pancreastatin and Carcinoma--Hepatocellular

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.

Topics: Aminoquinolines; Animals; Arginine; Calcium; Carbazoles; Carcinoma, Hepatocellular; Cell Enlargement; Cell Proliferation; Chromogranin A; Chromogranins; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; DNA; Enzyme Inhibitors; Guanylate Cyclase; Indoles; Isoenzymes; Leucine; Liver; MAP Kinase Signaling System; Nerve Tissue Proteins; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; omega-N-Methylarginine; Ornithine; Pancreatic Hormones; Peptides; Phospholipase C beta; Protein Isoforms; Rats; Receptors, Atrial Natriuretic Factor; Spermidine; Thymidine; Time Factors; Type C Phospholipases

Pancreastatin, a chromogranin A-derived peptide, has a counter-regulatory effect on insulin action. We have previously characterized pancreastatin receptor and signalling in rat liver and HTC hepatoma cells. A G alpha(q/11)-PLC-beta pathway leads to an increase in [Ca2+]i, PKC and mitogen activated protein kinase (MAPK) activation. These data suggested that pancreastatin might have a role in growth and proliferation, similar to other calcium-mobilizing hormones.. DNA and protein synthesis were measured as [3H]-thymidine and [3H]-leucine incorporation. Nitric oxide (NO) was determined by the Griess method and cGMP production was quantified by enzyme-linked immunoassay.. Contrary to the expected results, we have found that pancreastatin inhibits protein and DNA synthesis in HTC hepatoma cells. On the other hand, when the activity of NO synthase was inhibited by N-monomethyl-L-arginine (NMLA), the inhibitory effect of pancreastatin on DNA and protein synthesis was not only reverted, but a dose-dependent stimulatory effect was observed, probably due to MAPK activation, since it was prevented by PD98059. These data strongly suggested the role of NO in the inhibitory effect of pancreastatin on protein and DNA synthesis, which is overcoming the effect on MAPK activation. Moreover, pancreastatin dose-dependently increased NO production in parallel to cyclic guanosine monophosphate (cGMP). Both effects were prevented by NMLA. Finally, an indirect effect of pancreastatin through the induction of apoptosis was ruled out.. Therefore, the NO and the cGMP produced by the NO-activated guanylate cyclase may mediate the dose-dependent inhibitory effect of pancreastatin on growth and proliferation in HTC hepatoma cells.

Topics: Animals; Carcinoma, Hepatocellular; Cell Division; Chromogranin A; Chromogranins; Cyclic GMP; DNA; Dose-Response Relationship, Drug; Enzyme Inhibitors; Liver Neoplasms; Nitric Oxide; omega-N-Methylarginine; Pancreatic Hormones; Protein Synthesis Inhibitors; Rats; Tumor Cells, Cultured

Pancreastatin, a chromogranin A-derived peptide widely distributed throughout the neuroendocrine system, has a general inhibitory effect on endocrine secretion and a counterregulatory effect on insulin action. We have recently described the cross-talk of pancreastatin with insulin signaling in rat hepatoma cells (HTC), where it inhibits insulin action and signaling through the serine phosphorylation of the insulin receptor, thereby impairing tyrosine kinase activity. Here, we have characterized pancreastatin receptors and signaling in HTC cells. The pancreastatin effector systems were studied by determining phospholipase C activity in HTC membranes and mitogen-activated protein kinase (MAPK) phosphorylation activity in HTC cells. Binding studies with radiolabeled pancreastatin showed a population of high affinity binding sites, with a B(max) of 8 fmol/mg protein and a K(d) of 0.6 nM. Moreover, we assessed the coupling of the receptor with a G protein system by inhibiting the binding with guanine nucleotide and by measuring the GTP binding to HTC membranes. We found that pancreastatin receptor was coupled with a G alpha(q/11) protein which activates phospholipase C-beta(1) and phospholipase C-beta(3), in addition to MAPK via both beta gamma and alpha(q/11).

Topics: Animals; Binding, Competitive; Carcinoma, Hepatocellular; Cell Membrane; Chromogranin A; Dose-Response Relationship, Drug; Enzyme Activation; GTP-Binding Proteins; MAP Kinase Signaling System; Pancreatic Hormones; Protein Binding; Rats; Receptors, Gastrointestinal Hormone; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases