid play a crucial role in promoting carcinogenesis, in which the soluble epoxide hydrolase (sEH), as a pro-inflammatory enzyme, generally inactivates anti-inflammatory epoxyeicosatrienoic acids (EETs). Herein, we determined the effect of our newly-synthesized novel compound trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM), a dual inhibitor of sEH and RAF1 proto-oncogene serine/threonine kinase (c-RAF), on inhibiting the development of pancreatitis and pancreatic intraepithelial neoplasia (mPanIN) in LSL-Kras(G12D)/Pdx1-Cre mice. The results showed that t-CUPM significantly reduced the severity of chronic pancreatitis, as measured by the extent of acini loss, inflammatory cell infiltration and stromal fibrosis. The progression of low-grade mPanIN I to high-grade mPanIN II/III was significantly suppressed. Inhibition of mutant Kras-transmitted phosphorylation of mitogen-activated protein kinase's kinase/extracellular signal-regulated kinases was demonstrated in pancreatic tissues by western blots. Quantitative real-time polymerase chain reaction analysis revealed that t-CUPM treatment significantly reduced the levels of inflammatory cytokines including tumor necrosis facor-α, monocyte chemoattractant protein-1, as well as vascular adhesion molecule-1, and the levels of Sonic hedgehog and Gli transcription factor (Hedgehog pathway). Analysis of the eicosanoid profile revealed a significant increase of the EETs/dihydroxyeicosatrienoic acids ratio, which further confirmed sEH inhibition by t-CUPM. These results indicate that simultaneous inhibition of sEH and c-RAF by t-CUPM is important in preventing chronic pancreatitis and carcinogenesis.

Topics: Animals; Anti-Inflammatory Agents; Anticarcinogenic Agents; Carcinoma in Situ; Ceruletide; Chromatography, Liquid; Disease Models, Animal; Eicosanoids; Enzyme Inhibitors; Epoxide Hydrolases; Genetic Predisposition to Disease; Homeodomain Proteins; Immunohistochemistry; Inflammation Mediators; Integrases; Mice, Transgenic; Mutation; Neoplasm Grading; Niacinamide; Pancreas; Pancreatic Neoplasms; Pancreatitis, Chronic; Phenotype; Phenylurea Compounds; Proto-Oncogene Mas; Proto-Oncogene Proteins p21(ras); Severity of Illness Index; Signal Transduction; Tandem Mass Spectrometry; Trans-Activators

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an exuberant inflammatory desmoplastic response. The PDAC microenvironment is complex, containing both pro- and antitumorigenic elements, and remains to be fully characterized. Here, we show that sensory neurons, an under-studied cohort of the pancreas tumor stroma, play a significant role in the initiation and progression of the early stages of PDAC. Using a well-established autochthonous model of PDAC (PKC), we show that inflammation and neuronal damage in the peripheral and central nervous system (CNS) occurs as early as the pancreatic intraepithelial neoplasia (PanIN) 2 stage. Also at the PanIN2 stage, pancreas acinar-derived cells frequently invade along sensory neurons into the spinal cord and migrate caudally to the lower thoracic and upper lumbar regions. Sensory neuron ablation by neonatal capsaicin injection prevented perineural invasion (PNI), astrocyte activation, and neuronal damage, suggesting that sensory neurons convey inflammatory signals from Kras-induced pancreatic neoplasia to the CNS. Neuron ablation in PKC mice also significantly delayed PanIN formation and ultimately prolonged survival compared with vehicle-treated controls (median survival, 7.8 vs. 4.5 mo; P = 0.001). These data establish a reciprocal signaling loop between the pancreas and nervous system, including the CNS, that supports inflammation associated with oncogenic Kras-induced neoplasia. Thus, pancreatic sensory neurons comprise an important stromal cell population that supports the initiation and progression of PDAC and may represent a potential target for prevention in high-risk populations.

Topics: Adenocarcinoma in Situ; Afferent Pathways; Animals; Animals, Newborn; Capsaicin; Carcinoma, Pancreatic Ductal; Ceruletide; Denervation; Disease Progression; Female; Ganglia, Sympathetic; Genes, ras; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myelitis; Neoplasm Invasiveness; Pancreas; Pancreatic Neoplasms; Pancreatitis; Precancerous Conditions; Sensory Receptor Cells; Spinal Cord; Spinothalamic Tracts; Thoracic Vertebrae

The current paradigm of pancreatic neoplastic transformation proposes an initial step whereby acinar cells convert into acinar-to-ductal metaplasias, followed by progression of these lesions into neoplasias under sustained oncogenic activity and inflammation. Understanding the molecular mechanisms driving these processes is crucial to the early diagnostic and prevention of pancreatic cancer. Emerging evidence indicates that transcription factors that control exocrine pancreatic development could have either, protective or facilitating roles in the formation of preneoplasias and neoplasias in the pancreas. We previously identified that the homeodomain transcription factor Prox1 is a novel regulator of mouse exocrine pancreas development. Here we investigated whether Prox1 function participates in early neoplastic transformation using in vivo, in vitro and in silico approaches. We found that Prox1 expression is transiently re-activated in acinar cells undergoing dedifferentiation and acinar-to-ductal metaplastic conversion. In contrast, Prox1 expression is largely absent in neoplasias and tumors in the pancreas of mice and humans. We also uncovered that Prox1-heterozygosis markedly increases the formation of acinar-to-ductal-metaplasias and early neoplasias, and enhances features associated with inflammation, in mouse pancreatic tissues expressing oncogenic Kras. Furthermore, we discovered that Prox1-heterozygosis increases tissue damage and delays recovery from inflammation in pancreata of mice injected with caerulein. These results are the first demonstration that Prox1 activity protects pancreatic cells from acute tissue damage and early neoplastic transformation. Additional data in our study indicate that this novel role of Prox1 involves suppression of pathways associated with inflammatory responses and cell invasiveness.

Topics: Acinar Cells; Animals; Cell Transformation, Neoplastic; Ceruletide; Heterozygote; Homeodomain Proteins; Humans; Inflammation; Metaplasia; Mice; Pancreas; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras); Tumor Suppressor Proteins

Autophagy promotes tumor progression downstream of oncogenic KRAS, yet also restrains inflammation and dysplasia through mechanisms that remain incompletely characterized. Understanding the basis of this paradox has important implications for the optimal targeting of autophagy in cancer. Using a mouse model of cerulein-induced pancreatitis, we found that loss of autophagy by deletion of Atg5 enhanced activation of the IκB kinase (IKK)-related kinase TBK1 in vivo, associated with increased neutrophil and T-cell infiltration and PD-L1 upregulation. Consistent with this observation, pharmacologic or genetic inhibition of autophagy in pancreatic ductal adenocarcinoma cells, including suppression of the autophagy receptors NDP52 or p62, prolonged TBK1 activation and increased expression of CCL5, IL6, and several other T-cell and neutrophil chemotactic cytokines in vitro Defective autophagy also promoted PD-L1 upregulation, which is particularly pronounced downstream of IFNγ signaling and involves JAK pathway activation. Treatment with the TBK1/IKKε/JAK inhibitor CYT387 (also known as momelotinib) not only inhibits autophagy, but also suppresses this feedback inflammation and reduces PD-L1 expression, limiting KRAS-driven pancreatic dysplasia. These findings could contribute to the dual role of autophagy in oncogenesis and have important consequences for its therapeutic targeting. Cancer Immunol Res; 4(6); 520-30. ©2016 AACR.

Topics: Acute Disease; Adenocarcinoma; Animals; Autophagy; Autophagy-Related Protein 5; B7-H1 Antigen; Benzamides; Cell Transformation, Neoplastic; Ceruletide; Chemokine CCL5; Cytokines; Enzyme Activation; Gene Deletion; Mice; Pancreatic Neoplasms; Pancreatitis; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins p21(ras); Pyrimidines; Signal Transduction; Tumor Cells, Cultured

Surface CD24 has previously been described, together with CD44 and ESA, for the characterization of putative cancer stem cells in pancreatic ductal adenocarcinoma (PDAC), the most fatal of all solid tumors. CD24 has a variety of biological functions including the regulation of invasiveness and cell proliferation, depending on the tumor entity and subcellular localization. Genetically engineered mouse models (GEMM) expressing oncogenic KrasG12D recapitulate the human disease and develop PDAC. In this study we investigate the function of CD24 using GEMM of endogenous PDAC and a model of cerulein-induced acute pancreatitis. We found that (i) CD24 expression was upregulated in murine and human PDAC and during acute pancreatitis (ii) CD24 was expressed exclusively in differentiated PDAC, whereas CD24 absence was associated with undifferentiated tumors and (iii) membranous CD24 expression determines tumor subpopulations with an epithelial phenotype in grafted models. In addition, we show that CD24 protein is stabilized in response to WNT activation and that overexpression of CD24 in pancreatic cancer cells upregulated β-catenin expression augmenting an epithelial, non-metastatic signature. Our results support a positive feedback model according to which (i) WNT activation and subsequent β-catenin dephosphorylation stabilize CD24 protein expression, and (ii) sustained CD24 expression upregulates β-catenin expression. Eventually, membranous CD24 augments the epithelial phenotype of pancreatic tumors. Thus we link the WNT/β-catenin pathway with the regulation of CD24 in the context of PDAC differentiation.

Topics: Animals; Carcinoma, Pancreatic Ductal; CD24 Antigen; Cell Differentiation; Cell Membrane; Cell Proliferation; Ceruletide; Epithelial-Mesenchymal Transition; Epithelium; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Knockout; Mice, SCID; Neoplasm Transplantation; Pancreatic Neoplasms; Pancreatitis; Phenotype; Phosphorylation; Proto-Oncogene Proteins p21(ras); Up-Regulation

Chronic pancreatitis is a significant risk factor for pancreatic cancer. Previously, we demonstrated that the pancreatic cancer cells show enhanced expression of collapsin response mediator protein 4 (CRMP4) that strongly correlates with severe venous invasion, liver metastasis, and poor prognosis. However, involvement of CRMP4 in acute or chronic pancreatitis remains unknown.. Acute and chronic pancreatitis mice models were developed by periodic injection of caerulein. The expression levels of CRMP4 and its phosphorylation were examined.. Elevated CRMP4 levels were observed in the infiltrated lymphocytes as well as in the pancreas parenchyma of both acute and chronic pancreatitis. The expression pattern of phosphorylated CRMP4 was similar to that of CRMP4. Cdk5 partially co-localized with the phosphorylated CRMP4.. Pancreatitis induces CRMP4 expression in the pancreas parenchyma and in the infiltrated lymphocytes. Overlapping expression of CRMP4 and Cdk5 may suggest that the Cdk5 is at least, in part, responsible for the phosphorylation of CRMP4. The results suggest that CRMP4 is involved in the inflammatory response in pancreatitis. Understanding the mechanisms of CRMP4 would help us to develop novel therapeutic strategies against acute or chronic pancreatitis, and pancreatic cancer.

