h-89 and Pancreatic-Neoplasms

Cyclooxygenase-2 (COX-2) is constitutively expressed in most human primary carcinomas and with its synthesized product, prostaglandin E2 (PGE2), appears to play important roles in tumor invasion, angiogenesis, resistance to apoptosis and suppression of host immunity. However, the molecular mechanisms that control COX-2 expression are unclear. The purpose of this study was to clarify the mechanism of basal and PGE2-mediated COX-2 expression in the highly metastatic L3.6pl human pancreatic cancer cell line. Using RNA interference to disrupt the expression of CREB and the NF-kappaB p65 subunit, we found that both are involved in maintaining basal COX-2 expression in L3.6pl cells. We also demonstrated that PGE2 increased the cyclic AMP concentration, thereby activating protein kinase A (PKA), which in turn phosphorylated the cyclic AMP response element binding protein (CREB), leading to interaction with the cyclic AMP response element in the promoter region of the COX-2 gene. Immunocytochemical analysis confirmed that PGE2 stimulated the translocation of PKA to the nucleus and increased the immuno-reactivity of phosphorylated CREB. Pretreatment with the PKA selective inhibitor H 89 and the E-prostanoid receptor 2 inhibitor AH 6809 reduced COX-2 upregulation by PGE2. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay results further suggested a role for CREB in COX-2 transcriptional control. Understanding the pathways that control COX-2 expression may lead to a better understanding of its dysregulation in pancreatic carcinomas and facilitate the development of novel therapeutic approaches.

Topics: Cell Line, Tumor; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclooxygenase 2; Dinoprostone; Enzyme Activation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Isoquinolines; Models, Biological; Pancreatic Neoplasms; Phosphorylation; Prostaglandin Antagonists; Sulfonamides; Xanthones

Pancreatic cancer is the most lethal abdominal malignancy. Expression of the RIalpha subunit of protein kinase A (PKA) has been associated with neoplastic transformation and mitogenic signaling. The effect of PKA inhibition on pancreatic cancer cell growth and apoptosis is unknown. In pancreatic cancer cells, we sought to determine (1) whether inhibition of PKA can inhibit growth or induce apoptosis, and (2) whether growth can be inhibited by silencing of RIalpha expression.. Human pancreatic cancer cells (PANC-1, MIA PaCa-2, and SUIT-2) were treated with inhibitors of PKA (H89 or PKI) and cell growth, kinase activity, and induction of apoptosis measured. Small inhibitory RNA (siRNA) directed against the RIalpha subunit was synthesized and transfected into PANC-1 cells.. H89 decreased PKA activity and inhibited pancreatic cancer cell growth. Apoptosis was also induced by H89 in PANC-1 and MIA PaCa-2 cells. PANC-1 cells express high levels of the RIalpha subunit; transfection of siRNA decreased RIalpha protein expression and inhibited growth.. Inhibition of PKA in pancreatic cancer cells induces growth arrest and apoptosis; similar effects are noted in cells with siRNA used to block RIalpha expression. Inhibition of PKA may represent a novel therapeutic strategy for the adjuvant treatment of pancreatic cancer.

Topics: Apoptosis; Base Sequence; bcl-2-Associated X Protein; Carrier Proteins; Cell Division; Cell Survival; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Humans; Isoquinolines; Molecular Sequence Data; Pancreatic Neoplasms; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Sulfonamides; Tumor Cells, Cultured

Molecular mechanisms for the regulation of islet amyloid polypeptide (IAPP) gene expression remain unclear. In the present study, we investigated the effects of glucose and forskolin on IAPP gene regulation in the INS-1 islet beta-cell line. Both glucose and forskolin increased the level of expression of this gene, as measured by Northern blot analysis, and increased IAPP gene transcription in a time- and concentration-dependent manner, as demonstrated in a reporter gene assay. Although inhibition of protein kinase A activity with H-89 eliminated the effect of forskolin on this gene, the glucose effect was unaffected. This supported the predominant use of a protein kinase A-independent signaling pathway for glucose regulation of the IAPP gene. Electrophoretic mobility shift assay further indicated that glucose and forskolin regulated expression of this gene by targeting different elements of the promoter. Mutation of the cAMP regulatory element flanking the IAPP coding region resulted in the loss of most of the forskolin-stimulated IAPP gene promoter activity, whereas glucose-enhanced IAPP gene transcription was unaffected. These results demonstrate parallel and distinct regulatory pathways involved in glucose- and forskolin-induced IAPP gene expression in this model beta-cell system.

Topics: Adenylyl Cyclase Inhibitors; Amyloid; Blotting, Northern; Carcinoma, Pancreatic Ductal; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Glucose; Humans; Islet Amyloid Polypeptide; Isoquinolines; Pancreatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

An understanding of the role of CaM kinase II in the pancreatic beta-cell is dependent on the identification of its cellular targets. One of the best substrates of CaM kinase II in vitro that could function in secretory events is the microtubule-associated protein, MAP-2. By immunoblot analysis, a high molecular weight protein with electrophoretic properties characteristic of MAP-2, was identified in rat insulinoma betaTC3 cells and isolated rat islets. In immunoprecipitation experiments employing alpha-toxin-permeabilized betaTC3 cells, elevation of intracellular Ca2+ or addition of forskolin, an adenylate cyclase activator, induced significant phosphorylation of MAP-2 in situ. The effect of Ca2+ was rapid, concentration-dependent and closely correlated with activation of CaM kinase II under similar experimental conditions. H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (PKA), prevented forskolin-induced MAP-2 phosphorylation but had little effect on MAP-2 phosphorylation stimulated by elevated Ca2+. Phosphopeptide mapping revealed that the phosphorylation pattern observed in situ upon incubation of the betaTC3 cells with increased free Ca2+, was strikingly similar to that generated in vitro by CaM kinase II, most notably with regard to the increased phosphate incorporated into one prominent site. These data provide evidence that MAP-2 is phosphorylated by CaM kinase II in the pancreatic beta-cell in situ, and that this event may provide an important link in the mediation of Ca2+-dependent insulin secretion.

Topics: Adenylyl Cyclases; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Insulinoma; Islets of Langerhans; Isoquinolines; Kinetics; Microtubule-Associated Proteins; Pancreatic Neoplasms; Peptide Mapping; Phosphopeptides; Phosphorylation; Rats; Sulfonamides; Tumor Cells, Cultured