okadaic-acid and Pancreatic-Neoplasms

Serine/threonine protein phosphatase 2A (PP2A), is thought to be a cancer suppresser, as inhibition of PP2A can induce phosphorylation and activation of substrate kinases, most of which can accelerate growth. Interestingly, cantharidin potently inhibits PP2A but efficiently represses various cancer cells. In the present study, we found that PP2A inhibitors, cantharidin or Okadaic acid, inhibited cell viability and triggered apoptosis in PANC-1 pancreatic cancer cell line dependent on PP2A/IKKα/IκBα/p65 NF-κB pathway. The activation of NF-κB pathway up-regulated downstream pro-apoptotic genes, TNF-α, TRAILR1 and TRAILR2, and triggered apoptosis through the extrinsic pathway, indicating that PP2A is a potential target for pancreatic cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cantharidin; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Humans; I-kappa B Kinase; Mutagenesis, Site-Directed; NF-kappa B; Okadaic Acid; Pancreatic Neoplasms; Phosphorylation; Protein Phosphatase 2; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase that can dephosphorylate multiple kinases. It is generally considered to be a cancer suppressor as its inhibition can induce phosphorylation and activation of substrate kinases that mainly accelerate growth. We previously reported that cantharidin, an active constituent of a traditional Chinese medicine, potently and selectively inhibited PP2A, yet efficiently repressed the growth of pancreatic cancer cells through activation of the c-Jun N-terminal kinase (JNK) pathway. This suggested that activation of kinase pathways might also be a potential strategy for cancer therapy. In this study, we have confirmed that the basal activity of the phospatidylinositol 3-kinase (PI3K)/JNK/activator protein 1 (AP-1) pathway promoted pancreatic cancer cell growth when stimulated by growth factors. Interestingly, although treatment with the PP2A inhibitors, cantharidin or okadaic acid (OA), amplified the PI3K-dependent activation of JNK, cell growth was repressed. We therefore hypothesised that a specific level of activity of the JNK pathway might be required to maintain the promitogenic function, as both repression and overactivation of JNK could inhibit cell proliferation. It was found that the JNK-dependent growth inhibition was independent of the activation of AP-1, but dependent on the repression of Akt. Although the PP2A inhibitors triggered overactivation of JNK and inhibited cell growth, excessively activated protein kinase C (PKC) improved cell survival. Combined treatment with a PP2A inhibitor and a PKC inhibitor produced a synergistic effect, which indicates a potentially promising therapeutic approach to pancreatic cancer treatment.

Topics: Cantharidin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Enzyme Inhibitors; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Luciferases; Nerve Tissue Proteins; Nuclear Proteins; Okadaic Acid; Pancreatic Neoplasms; Polymerase Chain Reaction; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins

The sphingolipid ceramide (CER) and its metabolites have been recognized as important mediators of signal transduction processes leading to a variety of cellular responses, including survival and demise via apoptosis. Accumulating evidence implicates key regulatory roles for intracellularly generated CER in metabolic dysfunction of the islet beta cell. We have previously reported localization of an okadaic (OKA)-sensitive CER-activated protein phosphatase (CAPP) in the islet beta cell. We have also reported immunological identification of the structural A subunit, the regulatory B56alpha subunit, and the catalytic C subunit for CAPP holoenzyme complex in insulin-secreting INS-1 cells. Herein, we provide the first evidence to suggest that siRNA-mediated knockdown of the alpha isoform of the catalytic subunit of PP2Ac (PP2Acalpha) markedly reduces the CAPP activity in INS 832/13 cells. Potential significance of the functional activation of CAPP holoenzyme in the context of lipid-and glucose-induced metabolic dysfunction of the islet beta cell is discussed.