Topics: Acute Disease; Animals; Biopsy, Needle; Cell Transformation, Neoplastic; Ceruletide; Chronic Disease; Cyclin-Dependent Kinase 5; Disease Models, Animal; Gene Expression Regulation; Humans; Immunohistochemistry; Mice; Nerve Tissue Proteins; Pancreatic Neoplasms; Pancreatitis; Phosphorylation; Precancerous Conditions; RNA, Small Interfering

Proteasome activity is significantly increased in advanced cancers, but its role in cancer initiation is not clear, due to difficulties in monitoring this process in vivo. We established a line of transgenic mice that carried the ZsGreen-degron(ODC) (Gdeg) proteasome reporter to monitor the proteasome activity. In combination with Pdx-1-Cre;LSL-Kras(G12D) model, proteasome activity was investigated in the initiation of precancerous pancreatic lesions (PanINs). Normal pancreatic acini in Gdeg mice had low proteasome activity. By contrast, proteasome activity was increased in the PanIN lesions that developed in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice. Caerulein administration to Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice induced constitutive elevation of proteasome activity in pancreatic tissues and accelerated PanIN formation. The proteasome inhibitor markedly reduced PanIN formation in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice (P = 0.001), whereas it had no effect on PanIN lesions that had already formed. These observations indicated the significance of proteasome activity in the initiation of PanIN but not the maintenance per se. In addition, the expressions of pERK and its downstream factors including cyclin D1, NF-κB, and Cox2 were decreased after proteasome inhibition in PanINs. Our studies showed activation of proteasome is required specifically for the initiation of PanIN. The roles of proteasome in the early stages of pancreatic carcinogenesis warrant further investigation.

Topics: Animals; Carcinogenesis; Ceruletide; Cyclin D1; Cyclooxygenase 2; Disease Models, Animal; eIF-2 Kinase; Gene Expression Regulation, Neoplastic; Genes, Reporter; Homeodomain Proteins; Humans; Integrases; Mice; Mice, Transgenic; NF-kappa B; Pancreas; Pancreatic Neoplasms; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins p21(ras); Signal Transduction; Trans-Activators

Perturbation of pancreatic acinar cell state can lead to acinar-to-ductal metaplasia (ADM), a precursor lesion to the development of pancreatic ductal adenocarcinoma (PDAC). In the pancreas, Notch signaling is active both during development and in adult cellular differentiation processes. Hes1, a key downstream target of the Notch signaling pathway, is expressed in the centroacinar compartment of the adult pancreas as well as in both preneoplastic and malignant lesions. In this study, we used a murine genetic in vivo approach to ablate Hes1 in pancreatic progenitor cells (Ptf1a

Topics: Acinar Cells; Animals; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Differentiation; Cell Plasticity; Ceruletide; Disease Models, Animal; Female; Humans; Male; Metaplasia; Mice; Pancreas; Pancreas, Exocrine; Pancreatic Neoplasms; Pancreatitis; Regeneration; Signal Transduction; Stem Cells; Transcription Factor HES-1

Pancreatic cancer is one of the most aggressive cancers and has an extremely poor prognosis. Despite recent progress in both basic and clinical research, most pancreatic cancers are detected at an incurable stage owing to the absence of disease-specific symptoms. Thus, developing novel approaches for detecting pancreatic cancer at an early stage is imperative. Our in silico and immunohistochemical analyses showed that KIAA1199 is specifically expressed in human pancreatic cancer cells and pancreatic intraepithelial neoplasia, the early lesion of pancreatic cancer, in a genetically engineered mouse model and in human patient samples. We also detected secreted KIAA1199 protein in blood samples obtained from pancreatic cancer mouse models, but not in normal mice. Furthermore, we found that assessing KIAA1199 autoantibody increased the sensitivity of detecting pancreatic cancer. These results indicate the potential benefits of using KIAA1199 as a biomarker for early-stage pancreatic cancer.

Topics: Acute Disease; Animals; Autoantibodies; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Ceruletide; Databases, Genetic; Disease Models, Animal; Early Diagnosis; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hyaluronoglucosaminidase; Male; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Pancreatitis; Proteins; Tissue Array Analysis

Oncogenic mutations in KRAS contribute to the development of pancreatic ductal adenocarcinoma, but are not sufficient to initiate carcinogenesis. Secondary events, such as inflammation-induced signaling via the epidermal growth factor receptor (EGFR) and expression of the SOX9 gene, are required for tumor formation. Herein we sought to identify the mechanisms that link EGFR signaling with activation of SOX9 during acinar-ductal metaplasia, a transdifferentiation process that precedes pancreatic carcinogenesis.. We analyzed pancreatic tissues from Kras(G12D);pdx1-Cre and Kras(G12D);NFATc1(Δ/Δ);pdx1-Cre mice after intraperitoneal administration of caerulein, vs cyclosporin A or dimethyl sulfoxide (controls). Induction of EGFR signaling and its effects on the expression of Nuclear factor of activated T cells c1 (NFATc1) or SOX9 were investigated by quantitative reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemical analyses of mouse and human tissues and acinar cell explants. Interactions between NFATc1 and partner proteins and effects on DNA binding or chromatin modifications were studied using co-immunoprecipitation and chromatin immunoprecipitation assays in acinar cell explants and mouse tissue.. EGFR activation induced expression of NFATc1 in metaplastic areas from patients with chronic pancreatitis and in pancreatic tissue from Kras(G12D) mice. EGFR signaling also promoted formation of a complex between NFATc1 and C-JUN in dedifferentiating mouse acinar cells, leading to activation of Sox9 transcription and induction of acinar-ductal metaplasia. Pharmacologic inhibition of NFATc1 or disruption of the Nfatc1 gene inhibited EGFR-mediated induction of Sox9 transcription and blocked acinar-ductal transdifferentiation and pancreatic cancer initiation in mice.. EGFR signaling induces expression of NFATc1 and Sox9, leading to acinar cell transdifferentiation and initiation of pancreatic cancer. Strategies designed to disrupt this pathway might be developed to prevent pancreatic cancer initiation in high-risk patients with chronic pancreatitis.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line; Cell Transdifferentiation; Cell Transformation, Neoplastic; Ceruletide; Cyclosporine; Disease Models, Animal; ErbB Receptors; Gene Expression Regulation; Humans; Male; Metaplasia; Mice, Inbred C57BL; Mice, Knockout; Mutation; NFATC Transcription Factors; Pancreas, Exocrine; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Precancerous Conditions; Proto-Oncogene Proteins p21(ras); Signal Transduction; SOX9 Transcription Factor; Tissue Culture Techniques; Transcriptional Activation

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the Western world. The disease has the worst prognosis in the gastrointestinal malignancies with an overall 5-year survival rate of less than 5 %. Therefore, in the search for novel therapeutic targets, biomarkers for early detection and particularly adequate methods to develop and validate therapeutic strategies for this disease are still in urgent demand. Although significant progress has been achieved in understanding the genetic and molecular mechanisms, most approaches have not yet translated sufficiently for better outcome of the patients. In part, this situation is due to inappropriate or insufficient methods in modeling PDAC in laboratory settings. In the past several years, there has been an explosion of genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX) that recapitulate both genetic and morphological alterations that lead to the development of PDAC. Both models are increasingly used for characterization and validation of diagnostic and therapeutic strategies. In this chapter we will discuss state-of-the-art models to consider when selecting an appropriate in vivo system to study disease etiology, cell signaling, and drug development.

Topics: Animals; Cell Line, Tumor; Cell Separation; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Female; Humans; Mice; Mice, Transgenic; Pancreatic Neoplasms; Pancreatitis

Recent development of genetically engineered mouse models (GEMs) for pancreatic cancer (PC) that recapitulates human disease progression has helped to identify new strategies to delay/inhibit PC development. We first found that expression of the pancreatic tumor-initiating/cancer stem cells (CSC) marker DclK1 occurs in early stage PC and in both early and late pancreatic intraepithelial neoplasia (PanIN) and that it increases as disease progresses in GEM and also in human PC. Genome-wide next generation sequencing of pancreatic ductal adenocarcinoma (PDAC) from GEM mice revealed significantly increased DclK1 along with inflammatory genes. Genetic ablation of cyclo-oxygenase-2 (COX-2) decreased DclK1 in GEM. Induction of inflammation/pancreatitis with cerulein in GEM mice increased DclK1, and the novel dual COX/5-lipoxygenase (5-LOX) inhibitor licofelone reduced it. Dietary licofelone significantly inhibited the incidence of PDAC and carcinoma in situ with significant inhibition of pancreatic CSCs. Licofelone suppressed pancreatic tumor COX-2 and 5-LOX activities and modulated miRNAs characteristic of CSC and inflammation in correlation with PDAC inhibition. These results offer a preclinical proof of concept to target the inflammation initiation to inhibit cancer stem cells early for improving the treatment of pancreatic cancers, with immediate clinical implications for repositioning dual COX/5-LOX inhibitors in human trials for high risk patients.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Arachidonic Acid; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Proliferation; Ceruletide; Cyclooxygenase 2; Disease Models, Animal; Disease Progression; Doublecortin-Like Kinases; Lipoxygenase Inhibitors; Mice; Mice, Knockout; MicroRNAs; Neoplastic Stem Cells; Pancreatic Neoplasms; Pancreatitis; Protein Serine-Threonine Kinases; Pyrroles

