cyclic-gmp and Pancreatic-Neoplasms

Therapies using T cells that are programmed to express chimeric antigen receptors (CAR T cells) consistently produce positive results in patients with hematologic malignancies. However, CAR T cell treatments are less effective in solid tumors for several reasons. First, lymphocytes do not efficiently target CAR T cells; second, solid tumors create an immunosuppressive microenvironment that inactivates T cell responses; and third, solid cancers are typified by phenotypic diversity and thus include cells that do not express proteins targeted by the engineered receptors, enabling the formation of escape variants that elude CAR T cell targeting. Here, we have tested implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors, thereby exposing them to high concentrations of immune cells for a substantial time period. In immunocompetent orthotopic mouse models of pancreatic cancer and melanoma, we found that CAR T cells can migrate from biopolymer scaffolds and eradicate tumors more effectively than does systemic delivery of the same cells. We have also demonstrated that codelivery of stimulator of IFN genes (STING) agonists stimulates immune responses to eliminate tumor cells that are not recognized by the adoptively transferred lymphocytes. Thus, these devices may improve the effectiveness of CAR T cell therapy in solid tumors and help protect against the emergence of escape variants.

Topics: Adoptive Transfer; Animals; Antigen-Presenting Cells; Antineoplastic Agents; Biopolymers; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cyclic GMP; Drug Carriers; Female; Implants, Experimental; Melanoma, Experimental; Membrane Proteins; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Transplantation; Pancreatic Neoplasms; T-Lymphocytes

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in the world. Therefore, new therapeutic options are urgently needed to improve the survival of PDAC patients. Protein kinase G (PKG) conducts the interlude of cGMP signaling which is important for healthy as well as for cancer cells. DT3 is a specific inhibitor of PKG, and it has been shown to possess an anti-tumor cytotoxic activity in vitro. The main aim of this work was to investigate anti-tumor effects of DT3 upon PDAC in vivo.Expression of PKG was assessed with real-time PCR analysis in the normal and tumor pancreatic cells. In vitro cell viability, proliferation, apoptosis, necrosis, migration, and invasion of the murine PDAC cell line Panc02 were assessed after DT3 treatment. In vivo anti-tumor effects of DT3 were investigated in the murine Panc02 orthotopic model of PDAC. Western blot analysis was used to determine the phosphorylation state of the proteins of interest.Functional PKGI is preferentially expressed in PDAC cells. DT3 was capable to reduce viability, proliferation, and migration of murine PDAC cells in vitro. At the same time, DT3 treatment did not change the viability of normal epithelial cells of murine liver. In vivo, DT3 treatment reduced the tumor volume and metastases in PDAC-bearing mice, but it was ineffective to prolong the survival of the tumor-bearing animals. In addition, DT3 treatment decreased phosphorylation of GSK-3, P38, and CREB in murine PDAC.Inhibition of PKG could be a potential therapeutic strategy for PDAC treatment which should be carefully validated in future pre-clinical studies.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclic AMP Response Element-Binding Protein; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Epithelial Cells; Glycogen Synthase Kinase 3; Liver; Mice; Mice, Inbred C57BL; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Peptides; Protein Kinase Inhibitors; Tumor Burden

cAMP and cGMP signaling is important both for normal and cancer cells. This signaling is controlled by adenylyl and guanylyl cyclases and cyclic nucleotide phosphodiesterases. One of the direct targets for cGMP is protein kinase G (PKG). The main aim of this work was to investigate cGMP and PKG signaling in pancreatic adenocarcinoma (PDAC) cells.. The PKG activity, cGMP, and calcium level were measured with the CycLex Cyclic GMP dependent protein kinase (cGK) Assay Kit, the DetectX Cyclic GMP Colorimetric EIA Kit, and the Fluo-4 NW Calcium Assay Kit, respectively. The Proteome Profiler Array was done using Human Phospho-Kinase Array and Human Phospho-MAPK Array Kits.. This study shows for the first time that functional PKG1 is expressed in PDAC cells. It demonstrates that the specific PKG1 inhibitor, DT3, induces cytotoxicity through necrosis and reduces proliferation and migration of PDAC cells. Moreover, ERK1/2 and p38 can be considered as potential targets for PKG1 in PDAC cells. In addition, the study shows that phosphodiesterases and nitric oxide-guanylyl cyclases regulate the cGMP level in PDAC cells, affecting the proliferation of the cells.. The cGMP and PKG signaling may be a target for developing new therapeutic approaches for PDAC.