Topics: Animals; Cell Line, Tumor; Ceramides; Insulinoma; Okadaic Acid; Pancreatic Neoplasms; Phosphoprotein Phosphatases; Protein Phosphatase 2; Rats; RNA, Small Interfering

In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion is an early pathogenetic event. Glucose is the cardinal physiological stimulator of insulin secretion from the pancreatic beta-cell, but the mechanisms involved in glucose sensing are not fully understood. Specific ser/thr protein phosphatase (PPase) inactivation by okadaic acid promotes Ca(2+) entry and insulin exocytosis in the beta-cell. We now show that glycolytic and Krebs cycle intermediates, whose concentrations increase upon glucose stimulation, not only dose dependently inhibit ser/thr PPase enzymatic activities, but also directly promote insulin exocytosis from permeabilized beta-cells. Thus, fructose-1,6-bisphosphate, phosphoenolpyruvate, 3-phosphoglycerate, citrate, and oxaloacetate inhibit PPases and significantly enhance insulin exocytosis, nonadditive to that of okadaic acid, at micromolar Ca2+ concentrations. In contrast, the effect of GTP is potentiated by okadaic acid, suggesting that the action of GTP does not require PPase inactivation. We conclude that specific glucose metabolites and GTP inhibit beta-cell PPase activities and directly stimulate Ca2+-independent insulin exocytosis. The glucose metabolites, but not GTP, seem to require PPase inactivation for their stimulatory effect on exocytosis. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by metabolic intermediates, may be a novel regulatory mechanism linking glucose sensing to insulin exocytosis in the beta-cell.

Topics: Animals; Calcium; Citric Acid; Drug Synergism; Enzyme Inhibitors; Exocytosis; Fructosediphosphates; Glucose; Glyceric Acids; Guanosine Triphosphate; Insulin; Insulinoma; Islets of Langerhans; Okadaic Acid; Oxaloacetic Acid; Pancreatic Neoplasms; Phosphoenolpyruvate; Phosphoprotein Phosphatases; Phosphorylation; Rats; Tumor Cells, Cultured

Reversible protein phosphorylation is an important mechanism by which cells transduce external signals into biologic responses. Levels of protein phosphorylation are determined by the balanced actions of both protein kinases and protein phosphatases (PPases). However, compared with protein kinases, regulation of PPases has been relatively neglected. The insulin secretagogue L-arginine, an immediate metabolic precursor to polyamines, causes a rapid and transient decrease in PPase-1 activity in insulin-secreting RINm5F cells. We here show that polyamines dose-dependently suppress PPase-1-like activity when added to RINm5F cell homogenates at physiologic concentrations (spermine > spermidine > putrescine), while having minor and inconsistent effects on PPase-2A-like activity. The IC50 value for spermine on PPase-1-like activity was approximately 4 mM. The inhibitory effect was reproduced and of comparable magnitude on purified PPases types 1 and 2A. On the other hand, when endogenous polyamine pools were exhausted by 4 days of exposure to the specific L-ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine, there was an increase in PPase-2A-like activity. Quantitative Western analysis revealed that the amount of PPase-2A protein did not change after this treatment. It is concluded that polyamines cause time-and concentration-dependent inhibitory effects on RINm5F cell PPase activities, which may contribute to the increase in phosphorylation state that occurs after secretory stimulation.

Topics: Arginine; Eflornithine; Enzyme Activation; Enzyme Inhibitors; Histones; Humans; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Okadaic Acid; Pancreatic Neoplasms; Phosphoprotein Phosphatases; Phosphorylation; Polyamines; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Putrescine; Spermidine; Spermine; Tumor Cells, Cultured

Transforming growth factor beta (TGFbeta) is a tumor suppressor acting as inhibitor of cell cycle progression of epithelial cells. We show that treatment of the pancreatic carcinoma cell lines PANC-1 and BxPC-3 with TGFbeta1 inhibits both growth factor-induced activation of the extracellular signal-regulated kinase 2 (ERK2) and translocation of the kinase to the nucleus. TGFbeta1 causes a concentration-dependent reduction of cell proliferation in both cell lines. By measuring ERK activation, we can show that TGFbeta1 is able to repress ERK activation induced by mitogenic stimuli such as EGF. This inhibitory effect of TGFbeta1 is not mediated by suppression of Ras or c-Raf-1 activation, but mediated by TGFbeta1-induced activation of a serine-threonine phosphatase, as demonstrated by inhibition of phosphatases by treatment with okadaic acid. Results obtained in the Smad4-deficient pancreatic carcinoma cell line BxPC-3, demonstrate that TGFbeta1-induced growth inhibition is mediated by a Smad4-independent prevention of ERK2 activation. In contrast to the effects of TGFbeta1 on epithelial cells, mesenchymal NIH3T3 fibroblasts exhibit elevated ERK2 activation and increased cell proliferation in response to TGFbeta1 treatment. Smad4-independent phosphatase-mediated inhibition of mitogen-activated ERK2 represents a novel effector pathway contributing to suppression of epithelial pancreatic carcinoma cell proliferation by TGFbeta1, in addition to the well-known Smad-induced tumor suppressor activity of TGFbeta. Oncogene (2000) 19, 4531 - 4541.