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies, with a poor long-term prognosis, and effective therapeutic options are lacking. Observing the dynamics of the pathogenesis of pancreatic intraepithelial neoplasia (PanIN) and PDAC in tumor models can facilitate understanding of the molecular mechanisms involved in early PDAC. Furthermore, it can compensate for the research limitations associated with analyzing clinical specimens of late-stage PDAC. In this study, we orthotopically treated the pancreas with dimethylbenzanthracene (DMBA) combined with caerulein in wild-type C57BL/6 J mice to induce inflammation-related pancreatic carcinogenesis. We observed that DMBA and caerulein treatment induced a chronic consumptive disease, which caused a decrease in the relative body and pancreas weights, diminishing the health status of the mice and enhancing the inflammation-related histological changes. Moreover, mid-dose and high-frequency treatment with caerulein caused prolonged inflammatory damage to the pancreas and contributed to a permissive environment for the development of PDAC. CXCL12/CXCR4, CCL2/CCR2, and several cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were upregulated in the tumor tissue of DMBA and caerulein-induced PDAC mice. This orthotopic mouse pancreatic carcinogenesis model mimic human disease because it reproduces a spectrum of pathological changes observed in human PDAC, ranging from inflammatory lesions to pancreatic intraepithelial neoplasia. Thus, this mouse model may improve the understanding of molecular mechanisms underlying the injury-inflammation-cancer pathway in the early stages of pancreatic carcinogenesis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogenesis; Carcinoma, Pancreatic Ductal; Ceruletide; Female; Inflammation; Interleukin-1beta; Interleukin-6; Mice; Mice, Inbred C57BL; Pancreatic Neoplasms; Tumor Necrosis Factor-alpha; Up-Regulation

We describe the first mouse model of pancreatic intraepithelial neoplasia (PanIN) lesions induced by alcohol in the presence and absence of chronic pancreatitis.. Pdx1-Cre;LSL-K-ras mice were exposed to Lieber-DeCarli alcohol diet for 6 weeks with cerulein injections. The PanIN lesions and markers of fibrosis, inflammation, histone deacetylation, epithelial-to-mesenchymal transition (EMT), and cancer stemness were measured by immunohistochemistry and Western.. Exposure of Pdx1-Cre;LSL-K-ras mice to an alcohol diet significantly stimulated fibrosis and slightly but not significantly increased the level of PanIN lesions associated with an increase in tumor-promoting M2 macrophages. Importantly, the alcohol diet did not increase activation of stellate cells. Alcohol diet and cerulein injections resulted in synergistic and additive effects on PanIN lesion and M2 macrophage phenotype induction, respectively. Cerulein pancreatitis caused stellate cell activation, EMT, and cancer stemness in the pancreas. Pancreatitis caused histone deacetylation, which was promoted by the alcohol diet. Pancreatitis increased EMT and cancer stemness markers, which were not further affected by the alcohol diet.. The results suggest that alcohol has independent effects on promotion of PDAC associated with fibrosis formed through a stellate cell-independent mechanism and that it further promotes early PDAC and M2 macrophage induction in the context of chronic pancreatitis.

Topics: Acetylation; Acute Disease; Animals; Carcinoma in Situ; Cell Transformation, Neoplastic; Ceruletide; Disease Models, Animal; Epithelial-Mesenchymal Transition; Ethanol; Fibrosis; Histones; Macrophages; Mice, Transgenic; Neoplastic Stem Cells; Pancreas; Pancreatic Neoplasms; Pancreatic Stellate Cells; Pancreatitis; Pancreatitis, Alcoholic; Pancreatitis, Chronic; Time Factors

Nr5a2 participates in biliary acid metabolism and is a major regulator of the pancreatic exocrine programme. Single nucleotide polymorphisms in the vicinity of NR5A2 are associated with the risk of pancreatic ductal adenocarcinoma (PDAC).. To determine the role of Nr5a2 in pancreatic homeostasis, damage-induced regeneration and mutant KRas-driven pancreatic tumourigenesis.. Nr5a2+/- and KRas(G12V);Ptf1a-Cre;Nr5a2+/- mice were used to investigate whether a full dose of Nr5a2 is required for normal pancreas development, recovery from caerulein-induced pancreatitis, and protection from tumour development.. Adult Nr5a2+/- mice did not display histological abnormalities in the pancreas but showed a more severe acute pancreatitis, increased acino-ductal metaplasia and impaired recovery from damage. This was accompanied by increased myeloid cell infiltration and proinflammatory cytokine gene expression, and hyperactivation of nuclear factor κb and signal transducer and activator of transcription 3 signalling pathways. Induction of multiple episodes of acute pancreatitis was associated with more severe damage and delayed regeneration. Inactivation of one Nr5a2 allele selectively in pancreatic epithelial cells was sufficient to cause impaired recovery from pancreatitis. In comparison with Nr5a2+/+ mice, KRas(G12V);Ptf1a(Cre/+);Nr5a2+/- mice showed a non-statistically significant increase in the area affected by preneoplastic lesions. However, a single episode of acute pancreatitis cooperated with loss of one Nr5a2 allele to accelerate KRas(G12V)-driven development of preneoplastic lesions.. A full Nr5a2 dose is required to restore pancreatic homeostasis upon damage and to suppress the KRas(G12V)-driven mouse pancreatic intraepithelial neoplasia progression, indicating that Nr5a2 is a novel pancreatic tumour suppressor. Nr5a2 could contribute to PDAC through a role in the recovery from pancreatitis-induced damage.

Topics: Animals; Blotting, Western; Carcinoma, Pancreatic Ductal; Ceruletide; Heterozygote; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; NF-kappa B; Pancreatic Neoplasms; Pancreatitis; Polymerase Chain Reaction; Proto-Oncogene Proteins p21(ras); Receptors, Cytoplasmic and Nuclear; Signal Transduction; STAT3 Transcription Factor

Emerging evidence from mouse models suggests that mutant Kras can drive the development of pancreatic ductal adenocarcinoma (PDA) precursors from acinar cells by enforcing ductal de-differentiation at the expense of acinar identity. Recently, human genome-wide association studies have identified NR5A2, a key regulator of acinar function, as a susceptibility locus for human PDA. We investigated the role of Nr5a2 in exocrine maintenance, regeneration and Kras driven neoplasia.. To investigate the function of Nr5a2 in the pancreas, we generated mice with conditional pancreatic Nr5a2 deletion (PdxCre(late); Nr5a2(c/c)). Using this model, we evaluated acinar differentiation, regeneration after caerulein pancreatitis and Kras driven pancreatic neoplasia in the setting of Nr5a2 deletion.. We show that Nr5a2 is not required for the development of the pancreatic acinar lineage but is important for maintenance of acinar identity. Nr5a2 deletion leads to destabilisation of the mature acinar differentiation state, acinar to ductal metaplasia and loss of regenerative capacity following acute caerulein pancreatitis. Loss of Nr5a2 also dramatically accelerates the development of oncogenic Kras driven acinar to ductal metaplasia and PDA precursor lesions.. Nr5a2 is a key regulator of acinar plasticity. It is required for maintenance of acinar identity and re-establishing acinar fate during regeneration. Nr5a2 also constrains pancreatic neoplasia driven by oncogenic Kras, providing functional evidence supporting a potential role as a susceptibility gene for human PDA.

Topics: Animals; Carcinoma, Acinar Cell; Carcinoma, Pancreatic Ductal; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Ceruletide; Mice; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins p21(ras); Real-Time Polymerase Chain Reaction; Receptors, Cytoplasmic and Nuclear

Gene expression is affected by modifications to histone core proteins within chromatin. Changes in these modifications, or epigenetic reprogramming, can dictate cell fate and promote susceptibility to disease. The goal of this study was to determine the extent of epigenetic reprogramming in response to chronic stress that occurs following ablation of MIST1 (Mist1(-/-) ), which is repressed in pancreatic disease. Chromatin immunoprecipitation for trimethylation of lysine residue 4 on histone 3 (H3K4Me3) in purified acinar cells from wild type and Mist1(-/-) mice was followed by Next Generation sequencing (ChIP-seq) or ChIP-qPCR. H3K4Me3-enriched genes were assessed for expression by qRT-PCR in pancreatic tissue before and after induction of cerulein-induced pancreatitis. While most of H3K4Me3-enrichment is restricted to transcriptional start sites, >25% of enrichment sites are found within, downstream or between annotated genes. Less than 10% of these sites were altered in Mist1(-/-) acini, with most changes in H3K4Me3 enrichment not reflecting altered gene expression. Ingenuity Pathway Analysis of genes differentially-enriched for H3K4Me3 revealed an association with pancreatitis and pancreatic ductal adenocarcinoma in Mist1(-/-) tissue. Most of these genes were not differentially expressed but several were readily induced by acute experimental pancreatitis, with significantly increased expression in Mist1(-/-) tissue relative to wild type mice. We suggest that the chronic cell stress observed in the absence of MIST1 results in epigenetic reprogramming of genes involved in promoting pancreatitis to a poised state, thereby increasing the sensitivity to events that promote disease.

Topics: Acinar Cells; Animals; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Pancreatic Ductal; Ceruletide; Chromatin; Epigenesis, Genetic; Histones; Male; Metabolic Networks and Pathways; Methylation; Mice; Mice, Knockout; Pancreas; Pancreatic Neoplasms; Pancreatitis

Cancer-associated inflammation is a molecular key feature in pancreatic ductal adenocarcinoma. Oncogenic KRAS in conjunction with persistent inflammation is known to accelerate carcinogenesis, although the underlying mechanisms remain poorly understood. Here, we outline a novel pathway whereby the transcription factors NFATc1 and STAT3 cooperate in pancreatic epithelial cells to promote Kras(G12D)-driven carcinogenesis. NFATc1 activation is induced by inflammation and itself accelerates inflammation-induced carcinogenesis in Kras(G12D) mice, whereas genetic or pharmacologic ablation of NFATc1 attenuates this effect. Mechanistically, NFATc1 complexes with STAT3 for enhancer-promoter communications at jointly regulated genes involved in oncogenesis, for example, Cyclin, EGFR and WNT family members. The NFATc1-STAT3 cooperativity is operative in pancreatitis-mediated carcinogenesis as well as in established human pancreatic cancer. Together, these studies unravel new mechanisms of inflammatory-driven pancreatic carcinogenesis and suggest beneficial effects of chemopreventive strategies using drugs that are currently available for targeting these factors in clinical trials.. Our study points to the existence of an oncogenic NFATc1-STAT3 cooperativity that mechanistically links inflammation with pancreatic cancer initiation and progression. Because NFATc1-STAT3 nucleoprotein complexes control the expression of gene networks at the intersection of inflammation and cancer, our study has significant relevance for potentially managing pancreatic cancer and other inflammatory-driven malignancies.