Topics: Adenocarcinoma; Apoptosis; Blotting, Western; Calcium; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Gene Expression Regulation, Neoplastic; Guanylate Cyclase; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitric Oxide; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Peptides; Phosphoric Diester Hydrolases; Phosphorylation; Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction

Long-acting natriuretic peptide, vessel dilator, kaliuretic peptide and atrial natriuretic peptide are four peptide hormones synthesized by the same gene. Their main known biologic properties are sodium and water excretion and blood pressure lowering in both animals and humans.. These four peptide hormones, each at their 1-microm concentrations, were evaluated for their ability to decrease the number and/or proliferation of human pancreatic adenocarcinoma cells in culture at 24, 48, 72 and 96 h.. Vessel dilator, long-acting natriuretic peptide, kaliuretic peptide and atrial natriuretic peptide decreased the number of human pancreatic adenocarcinoma cells in culture by 65% (P<0.001), 47% (P<0.01), 37% (P<0.05) and 34% (P<0.05), respectively, within 24 h. This decrease was sustained without any proliferation of the cancer cells occurring in the 3 days following this decrease in number. The mechanism of these peptide hormones' decrease in cancer cell number and antiproliferative effects was a 83% (P<0.001) or greater inhibition of DNA synthesis but not owing to enhanced apoptosis, i.e. programmed cell death. The two known mediators of these peptide hormones' mechanism(s) of action, i.e. cyclic GMP and prostaglandin E2, inhibited DNA synthesis in these adenocarcinoma cells by 51% and 23%, respectively.. Four peptide hormones significantly decrease the number of pancreatic adenocarcinoma cells within 24 h and inhibit the proliferation of these cells for at least 96 h. Their mechanism of doing so is via inhibition of DNA synthesis mediated in part by cyclic GMP.

Topics: Adenocarcinoma; Apoptosis; Atrial Natriuretic Factor; Cell Division; Cyclic GMP; Dinoprostone; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Pancreatic Neoplasms; Peptide Fragments; Protein Precursors; Tumor Cells, Cultured

The effect of nitric oxide (NO) donors on high-voltage-activated Ca2+ channels in insulin-secreting RINm5F cells was investigated using the patch-clamp technique in the whole-cell configuration. Sodium nitroprusside (SNP, 2-400 microM) induced a dose-dependent reduction in Ba2+ currents with maximal inhibition of 58%. The IC50 for SNP was 45 microM. A different NO donor, (+/-)S-nitroso-N-acetylpenicillamine (SNAP, 500 microM), also produced a 50% decrease in current amplitude. When 200 microM SNP was administered together with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidozoline-1-oxyl-3-oxide (carboxy-PTIO, 300 microM), the Ba2+ current inhibition was lowered to 7%. Administration of 500 microM 8-bromoguanosine 3':5'-cyclic monophosphate sodium salt (8-Br-cGMP) mimicked the effects of SNP, causing a comparable decrease (56%) in peak-current amplitude. When soluble guanylyl cyclase was blocked by 10 microM 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), the inhibitory effect of 200 microM SNP was reduced from 39% to 15%. The SNP-induced current decrease was 36% of controls after the blockade of L-type Ca2+ channels and 30% in the presence of 2.5 microM omega-conotoxin-MVIIC. These data indicate that NO inhibits both L-type and P/Q-type Ca2+ channels in RINm5F cells, probably by an increase in the intracellular levels of cGMP. NO may then significantly influence the Ca2+-dependent release of hormones from secretory cells as well as that of neurotransmitters from nerve terminals.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Cyclic GMP; Electrophysiology; Enzyme Inhibitors; Insulinoma; Membrane Potentials; Nitric Oxide; Pancreatic Neoplasms; Patch-Clamp Techniques; Penicillamine; Rats; S-Nitroso-N-Acetylpenicillamine; Sodium-Potassium-Exchanging ATPase; Tumor Cells, Cultured

Gastroenteropancreatic neuroendocrine cells secrete chemical messengers in a calcium-dependent fashion. So far, other second messenger systems involved in regulated secretion have gained little attention. The aim of this study was to characterize guanosine 3',5'-cyclic monophosphate (cGMP)-mediated vesicular secretion in pancreatic neuroendocrine cells.. In a human pancreatic cell line, BON, cyclic nucleotide levels and chromogranin A release were monitored with specific immunoassays. Uptake and release of gamma-aminobutyric acid were measured. Intracellular Ca2+ concentration was monitored with fura-2. Guanylyl cyclase C was analyzed by reverse-transcription polymerase chain reaction.. Guanylin increased cGMP concentrations in BON cells via guanylyl cyclase C. Stimulation of the cGMP pathway by guanylin or Escherichia coli heat-stable enterotoxin increased the release of chromogranin A and gamma-aminobutyric acid from BON cells. This effect was mimicked by the cGMP analogue 8-bromo-cGMP.. Guanylin and STa stimulate the regulated secretion from BON cells via guanylyl cyclase C and cGMP. Our study yields novel information about secretory properties of guanylin, mediated via a signal transduction pathway, increasing cGMP and leading to regulated secretion of neuroendocrine cells.