Topics: Animals; Cell Line, Tumor; Ceruletide; Gene Expression Regulation, Neoplastic; Mice, Transgenic; NFATC Transcription Factors; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins p21(ras); STAT3 Transcription Factor

Pancreas cancer, or pancreatic ductal adenocarcinoma, is the deadliest of solid tumors, with a five-year survival rate of <5%. Detection of resectable disease improves survival rates, but access to tissue and other biospecimens that could be used to develop early detection markers is confounded by the insidious nature of pancreas cancer. Mouse models that accurately recapitulate the human condition allow disease tracking from inception to invasion and can therefore be useful for studying early disease stages in which surgical resection is possible. Using a highly faithful mouse model of pancreas cancer in conjunction with a high-density antibody microarray containing ∼2500 antibodies, we interrogated the pancreatic tissue proteome at preinvasive and invasive stages of disease. The goal was to discover early stage tissue markers of pancreas cancer and follow them through histologically defined stages of disease using cohorts of mice lacking overt clinical signs and symptoms and those with end-stage metastatic disease, respectively. A panel of seven up-regulated proteins distinguishing pancreas cancer from normal pancreas was validated, and their levels were assessed in tissues collected at preinvasive, early invasive, and moribund stages of disease. Six of the seven markers also differentiated pancreas cancer from an experimental model of chronic pancreatitis. The levels of serine/threonine stress kinase 4 (STK4) increased between preinvasive and invasive stages, suggesting its potential as a tissue biomarker, and perhaps its involvement in progression from precursor pancreatic intraepithelial neoplasia to pancreatic ductal adenocarcinoma. Immunohistochemistry of STK4 at different stages of disease revealed a dynamic expression pattern further implicating it in early tumorigenic events. Immunohistochemistry of a panel of human pancreas cancers confirmed that STK4 levels were increased in tumor epithelia relative to normal tissue. Overall, this integrated approach yielded several tissue markers that could serve as signatures of disease stage, including early (resectable), and therefore clinically meaningful, stages.

Topics: Animals; Antibodies; Biomarkers, Tumor; Carcinoma, Pancreatic Ductal; Ceruletide; Diagnosis, Differential; Disease Models, Animal; Disease Progression; Early Diagnosis; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Molecular Sequence Annotation; Neoplasm Proteins; Pancreatic Neoplasms; Pancreatitis, Chronic; Protein Array Analysis; Protein Serine-Threonine Kinases; Proteome; Signal Transduction; Time Factors

Diabetes mellitus type 2 and chronic pancreatitis are regarded as risk factors for pancreatic cancer. Pancreatic duct glands (PDGs) were recently described as a new compartment of the major duct in humans and mice. To evaluate the influence of diabetes and chronic pancreatitis on PDGs, cerulein was injected i.p., repetitively over 10 weeks, in mice exhibiting obesity and a type 2 diabetes-like syndrome (B6.V-Lep(ob/ob)) and in lean littermates. By using 5-bromo-2'-deoxyuridine (BrdU), a label-retaining cell population was characterized in PDGs. Cerulein administration led to more BrdU(+) cells in PDGs of obese mice compared with lean mice. The observed increase was specific to PDGs, because BrdU incorporation in cells of the pancreatic duct was not increased. In addition, the expression of distinct tumor markers in PDGs was characterized by Muc5ac, S100P, regenerating islet-derived 3β, 14-3-3 σ, and prostate stem cell antigen immunochemistry. Type 2 diabetes-like syndrome, accompanied by chronic pancreatitis, enhanced nuclear localization of S100P. Both risk factors for pancreatic cancer also induced the production of Muc5ac and the nuclear localization of S100P [corrected]. These results demonstrate that diabetes and chronic pancreatitis jointly enhance BrdU incorporation and production of pancreatic cancer-specific proteins in PDGs. The observed alterations suggest that pancreatic tumors might originate from the newly discovered histomorphological structures, called PDGs, which could represent a target for future anticancer therapies.

Topics: Animals; Bromodeoxyuridine; Carcinoma, Pancreatic Ductal; Ceruletide; Diabetes Mellitus, Type 2; Humans; Male; Metaplasia; Mice; Mice, Obese; Mucins; Neoplasm Proteins; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis, Chronic; Risk Factors

Patients with pancreatic cancer, which is characterized by an extensive collagen-rich fibrotic reaction, often present with metastases. A critical step in cancer metastasis is epithelial-to-mesenchymal transition (EMT), which can be orchestrated by the Snail family of transcription factors. To understand the role of Snail (SNAI1) in pancreatic cancer development, we generated transgenic mice expressing Snail in the pancreas. Because chronic pancreatitis can contribute to pancreatic cancer development, Snail-expressing mice were treated with cerulein to induce pancreatitis. Although significant tissue injury was observed, a minimal difference in pancreatitis was seen between control and Snail-expressing mice. However, because Kras mutation is necessary for tumor development in mouse models of pancreatic cancer, we generated mice expressing both mutant Kras(G12D) and Snail (Kras(+)/Snail(+)). Compared with control mice (Kras(+)/Snai(-)), Kras(+)/Snail(+) mice developed acinar ectasia and more advanced acinar-to-ductal metaplasia. The Kras(+)/Snail(+) mice exhibited increased fibrosis, increased phosphorylated Smad2, increased TGF-β2 expression, and activation of pancreatic stellate cells. To further understand the mechanism by which Snail promoted fibrosis, we established an in vitro model to examine the effect of Snail expression in pancreatic cancer cells on stellate cell collagen production. Snail expression in pancreatic cancer cells increased TGF-β2 levels, and conditioned media from Snail-expressing pancreatic cancer cells increased collagen production by stellate cells. Additionally, inhibiting TGF-β signaling in stellate cells attenuated the conditioned media-induced collagen production by stellate cells. Together, these results suggest that Snail contributes to pancreatic tumor development by promoting fibrotic reaction through increased TGF-β signaling.. Expression of the EMT regulator Snail in the context of mutant Kras provides new insight into pancreatic cancer progression.

Topics: Animals; Cell Proliferation; Ceruletide; Collagen; Epithelial-Mesenchymal Transition; Fibrosis; Mice; Mice, Transgenic; Pancreas; Pancreatic Neoplasms; Pancreatic Stellate Cells; Pancreatitis, Chronic; Proto-Oncogene Proteins p21(ras); Signal Transduction; Smad2 Protein; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta2; Tumor Cells, Cultured

This study aims to determine the differentially expressed proteins in the pancreatic acinar cells undergoing apoptosis and oncosis stimulated with caerulein to explore different cell death process of the acinar cell. AR42J cells were treated with caerulein to induce cell model of acute pancreatitis. Cells that were undergoing apoptosis and oncosis were separated by flow cytometry. Then differentially expressed proteins in the two groups of separated cells were detected by shotgun liquid chromatography-tandem mass spectrometry. The results showed that 11 proteins were detected in both apoptosis group and oncosis group, 17 proteins were detected only in apoptosis group and 29 proteins were detected only in oncosis group. KEGG analysis showed that proteins detected only in apoptosis group were significantly enriched in 10 pathways, including ECM-receptor interaction, cell adhesion molecules, and proteins detected only in oncosis group were significantly enriched in three pathways, including endocytosis, base excision repair, and RNA degradation. These proteins we detected are helpful for us to understand the process of cell death in acute pancreatitis and may be useful for changing the death mode of pancreatic acinar cells, thus attenuating the severity of pancreatitis.

Topics: Acinar Cells; Animals; Apoptosis; Cell Line; Ceruletide; Chromatography, Liquid; Flow Cytometry; Gene Ontology; Mass Spectrometry; Molecular Sequence Annotation; Pancreas; Pancreatic Neoplasms; Proteome; Proteomics; Rats; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Software; Time Factors

Pancreatic cancer is a uniformly lethal disease characterized by a strong stromal reaction called desmoplasia. Organ fibrosis is also a feature of chronic pancreatitis a known risk factor for pancreatic cancer. Here we describe a transplantation approach to investigate bone marrow-derived cells in murine models of chronic pancreatitis and pancreatic cancer.

Topics: Animals; Bone Marrow Cells; Bone Marrow Transplantation; Cell Separation; Cell Tracking; Ceruletide; Disease Models, Animal; Humans; Mice; Pancreatic Neoplasms; Pancreatitis, Chronic; Whole-Body Irradiation

Genetic mutations that give rise to active mutant forms of Ras are oncogenic and found in several types of tumor. However, such mutations are not clear biomarkers for disease, since they are frequently detected in healthy individuals. Instead, it has become clear that elevated levels of Ras activity are critical for Ras-induced tumorigenesis. However, the mechanisms underlying the production of pathological levels of Ras activity are unclear. Here, we show that in the presence of oncogenic Ras, inflammatory stimuli initiate a positive feedback loop involving NF-κB that further amplifies Ras activity to pathological levels. Stimulation of Ras signaling by typical inflammatory stimuli was transient and had no long-term sequelae in wild-type mice. In contrast, these stimuli generated prolonged Ras signaling and led to chronic inflammation and precancerous pancreatic lesions (PanINs) in mice expressing physiological levels of oncogenic K-Ras. These effects of inflammatory stimuli were disrupted by deletion of inhibitor of NF-κB kinase 2 (IKK2) or inhibition of Cox-2. Likewise, expression of active IKK2 or Cox-2 or treatment with LPS generated chronic inflammation and PanINs only in mice expressing oncogenic K-Ras. The data support the hypothesis that in the presence of oncogenic Ras, inflammatory stimuli trigger an NF-κB-mediated positive feedback mechanism involving Cox-2 that amplifies Ras activity to pathological levels. Because a large proportion of the adult human population possesses Ras mutations in tissues including colon, pancreas, and lung, disruption of this positive feedback loop may be an important strategy for cancer prevention.

Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Ceruletide; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Enzyme Induction; Esters; Feedback, Physiological; Gabexate; Gene Expression Regulation, Neoplastic; Gene Knock-In Techniques; Genes, ras; Guanidines; Humans; I-kappa B Kinase; Inflammation; Inflammation Mediators; Lipopolysaccharides; Mice; Mice, Transgenic; Neoplasm Proteins; NF-kappa B; Pancreas; Pancreatic Neoplasms; Pancreatitis, Chronic; Precancerous Conditions; Proto-Oncogene Proteins p21(ras); Sincalide

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.. Pancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.. Inhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.. The utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cell Proliferation; Ceruletide; Collagen; Extracellular Matrix; Fibrosis; Humans; Male; Mice; Mice, Transgenic; Myofibroblasts; Neoplasm Invasiveness; Pancreatic Neoplasms; Piperidines; Quinazolinones; Stromal Cells

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease without effective chemopreventive or therapeutic approaches. Although the role of oncogenic Kras in initiating development of PDAC is well established, downstream targets of aberrant Ras signaling are poorly understood. Acinar-ductal metaplasia (ADM) appears to be an important prerequisite for development of pancreatic intraepithelial neoplasia (PanIN), a common precursor to PDAC. RAS-related C3 botulinum substrate 1 (Rac1), which controls actin reorganization, can be activated by Ras, is up-regulated in several human cancers, and is required for cerulein-induced morphologic changes in acini. We investigated effects of loss of Rac1 in Kras-induced pancreatic carcinogenesis in mice.. Using a Cre/lox approach, we deleted Rac1 from pancreatic progenitor cells in different mouse models of PDAC and in mice with cerulein-induced acute pancreatitis. Acinar epithelial explants of mutant mice were used to investigate the role of Rac1 in vitro.. Rac1 expression increased in mouse and human pancreatic tumors, particularly in the stroma. Deletion of Rac1 in Kras(G12D)-induced PDAC in mice reduced formation of ADM, PanIN, and tumors and significantly prolonged survival. Pancreatic epithelial metaplasia was accompanied by apical-basolateral redistribution of F-actin, along with basal expression of Rac1. Acinar epithelial explants that lacked Rac1 or that were incubated with inhibitors of actin polymerization had a reduced ability to undergo ADM in 3-dimensional cultures.. In mice, Rac1 is required for early metaplastic changes and neoplasia-associated actin rearrangements in development of pancreatic cancer. Rac1 might be developed as a diagnostic marker or therapeutic target for PDAC.

Topics: Actins; Animals; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Transformation, Neoplastic; Ceruletide; Genes, ras; Humans; Kaplan-Meier Estimate; Keratin-19; Metaplasia; Mice; Models, Animal; Pancreas; Pancreatic Neoplasms; Pancreatitis; rac1 GTP-Binding Protein; Signal Transduction; Survival Rate

Capsaicin is a major biologically active ingredient of chili peppers. Extensive studies indicate that capsaicin is a cancer-suppressing agent via blocking the activities of several signal transduction pathways including nuclear factor-kappaB, activator protein-1 and signal transducer and activator of transcription 3. However, there is little study on the effect of capsaicin on pancreatic carcinogenesis. In the present study, the effect of capsaicin on pancreatitis and pancreatic intraepithelial neoplasia (PanIN) was determined in a mutant Kras-driven and caerulein-induced pancreatitis-associated carcinogenesis in LSL-Kras(G12D)/Pdx1-Cre mice. Forty-five LSL-Kras(G12D)/Pdx1-Cre mice and 10 wild-type mice were subjected to one dose of caerulein (250 μg/kg body wt, intraperitoneally) at age 4 weeks to induce and synchronize the development of chronic pancreatitis and PanIN lesions. One week after caerulein induction, animals were randomly distributed into three groups and fed with either AIN-76A diet, AIN-76A diet containing 10 p.p.m. capsaicin or 20 p.p.m. capsaicin for a total of 8 weeks. The results showed that capsaicin significantly reduced the severity of chronic pancreatitis, as determined by evaluating the loss of acini, inflammatory cell infiltration and stromal fibrosis. PanIN formation was frequently observed in the LSL-Kras(G12D)/Pdx1-Cre mice. The progression of PanIN-1 to high-grade PanIN-2 and -3 were significantly inhibited by capsaicin. Further immunochemical studies revealed that treatment with 10 and 20 p.p.m. capsaicin significantly reduced proliferating cell nuclear antigen-labeled cell proliferation and suppressed phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun as well blocked Hedgehog/GLI pathway activation. These results indicate that capsaicin could be a promising agent for the chemoprevention of pancreatic carcinogenesis, possibly via inhibiting pancreatitis and mutant Kras-led ERK activation.

Topics: Animals; Blotting, Western; Capsaicin; Capsicum; Carcinoma in Situ; Cell Transformation, Neoplastic; Ceruletide; Extracellular Signal-Regulated MAP Kinases; Female; Homeodomain Proteins; Immunoenzyme Techniques; Integrases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Pancreatic Neoplasms; Pancreatitis, Chronic; Proto-Oncogene Proteins p21(ras); Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensory System Agents; Trans-Activators; Transcription Factor AP-1

Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing cells is sufficient for the development of low grade pancreatic intraepithelial neoplasia (PanIN) between 2 and 4 months. The mice die around 6 month-old of unrelated causes, and it is therefore not possible to assess whether the lesions will progress to carcinoma. We now report that two brief episodes of caerulein-induced acute pancreatitis in 2 month-old mice causes rapid PanIN progression and pancreatic ductal adenocarcinoma (PDAC) development by 4 months of age. These events occur with similar frequency as observed in animals where the oncogene is targeted during embryogenesis to all pancreatic cell types. Thus, these data show that oncogenic Kras-driven PanIN originating in a non-ductal compartment can rapidly progress to PDAC when subjected to a brief inflammatory insult.

Topics: Animals; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Lineage; Ceruletide; Disease Progression; Gene Targeting; Humans; Integrases; Intermediate Filament Proteins; Mice; Mice, Transgenic; Nerve Tissue Proteins; Nestin; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis; Precancerous Conditions; Proto-Oncogene Proteins p21(ras); STAT3 Transcription Factor; Stem Cells; Transgenes

Pancreatic intraepithelial neoplasia (PanIN) are pancreatic cancer precursor lesions of unclear origin and significance. PanIN aberrantly express sonic hedgehog (Shh), an initiator of pancreatic cancer, and gastrointestinal mucins. A majority of PanIN are thought to arise from ducts. We identified a novel ductal compartment that is gathered in gland-like outpouches (pancreatic duct glands [PDG]) of major ducts and characterized its role in injury and metaplasia.. The ductal system was analyzed in normal pancreata and chronic pancreatitis in humans and mice. Anatomy was assessed by serial hematoxylin and eosin sections and scanning electron microscopy of corrosion casts. Expression of mucins and developmental genes and proliferation were assessed by immunohistochemistry or real-time quantitative polymerase chain reaction. Effects of Shh on ductal cells were investigated by exposure to Shh in vitro and transgenic misexpression in vivo.. Three-dimensional analysis revealed blind-ending outpouches of ducts in murine and human pancreata. These PDG are morphologically and molecularly distinct from normal ducts; even in normal pancreata they display PanIN and metaplastic features, such as expression of Shh and gastric mucins. They express other developmental genes, such as Pdx-1 and Hes-1. In injury, Shh is up-regulated along with gastric mucins. Expansion of the PDG compartment results in a mucinous metaplasia. Shh promotes this transformation in vitro and in vivo.. PDG are distinct gland-like mucinous compartments with a distinct molecular signature. In response to injury, PDG undergo an Shh-mediated mucinous gastrointestinal metaplasia with PanIN-like features. PDG may provide a link between Shh, mucinous metaplasia, and neoplasia.

Topics: Animals; Carcinoma in Situ; Cells, Cultured; Ceruletide; Corrosion Casting; Disease Models, Animal; Epithelial Cells; Gastric Mucins; Gene Expression Regulation, Developmental; Hedgehog Proteins; Humans; Immunohistochemistry; Metaplasia; Mice; Mice, Transgenic; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis, Chronic; Precancerous Conditions; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors

Oxidative stress is regarded as a major pathogenic factor in acute pancreatitis. Inflammation and apoptosis linked to oxidative stress has been implicated in cerulein-induced pancreatitis as an experimental model of acute pancreatitis. Recently, we found that reactive oxygen species mediate inflammatory cytokine expression and apoptosis of pancreatic acinar cells stimulated with cerulein. Omega-3 fatty acids show antioxidant action in various cells and tissues. In the present study, we investigated whether omega-3 fatty acids inhibit cytokine expression in cerulein-stimulated pancreatic acinar cells and whether omega-3 fatty acids suppress apoptotic cell death in pancreatic acinar cells exposed to hydrogen peroxide. We found that omega-3 fatty acids, such as docosahexaenoic acid (DHA) and alpha-linolenic acid (ALA), suppressed the expression of inflammatory cytokines (IL-1beta, IL-6) and inhibited the activation of transcription factor activator protein-1 in cerulein-stimulated pancreatic acinar cells. DHA and ALA inhibited DNA fragmentation, inhibited the decrease in cell viability, and inhibited the expression of apoptotic genes (p53, Bax, apoptosis-inducing factor) induced by hydrogen peroxide in pancreatic acinar cells. In conclusion, omega-3 fatty acids may be beneficial for preventing oxidative stress-induced pancreatic inflammation and apoptosis by inhibiting inflammatory cytokine and apoptotic gene expression of pancreatic acinar cells.

Topics: alpha-Linolenic Acid; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Line, Tumor; Cell Survival; Ceruletide; Cytokines; DNA Fragmentation; Docosahexaenoic Acids; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Fatty Acids, Omega-3; Gene Expression; Hydrogen Peroxide; Inflammation; Interleukin-1beta; Interleukin-6; Pancreatic Neoplasms; Protein Binding; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor AP-1; Tumor Suppressor Protein p53

To study the molecular mechanism of arsenic trioxide-induced cell death on cerulein-stimulated AR42J cells.. AR42J cells were incubated for 24 hours, and cerulein (10 nmol/L) and different concentrations of arsenic trioxide were added for another 24 hours. The cells were collected and analyzed for apoptosis and oncosis by using rhodamine 123 and propidium iodide staining, and the changes in the genes that related to cell death were detected by a gene chip.. After cerulein stimulation, apoptosis was significantly increased in the AR42J cells. The addition of arsenic trioxide increased the number of apoptotic cells, and the apoptotic index reached its peak in the 1-micromol/L group. Regarding oncosis, low concentrations of arsenic trioxide (0.5, 1, 2, and 4 micromol/L) reduced the development of oncosis, and in the 2-micromol/L group, it was most significant, whereas high concentration of arsenic trioxide (8 micromol/L) promoted the development of oncosis. A total of 96 genes related to apoptosis were detected, and 36 genes were differentially expressed.. Appropriate concentrations of arsenic trioxide can induce apoptosis in AR42J cells that were induced by cerulein and lead to changes in the expressions of certain genes.