Topics: Bacterial Toxins; Calcium; Chromogranin A; Chromogranins; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; gamma-Aminobutyric Acid; Gastrointestinal Hormones; Humans; Natriuretic Peptides; Neurosecretory Systems; Pancreatic Neoplasms; Peptides; RNA, Messenger; Tumor Cells, Cultured

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; Guanylate Cyclase; In Vitro Techniques; Insulinoma; Islets of Langerhans; Molecular Sequence Data; Nitroprusside; Pancreatic Neoplasms; Point Mutation; Rats; RNA, Messenger; Sequence Deletion; Signal Transduction; Transfection; Tumor Cells, Cultured

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cyclic GMP; Diabetes Mellitus, Type 1; Free Radicals; Humans; Insulinoma; Interferon-gamma; Interleukin-1; Islets of Langerhans; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Pancreatic Neoplasms; Rats; Tumor Cells, Cultured

5-hydroxytryptamine (5-HT) is a mitogen for fibroblasts, vascular smooth muscle cells, renal mesangial cells, and jejunal crypt cells. The human carcinoid cell line (termed BON) that we established in our laboratory from a pancreatic carcinoid tumor produces and secretes 5-HT. In this study, therefore, we examined the effect of 5-HT on growth of BON cells. Furthermore, by use of selective 5-HT receptor antagonists, we examined receptor and post-receptor mechanisms by which 5-HT-induced responses were produced. 5-HT stimulated growth of BON cells. 5-HT stimulated phosphatidylinositol (PI) hydrolysis in a dose-dependent fashion and inhibited cyclic AMP production in a dose-dependent fashion. The 5-HT1A/1B receptor antagonist, SDZ 21-009, prevented the reduction of cyclic AMP production evoked by 5-HT and inhibited the mitogenic action of 5-HT. The 5-HT1C/2 receptor antagonist, mesulergine, competitively inhibited PI hydrolysis, but did not affect the mitogenic action of 5-HT. The mitogenic action of 5-HT and the reduction of cyclic AMP production evoked by 5-HT were also inhibited by pertussis toxin. These results suggest that 5-HT is an autocrine growth factor for BON cells and that mitogenic mechanism of 5-HT involves receptor-mediated inhibition of the production of cyclic AMP which may be linked to pertussis toxin-sensitive GTP binding protein. 8-bromo-cyclic AMP inhibited growth of BON cells whereas 8-bromo-cyclic GMP had no effect on cell growth. Involvement of protein kinase A in BON cell growth regulation was confirmed by the observation that a cAMP-dependent protein kinase antagonist (Rp-cAMPS) could stimulate BON cell growth.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Cell Division; Cyclic AMP; Cyclic GMP; Ergolines; Humans; Pancreatic Neoplasms; Pertussis Toxin; Phosphatidylinositols; Pindolol; Receptors, Serotonin; Second Messenger Systems; Serotonin; Serotonin Antagonists; Tumor Cells, Cultured; Virulence Factors, Bordetella

L-Arginine (L-Arg) is metabolized by nitric oxide synthase to the reactive intermediate nitric oxide. Since nitric oxide stimulates guanylyl cyclase and cGMP synthesis, L-Arg effects on cGMP accumulation in isolated pancreatic islets of the rat and RINm5F insulinoma cells were determined. Both L-Arg and glucose stimulation increased islet cGMP levels, and glucose potentiated the response to L-Arg alone. A competitive inhibitor of L-Arg metabolism to nitric oxide, NG-monomethyl-L-arginine, reduced glucose- and L-Arg-stimulated insulin release and glucose-induced increases in cGMP; however, basal insulin release was slightly increased. D-Arg and L-ornithine did not affect islet cGMP levels, although insulin release was stimulated. RINm5F cell cGMP levels and insulin release increased in response to L-Arg in a concentration- and time-related manner, whereas glucose and L-histidine were without effect. 8-Bromo-cGMP also slightly increased RINm5F cell insulin release. Sodium nitroprusside as a source of nitric oxide increased RINm5F cell cGMP production. Methylene blue and LY83583, inhibitors of soluble guanylyl cyclase activation, reduced RINm5F cell cGMP levels in the presence and absence of L-Arg; LY83583 also reduced glucose-stimulated cGMP levels in islets. Insulin release by glucose and L-Arg was also inhibited by methylene blue and LY83583 in islets. We conclude that glucose and L-Arg stimulate guanylyl cyclase activity and cGMP formation in beta-cells at least in part through metabolism to the reactive intermediate nitric oxide. However, neither nitric oxide nor cGMP synthesis is obligatory for insulin secretion.

Topics: Aminoquinolines; Animals; Arginine; Cyclic GMP; Glucose; Guanylate Cyclase; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Male; Methylene Blue; Nitroprusside; Pancreatic Neoplasms; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

Topics: Adenoma, Islet Cell; Animals; Atrial Natriuretic Factor; Cyclic GMP; Insulin; Insulin Secretion; Insulinoma; Pancreatic Neoplasms; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Tumor Cells, Cultured

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

Pancreas cancer was induced in noninbred male Holtzman rats by the implantation of beeswax containing 7.12-dimethylbenz[a]anthracene (DMBA) into the "head" of the pancreas. The tumors that developed 4--6 months later were examined for their cyclic AMP and cyclic GMP levels. The lesions could be considered in one of two categories according to their cyclic nucleotide contents: lesions with significantly smaller amounts and those with greater amounts, compared with levels measured in the pancreas tissues of the control rats. The existence of two biochemically distinct groups may indicate different growth patterns of the DMBA-induced pancreatic neoplasia.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Benz(a)Anthracenes; Cyclic AMP; Cyclic GMP; Male; Neoplasms, Experimental; Pancreas; Pancreatic Neoplasms; Rats