Topics: Amylases; Animals; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Cell Death; Cell Line, Tumor; Ceruletide; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; L-Lactate Dehydrogenase; Oligonucleotide Array Sequence Analysis; Oxides; Pancreatic Neoplasms

The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice.. Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays.. Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain.. Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

Topics: Acute Disease; Amyloid Precursor Protein Secretases; Animals; beta Catenin; Cell Line, Tumor; Ceruletide; Dibenzazepines; Disease Models, Animal; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreas, Exocrine; Pancreatic Neoplasms; Pancreatitis; Receptor, Notch1; Regeneration; Signal Transduction; Wnt Proteins

In this issue of Cancer Cell, Guerra and colleagues provide important new insights regarding the ability of specific pancreatic cell types to generate invasive pancreatic cancer. First, they demonstrate that classical pancreatic "ductal" neoplasia can be induced by activation of oncogenic Kras in nonductal exocrine cells. Second, they show that, while Kras activation in immature acinar and centroacinar cells is readily able to induce ductal neoplasia, Kras-mediated tumorigenesis in mature exocrine pancreas requires the induction of chronic epithelial injury. The results shed new light on the "cell of origin" of pancreatic ductal cancer and demonstrate that chronic pancreatitis provides a permissive environment for Kras-induced pancreatic neoplasia.

Topics: Animals; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Lineage; Cell Transformation, Neoplastic; Ceruletide; Genes, ras; Humans; Mice; Mutation; Neoplasm Invasiveness; Pancreatic Neoplasms; Pancreatitis, Chronic

Pancreatic ductal adenocarcinoma (PDA), one of the deadliest human cancers, often involves somatic activation of K-Ras oncogenes. We report that selective expression of an endogenous K-Ras(G12V) oncogene in embryonic cells of acinar/centroacinar lineage results in pancreatic intraepithelial neoplasias (PanINs) and invasive PDA, suggesting that PDA originates by differentiation of acinar/centroacinar cells or their precursors into ductal-like cells. Surprisingly, adult mice become refractory to K-Ras(G12V)-induced PanINs and PDA. However, if these mice are challenged with a mild form of chronic pancreatitis, they develop the full spectrum of PanINs and invasive PDA. These observations suggest that, during adulthood, PDA stems from a combination of genetic (e.g., somatic K-Ras mutations) and nongenetic (e.g., tissue damage) events.

Topics: Animals; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Cell Lineage; Cell Transformation, Neoplastic; Ceruletide; Doxycycline; Genes, ras; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Mutant Strains; Mutation; Neoplasm Invasiveness; Pancreas; Pancreatic Neoplasms; Pancreatitis, Chronic; Signal Transduction

Somatostatin, or its structural analog SMS 201-995 (SMS), is recognized to exert a growth-inhibitory action in rat pancreas, but the cellular mechanisms are not completely understood. This study was undertaken to evaluate the effect of SMS on p42/p44 MAP kinases and phosphatidylinositol 3-kinase activation and to analyze expression of some cell cycle regulatory proteins in relation to pancreatic acinar cell proliferation in vivo (rat pancreas), as well as in the well-established tumoral cell line AR4-2J. We herein report that: 1) SMS inhibits caerulein-induced pancreatic weight and DNA content and abolishes epidermal growth factor (EGF)-stimulated AR4-2J proliferation; 2) SMS only moderately reduces the stimulatory effect of caerulein on p42/p44 MAP kinase activities in pancreas and has no effect on EGF-stimulated MAP kinase activities in AR4-2J cells; 3) SMS repressed caerulein-induced Akt activity in normal pancreas; 4) SMS has a strong inhibitory action on cyclin E expression induced by caerulein in pancreas and EGF in AR4-2J cells and as expected, the resulting cyclin E-associated cyclin-dependent kinase (cdk)2 activity, as well as pRb phosphorylation, are blunted by SMS treatment in both models; and 5) SMS suppresses mitogen-induced p27(Kip1) down-regulation, as well as marginally induces p21(Cip) expression. Thus, our data suggest that somatostatin-induced growth arrest is mediated by inhibition of phosphatidylinositol 3-kinase pathway and by enhanced expression of p21(Cip) and p27(Kip1), leading to repression of pRb phosphorylation and cyclin E-cdk2 complex activity.

Topics: Animals; Cell Cycle; Cell Division; Cell Line; Ceruletide; Enzyme Activation; Epidermal Growth Factor; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Octreotide; Pancreas; Pancreatic Neoplasms; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Somatostatin; Tumor Cells, Cultured

Activation of the receptor c-met stimulates motility, mitosis, morphogenesis, processes involved in organ regeneration, or progression of malignancies. In the present study we investigated the expression of c-met protein in the regenerating pancreas and characterized the influence of cytokines on c-met expression.. Acute pancreatitis was induced in rats by cerulein injection. Rat acini and rat and human pancreatic cancer cells were stimulated with interleukin-1alpha (IL-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-beta1 (TGF-beta1). C-met expression was analyzed by means of Western blotting and localization in pancreatic tissue by immunohistochemistry.. C-met protein expression was significantly upregulated in the regenerating pancreas and localized in areas of regenerating tissue. Stimulation with cytokines resulted in a two- to threefold increase of c-met expression in vitro.. Enhanced c-met expression after acute pancreatitis suggests that HGF/met has an important role in pancreatic regeneration, which is probably mediated by cytokines. This regulatory mechanism is also of importance in pancreatic cancer.

Topics: Acute Disease; Animals; Blotting, Western; Cells, Cultured; Ceruletide; Cytokines; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Interleukin-1; Interleukin-6; Male; Pancreas; Pancreatic Neoplasms; Pancreatitis; Proto-Oncogene Proteins c-met; Rats; Rats, Wistar; Regeneration; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Gastrointestinal hormones influence the microcirculation in the normal pancreas. In the present study, we studied the effect of cerulein and somatostatin on pancreatic cancer microcirculation after orthotopic and nonorthotopic tumor implantation.. In 36 male Lewis rats (150-180 g) induction of a ductlike pancreatic cancer was achieved by intrapancreatic or intraperitoneal tumor fragment interposition between two inert transparent polymethyl methacrylate plates. After 4 weeks, intravital microscopy of the tumor microcirculation was performed in a temperature-controlled immersion chamber. The animals received 5 microg/kg cerulein or 3 mg/kg somatostatin for 1 h intravenously. The erythrocyte velocity in normal pancreatic capillaries or in tumor vessels was measured.. The erythrocyte velocity in the capillaries of the normal pancreas was 1.01 +/- 0.11 mm/s at baseline and increased to 1.64 +/- 0.09 mm/s after cerulein stimulation (p = 0.007). Pancreatic cancer vessels demonstrated no increase in erythrocyte velocity after orthotopic (baseline 0.95 +/- 0.14 mm/s, after 1 h 0.86 +/- 0.13 mm/s; n.s.) and nonorthotopic tumor implantation (baseline 0.91 +/- 0.12 mm/s, after 1 h 0.95 +/- 0.14 mm/s; n.s.) after cerulein stimulation. Somatostatin decreased the erythrocyte velocity both in normal pancreas (baseline 0.87 +/- 0.02 mm/s, after 1 h 0.60 +/- 0.07 mm/s; p = 0.01) and in pancreatic cancer (baseline 0.85 +/- 0.20 mm/s, after 1 h 0.63 +/- 0.18 mm/s; p = 0.02) after orthotopic tumor implantation. There was no effect of somatostatin after nonorthotopic tumor implantation (baseline 0.90 +/- 0.10 mm/s, after 1 h 0.88 +/- 0.14 mm/s; n.s.).. These data suggest that pancreatic cancer microcirculation lacks physiological blood flow control by stimulatory hormones, in contrast to the normal pancreas.

Topics: Animals; Ceruletide; Erythrocyte Indices; Gastrointestinal Hormones; Hemodynamics; Male; Microcirculation; Pancreatic Neoplasms; Rats; Rats, Inbred Lew; Somatostatin

The intracellular events involved in normal pancreatic growth have been extensively investigated in response to cholecystokinin. Recent data indicate that tyrosine kinase, phospholipase D, phosphatidylinositol 3-kinase, and p42/p44 MAPK are stimulated in rat pancreatic acinar cells. Although we begin to understand the intracellular signaling pathways activated in normal pancreas, such information is not yet available in pancreatic cancer cells. This study was undertaken to identify the growth factors and hormones involved in cell proliferation of two human pancreatic cancer cell lines of ductal origin, the MIA PaCa-2, and PANC-1 cells, and to establish the intracellular events involved in the control of their growth. We demonstrated that FGF-2, IGF-1, cerulein, and gastrin but not FGF-1, HGF, secretin, and PACAP, stimulated proliferation of MIA PaCa-2 and PANC-1 cells. Autocrine factors such as gastrin and IGF-1 were also responsible for their proliferation. In response to EGF, FGF-2, IGF-1, cerulein, gastrin and bombesin, tyrosine kinase, and tyrosine phosphatase activities were stimulated in both cell lines. The close relationship established between cell growth and tyrosine kinase activation results from the observation that maximal growth stimulation paralleled with maximal enzyme activation and that genistein, the tyrosine kinase inhibitor, blocked cell growth and enzyme activation. The implication of PLD in growth-stimulated processes is doubtful since all growth factors and hormones tested failed to stimulate an already very active PLD activity. We finally observed a constitutive activity of p44 MAPK in both cell lines and of p42 in MIA PaCa-2 cells. However, p38 and p42 were stimulated in MIA PaCa-2 and PANC-1 cells, respectively, by all growth factors and hormones.

Topics: Animals; Bombesin; Cell Division; Ceruletide; Enzyme Activation; Fibroblast Growth Factor 2; Gastrins; Growth Substances; Hormones; Humans; Insulin-Like Growth Factor I; Pancreatic Neoplasms; Phospholipase D; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Signal Transduction; Tumor Cells, Cultured

AR4-2J pancreatoma cells were stably transfected with an expression vector containing the cDNA for PKC-alpha in the antisense orientation. Transfectants designated antisense-alpha AA1, AA2, and AA3 exhibited marked reductions in PKC-alpha expression and decrements in cell growth. The magnitude of the decrement in cell growth paralleled the reduction in PKC-alpha expression, i.e., AA3 > AA1 > AA2. The ability of dexamethasone to induce cell differentiation as assessed by a rise in cellular amylase levels was not markedly affected by the reduction in PKC-alpha expression. Unstimulated amylase release was attenuated in AA1 cells and almost completely blocked in AA2 transfectants. The AA2 transfectant cell line failed to elicit a secretory response to caerulein, and the AA1 transfectant exhibited a lack of the secondary phase of stimulated amylase secretion. These findings demonstrate that PKC-alpha is involved in the mechanisms regulating growth and secretion in AR4-2J cells, but is not necessary for the induction of amylase stores following differentiation.

Topics: Amylases; Animals; Cell Differentiation; Cell Division; Ceruletide; Dexamethasone; Isoenzymes; Pancreas; Pancreatic Neoplasms; Protein Kinase C; Protein Kinase C-alpha; Rats; RNA, Antisense; RNA, Messenger; Transfection; Tumor Cells, Cultured

Expression of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) was studied in normal pancreatic tissue and in (pre)neoplastic pancreatic lesions of azaserine-treated rats. They were given either a low fat, high fiber (low caloric) diet, to inhibit carcinogenesis, or a low fat diet combined with injections of the cholecystokinin analog caerulein to enhance carcinogenesis. The control groups, maintained on a low fat diet, were injected with azaserine or were not treated at all. Autopsy was performed at 6 and 15 months after the last azaserine injection. After both 6 and 15 months immunohistochemistry revealed a weak expression of EGF and TGF-alpha peptides in the acinar cells, and a stronger expression in the ductular and centroacinar cells. TGF-alpha peptide expression was reduced in both putative preneoplastic and neoplastic acinar cell lesions, but no differences in EGF peptide expression were observed between the various stages of exocrine pancreatic carcinogenesis. After 16 months an increase in TGF-alpha mRNA due to treatment with azaserine was detected by semi-quantitative PCR in total pancreatic homogenates, whereas EGF mRNA expression had decreased. TGF-alpha mRNA levels in macroscopically isolated tumors were significantly lower, but EGF mRNA levels were significantly higher, than in total pancreatic homogenates from azaserine-treated rats. Furthermore, EGF and TGF-alpha mRNA levels in isolated tumors did not differ significantly from mRNA levels in non-carcinogen-treated rats. Neither with immunohistochemistry nor with PCR were differences in EGF or TGF-alpha expression observed due to either inhibition or stimulation of carcinogenesis. It is concluded that putative preneoplastic acinar cell lesions induced in rat pancreas by azaserine may develop into acinar adenocarcinomas independently of TGF-alpha and EGF. The results suggest involvement of these growth factors at the early stage of the carcinogenic process, during the initiation of normal acinar cells into putative preneoplastic cells. However, modulation of azaserine-induced pancreatic carcinogenesis by cholecystokinin or a low fat, high fiber (caloric restricted) diet appeared not to be regulated by EGF or TGF-alpha.

Topics: Animals; Azaserine; Base Sequence; Body Weight; Carcinogens; Ceruletide; Cocarcinogenesis; Diet, Fat-Restricted; Dietary Fiber; Drug Synergism; Energy Intake; Epidermal Growth Factor; Immunohistochemistry; Male; Molecular Sequence Data; Organ Size; Pancreas; Pancreatic Neoplasms; Polymerase Chain Reaction; Rats; Rats, Wistar; RNA, Messenger; Transforming Growth Factor alpha

The novel 38-amino acid neuropeptide PACAP (pituitary adenylate activating peptide) has recently been shown to induce the pancreatic acinar tumour AR4-2J cell growth. This growth promoting effect of PACAP was, however, independent of adenylate cyclase activation but suppressed by pertussis toxin and the somatostatin analog SMS 201-995. This study was undertaken to search for potential cell signalling pathways involved in the growth promoting effect of PACAP on AR4-2J cells. The AR4-2J cells were grown in Dulbecco's Modified Eagle's Medium containing 10% foetal calf serum. For studies on cell signalling pathways, all experiments were carried out on cells which have reached 50 to 75% confluency. At that point, they were transferred to serum free medium overnight with or without 1 microCi/ml myristic acid. The next morning, cells were harvested, washed and used for tyrosine kinase and phospholipase D (PLD) activities. For studies on growth, cells were grown for 2 days in the presence of 1 nM PACAP +/- the different inhibitors of tyrosine kinase and PLD. PACAP-38 and -27 caused a dose-dependent and parallel activation of tyrosine kinase and PLD an effect prevented by the antagonist PACAP 7-38. PACAP-38-stimulated tyrosine kinase and PLD activation are both dose-dependently inhibited by SMS 201-995. Finally, PACAP-stimulated tyrosine kinase and PLD activities are both inhibited by cell's preincubation with genistein and pertussis toxin. After 2 days, the PACAP-induced increase in AR4-2J cell growth was significantly inhibited by increasing concentrations of genistein and wortmannin, inhibitors of tyrosine kinase, PLD and phosphatidylinositol 3-kinase, respectively. PACAP can induce concomitant activation of tyrosine kinase and PLD; this finding and the observation that inhibition of these two enzymes inhibited PACAP-induced AR4-2J cell growth strongly suggests that they are intimately involved in the overall process of PACAP-induced AR4-2J cell proliferation.

Topics: Androstadienes; Cell Division; Cell Line; Ceruletide; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Genistein; Humans; Isoflavones; Models, Biological; Neuropeptides; Neurotransmitter Agents; Pancreatic Neoplasms; Peptide Fragments; Phospholipase D; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein-Tyrosine Kinases; Signal Transduction; Tumor Cells, Cultured; Wortmannin

The effects of the cholecystokinin (CCK)-analogue, caerulein, and CCK-receptor antagonist lorglumide (CR-1409) on pancreatic carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA) were studied. One hundred thirty rats were divided into the following 10 treatment groups: group 1, DMBA (2-3 mg); group 2, DMBA + caerulein (5 micrograms/kg); group 3, DMBA + caerulein + CR-1409 (12 mg/kg); group 4, caerulein + DMBA; group 5, caerulein + CR-1409 + DMBA; group 6, DMBA + CR-1409; group 7, CR-1409 + DMBA; group 8, caerulein; group 9, CR-1409; and group 10, sham operation + saline. DMBA was surgically implanted into the pancreas. Caerulein and/or CR-1409 was administered twice daily for 15 days after (in groups 2, 3, and 6) or before (in groups 4, 5, and 7) DMBA implantation. Six months after carcinogen administration, all rats were sacrificed and autopsied. The incidence of pancreatic cancer appeared significantly (P < 0.001) increased when caerulein was administered following DMBA implantation. CR-1409 significantly inhibited (P < 0.02) caerulein effects and reduced tumor growth when injected after carcinogen exposure.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Adenocarcinoma; Animals; Body Weight; Ceruletide; Chi-Square Distribution; DNA, Neoplasm; Male; Organ Size; Pancreas; Pancreatic Neoplasms; Proglumide; Random Allocation; Rats; Receptors, Cholecystokinin

The purpose of the present study was to determine whether vasoactive intestinal polypeptide (VIP) is released from the tumor cells of VIPoma and if so then to attempt to show how its release is regulated by cultured human VIPoma cells.. A resected specimen of a pancreatic tumor from a patient with watery diarrhea, hypokalemia, and achrohydria syndrome was examined. The dissociated cells were obtained by collagenase digestion of the tumor tissue and were cultured in vitro.. The extraction of tumor cells disclosed that the cells contained VIP and pancreatic polypeptide (PP). Neither insulin, glucagon, somatostatin nor pancreastatin was detected. Immunohistochemically, 40% to 60% of the cells in the tumor stained positively for VIP and 1% to 5% stained positively for PP. The dissociated cells became reaggregated in the culture (50 to 300 microns) and could be maintained in vitro. Incubation experiments revealed a simultaneous in vitro release of VIP and PP with a significant increase by either carbachol or phorbol myristate acetate but not by theophylline or caerulein. Atropine completely abolished the stimulatory effects of carbachol on VIP and PP release. Octreotide (somatostatin analogue [SMS 201-995]) significantly inhibited the carbachol and phorbol myristate acetate-stimulated VIP and PP release.. These findings show the in vitro release of VIP and PP from the VIPoma cells and also provide evidence for the direct inhibitory effect of somatostatin analogue on both the VIP and PP release from the tumor cells.

Topics: Adult; Atropine; Carbachol; Ceruletide; Dose-Response Relationship, Drug; Female; Humans; Immunohistochemistry; Microscopy, Phase-Contrast; Octreotide; Pancreatic Neoplasms; Pancreatic Polypeptide; Tetradecanoylphorbol Acetate; Theophylline; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Vipoma

The Human pancreatic carcinoma cell line KP-1N and its clone KP-1NL which has a high rate of liver metastasis were established. Ki-ras DNA point mutation on the codon 12 was found. The growth of KP-1N was stimulated by a physiological range of concentration (10(-11)-10(-10) M) of cholecystokinin and the increase was inhibited by the addition of a cholecystokinin receptor antagonist (CR 1505). Daily injections of CR 1505 (35 mg/kg) diminished the number of tumor colonies in the liver that were formed after an intrasplenic injection of the highly liver metastatic KP-1NL cells. These results suggest that cholecystokinin antagonists may be useful as growth inhibitors for some pancreatic cancer.

Topics: Adenocarcinoma; Animals; Ceruletide; Cholecystokinin; Humans; In Vitro Techniques; Liver Neoplasms; Mice; Mice, Nude; Pancreatic Neoplasms; Proglumide; Tumor Cells, Cultured

This study was designed to analyze the effect of two pancreaticotrophic peptides on pancreatic carcinogenesis in the azaserine-rat model. The rats were treated with bombesin or caerulein for 16 weeks after initiation with azaserine. Two-week-old Lewis rats were given injections of a single dose of azaserine (30 mg/kg) and the control pups received an injection of saline. They were divided into ten groups for peptide treatment as follows: Group 1, azaserine-saline; Group 2, azaserine-bombesin, 10 micrograms/kg; Group 3, azaserine-bombesin, 30 micrograms/kg; Group 4, azaserine-caerulein, 5 micrograms/kg; Group 5, azaserine-caerulein, 15 micrograms/kg; Group 6, control-saline; Group 7, control-bombesin, 10 micrograms/kg; Group 8, control-bombesin, 30 micrograms/kg; Group 9, control-caerulein, 5 micrograms/kg; and Group 10, control-caerulein, 15 micrograms/kg. At 3 weeks of age, they were weaned. Peptides or saline were injected 3 consecutive days a week for 16 weeks. Rats were autopsied 4 months after the administration of azaserine. Pancreatic weight was increased by bombesin and decreased by caerulein treatment. Quantitative histological analysis of azaserine-induced atypical acinar cell nodules in the pancreas showed that the size and number of atypical acinar cell nodules were increased in both bombesin- and caerulein-treated groups. Thus, these peptides appear to stimulate the growth of preneoplastic acinar cell lesions.

Topics: Animals; Azaserine; Bombesin; Ceruletide; Drug Interactions; Pancreas; Pancreatic Neoplasms; Rats; Rats, Inbred Strains

The effects of cerulein on normal pancreas and on N-nitrobis (2-hydroxypropyl) amine (BHP)-induced experimental pancreatic carcinoma in Syrian golden hamsters were studied. Twenty hamsters received a subcutaneous injection of cerulein (20 micrograms/kg). The results showed that when cerulein was injected subcutaneously for 10 days, pancreatic weight and amylase increased. DNA and the pancreatic weight/DNA ratio were also increased significantly in treated hamsters compared with controls (p less than 0.02 versus p less than 0.01). These results indicated that chronic cerulein injection had hypertrophic and hyperplastic effects. DNA synthesis, as measured by histoautoradiography of tritiated thymidine-labeled tissue, increased in pancreatic acinar cells (p less than 0.01) and increased slightly in islet cells and in ductal cells. Tritiated thymidine uptake in the pancreas of the treated group indicated a rather selective exocrine gland incorporation by acinar rather than ductal cells. Sixty hamsters received a subcutaneous injection of BHP (500 mg/kg) once a week, while 63 hamsters received BHP (500 mg/kg) plus cerulein (20 micrograms/kg). Twenty-seven hamsters received cerulein (20 micrograms/kg) alone. All animals were killed from 8 to 27 weeks later, and no cancer-bearing hamsters were observed during the eighth and ninth week following administration. From the 10th to 14th weeks after administration of BHP and cerulein, 87.9% (13 of 15) had tumors compared with 18.7% (3 of 16) after BHP alone (p less than 0.01). One of three and two of 13 tumors were adenoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenocarcinoma; Amylases; Animals; Ceruletide; Cricetinae; Disease Models, Animal; DNA; Male; Mesocricetus; Nitrosamines; Organ Size; Pancreas; Pancreatic Neoplasms

The effect of glucagon on human exocrine pancreatic secretion was evaluated in ten patients by analysis of pure pancreatic juice. Pancreatic juice was obtained by endoscopic cannulation of the pancreatic duct at 2-min intervals during constant intravenous infusion of secretin (1 U per kg of body weight per hr) plus caerulein (0.04 micrograms per kg of body weight per hr). Since steady secretion was established 20 minutes after the start of juice collection, a further five 2-min fractions were collected as controls, then constant intravenous infusion of glucagon (15 micrograms per kg of body weight per hr) was commenced. Pancreatic juice was collected for a further 20 minutes. The control fractions and post-glucagon fractions were compared in each patient using Student's test. Glucagon depressed secretin-caerulein-stimulated pancreatic secretions. More uniform reductions were observed in the concentration and output of protein and enzymes. Individual variations were observed in the secretory volume and bicarbonate concentration and output. Amylase and lipase were depressed in a parallel fashion in seven patients and in the remaining three, amylase was more depressed than lipase. The post-glucagon reduction in pancreatic secretion was not proportional to the rise in plasma glucagon and blood glucose.

Topics: Adenocarcinoma; Adult; Aged; Ceruletide; Chronic Disease; Depression, Chemical; Female; Glucagon; Humans; Male; Middle Aged; Pancreas; Pancreatic Juice; Pancreatic Neoplasms; Pancreatitis; Secretin; Secretory Rate; Time Factors

The author investigated the hyperplastic and hypertrophic effect of caerulein in the pancreas of normal syrian golden hamsters, and the promoting effect of experimental pancreatic cancer in hamster induced by N-nitroso-bis (2-hydroxypropyl) amine (BHP). The results are as follow: Repeated subcutaneous injections of caerulein in every 12 hours for 10 days elicited a marked trophic effect on the pancreas, characterized by increased pancreatic weight and pancreatic weight/DNA ratios with an enhanced content of DNA and amylase in the pancreas in treated hamsters. DNA synthesis, as measured by histoautoradiography of tritiated thymidine labeled tissues, was increased in pancreatic acinar but little in islet nor in ductal cells. Weekly subcutaneous administration of BHP with caerulein brought pancreatic carcinomas earlier and in higher incidence than BHP alone. The majority of induced carcinomas were well differentiated adenocarcinomas, and acinar cell carcinoma was seen in neither groups. In addition, a further investigation was performed in search target cells of both BHP and caerulein. Repeated injections of caerulein in every 12 hours for 5 days before one shot of subcutaneous BHP administration led to increase both mitotic and labeling index using tritiated thymidine in most acinar cells. These results suggested that caerulein has a trophic action on the pancreas, and acts as a promotor in experimental pancreatic carcinoma.

Topics: Animals; Ceruletide; Cricetinae; DNA, Neoplasm; Male; Mesocricetus; Mitosis; Nitrosamines; Pancreas; Pancreatic Neoplasms

The influence of the cholecystokinin analogue cerulein on induced pancreatic cancer in the Syrian golden hamster was investigated. Of hamsters given weekly subcutaneous injection of N-nitrosobis(2-hydroxypropyl)amine (BHP) in initial experiments 50% succumbed within 30 weeks when a dose of 125 mg BHP per kg body weight was used and within 25 weeks after the double dose. An induction time of at most 24 weeks was therefore used in the subsequent experiments. Administration of cerulein (2 micrograms twice daily for 5 days a week) for 18 or 22 weeks caused an increase of pancreatic wet weight by about 100% and of pancreatic protein content by 73% (18 weeks). BHP did not influence the pancreatic weight either in hamsters given cerulein or in those given saline injections. BHP (125 mg/kg) caused tumors in 44% of the animals after 18 weeks and in 73% after 22 weeks. When BHP was given in a dose of 250 mg/kg, 100% of the animals had pancreatic tumors after 22 weeks. At neither dose and neither time interval did cerulein influence the number of tumor-bearing animals, number of cancer-bearing animals, or number of tumors per tumor-bearing animal or cancers per cancer-bearing animal. No morphological differences were found within the lesions of animals given only BHP as compared with those given cerulein in addition. All lesions were of ductal appearance. The distribution of tumors was also similar irrespective of the treatment given. The results show that cerulein does not influence experimental pancreatic carcinogenesis in the Syrian golden hamster, possibly reflecting that cerulein and BHP primarily act on different target cells.

Topics: Animals; Ceruletide; Cricetinae; Mesocricetus; Neoplasms, Experimental; Nitrosamines; Organ Size; Pancreas; Pancreatic Neoplasms

We have compared responsiveness of serum lipase and amylase activity to the pancreatic exocrine stimulation with cerulein and secretin (CS test) in normal subjects and patients with pancreas-related and other diseases. The lipase and amylase activities were measured by a sensitive colorimetric method, the BALB-DTNB method and the Caraway method, respectively. The percentage of positive lipase and amylase response cases was as follows: confirmed chronic pancreatitis (N = 22), 27 and 14%; suspected chronic pancreatitis (N = 37), 46 and 32%; pancreatic cancer (N = 16), 44 and 25%; biliary tract diseases (N = 11), 14 and 14%; miscellaneous (N = 11), 0 and 18%; normal subjects (N = 13), and partial pancreatectomy (N = 5), 0 and 0%, respectively. The serum lipase response cannot be regarded as specific for pancreatic diseases because the lipase response cases were found in biliary tract diseases as well. However, in view of frequent, fast, and intense responsiveness to the CS test, the serum lipase activity measured by the BALB-DTNB method may be more useful than the serum amylase as an auxiliary diagnostic aid for suspected pancreatitis which might develop into confirmed chronic pancreatitis or cancer of the head or body of the pancreas.

Topics: Adolescent; Adult; Aged; alpha-Amylases; Biliary Tract Diseases; Ceruletide; Clinical Enzyme Tests; Colorimetry; Female; Humans; Lipase; Male; Middle Aged; Pancreas; Pancreatic Diseases; Pancreatic Function Tests; Pancreatic Neoplasms; Secretin

Topics: Animals; Cell Differentiation; Ceruletide; Cholecystokinin; Humans; Ornithine Decarboxylase; Pancreas; Pancreatic Neoplasms; Rats

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; Adenocarcinoma; Animals; Ceruletide; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Humans; Mice; Pancreatic Neoplasms; Receptors, Cell Surface; Receptors, Cholecystokinin; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Secretin; Theophylline

Comparative studies between activity of serumlipase, lipaseevocationstest and endoscopic retrograde cholangiopancreaticography (ERCP) in 45 patients with chronic pancreatitis, pancreatic cysts or pancreatic carcinoma confirm, that elevated enzyme values after caerulein stimulation indicate an alteration of the pancreatic parenchyma. However, this findings does not give information on the etiology of the damage. On the basis of the presented results it seems that the lipase-evocationstest has only limited value as screening method within the total of clinical methods for the assessment of light and medium severe stages of chronic pancreatitis and accompanying forms of pancreatitis. There is no evidence that this method may be used for screening for carcinoma of the pancreas. A positive result gives a valuable information, but the lack of an increase of lipase activity does not exclude the existence of pancreatic affection. In each case with clinical suspicion for pancreatic disease further diagnostic investigation by the ERCP and additional radiographic methods should be performed.

Topics: Adult; Aged; Amylases; Ceruletide; Cholangiography; Chronic Disease; Clinical Enzyme Tests; Endoscopy; Humans; Lipase; Male; Middle Aged; Pancreatic Cyst; Pancreatic Diseases; Pancreatic Neoplasms; Pancreatitis

Angiography of the pancreas was performed in 55 patients after the administration of different drugs stimulating the blood flow in the pancreas and the pancreatographic effect in this group was compared with that in a series of 174 celiac angiographies without drugs. With drugs the pancreatographic effect appeared more frequently (74.5 per cent) than without (18.4 per cent). The small pancreatic arteries and veins were better demonstrated with drugs. The pancreatographic effect seems to be of value for the differential diagnosis of chronic pancreatitis and carcinoma of the pancreas.

Topics: Adult; Angiography; Ceruletide; Chronic Disease; Diagnosis, Differential; Female; Humans; Male; Middle Aged; Pancreatic Neoplasms; Pancreatitis; Secretin; Serotonin; Tolazoline

Topics: Adenoma; Blood Glucose; Ceruletide; Cholecystokinin; Gastrointestinal Hormones; Humans; Insulin; Insulin Secretion; Islets of Langerhans; Kinetics; Pancreatic Neoplasms; Peptides