Topics: 3T3 Cells; Animals; Biological Transport; Carcinoma; Cell Cycle; Cell Division; Cell Nucleus; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Humans; Mice; Mitogen-Activated Protein Kinase 1; Okadaic Acid; Pancreatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; ras Proteins; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Intracellular movement of secretory granules is a proximal stage in the secretory cascade that ends in the release product from cells. We investigated mechanisms underlying the control of this movement by acetylcholine using an insulinoma cell line, MIN6, in which acetylcholine increases both insulin secretion and granule movement. The peak activation of movement was observed 3 min after an acetylcholine challenge. The effects were nullified by the muscarinic inhibitor atropine, phospholipase C (PLC) inhibitors (D 609 and compound 48/80), and pretreatment with the Ca2+ pump inhibitor, thapsigargin. Inhibitors of Ca2+-dependent phospholipase A2 (arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphate) also partially inhibited the movement caused by acetylcholine, but downregulation of protein kinase C by overnight incubation with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate failed to exert any influence. Acetylcholine stimulation of granule movement was not reproduced by membrane depolarization with high K+. Phosphorylation of the endogenous myosin light chain in MIN6 cells was increased by addition of acetylcholine and decreased by the Ca2+ chelator BAPTA (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid). The calmodulin inhibitor W-7 and the myosin light-chain kinase inhibitor ML-9 decreased the motile events in the beta-cells under both nonstimulated and acetylcholine-stimulated conditions. These findings led us to conclude that inositol trisphosphate [corrected] causes Ca2+ mobilization by muscarinic activation of PLC, leading to intracellular translocation of insulin granules to the ready-releasable pool in pancreatic beta-cells via Ca2+/calmodulin-dependent phosphorylation of myosin light chains.

Topics: Acetylcholine; Animals; Atropine; Calcium; Calcium-Transporting ATPases; Cytoplasmic Granules; Enzyme Inhibitors; Inositol Phosphates; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Kinetics; Mice; Microscopy, Electron; Microscopy, Fluorescence; Muscarinic Antagonists; Okadaic Acid; Pancreatic Neoplasms; Phosphorylation; Rats; Tumor Cells, Cultured; Type C Phospholipases

It has been shown that okadaic acid (OA) diminishes insulin secretion of rat pancreatic islets in response to glucose, glyceraldehyde and KCl. Glucose, glyceraldehyde and KCl cause release of insulin by depolarization and subsequent opening of L-type calcium channels. Calcium entry into cells is thought to be related to protein phosphorylation. To evaluate whether or not OA mediated inhibition of insulin secretion in response to depolarization might be due to an interference with calcium uptake, we studied its effect on KCl (30 mM)-induced increases of cytosolic calcium and discharge of insulin in the insulin secreting clonal tumor cell line RINm5F. OA inhibited KCl-stimulated insulin release in concentrations > or = 1 microM. In intact RINm5F cells similar concentrations of OA decreased the activity of protein phosphates PP-1/PP-2A and inhibited the depolarization-induced rise of cytosolic calcium ([Ca2+]i). The latter action could also be achieved with the protein phosphatase inhibitor calyculin A, whereas the OA analogue 1-nor-okadaone, which is without effect on phosphatases, did not affect [Ca2+]i or insulin release. It is concluded that depression of depolarization-induced insulin secretion by OA is due to inhibition of calcium entry along voltage dependent calcium channels. The data also suggest that in RINm5F cells protein phosphatases PP-1/PP-2A are related to the function of voltage-dependent calcium channels.

Topics: Animals; Calcium; Enzyme Inhibitors; Insulin; Insulin Secretion; Insulinoma; Membrane Potentials; Okadaic Acid; Pancreatic Neoplasms; Phosphoprotein Phosphatases; Phosphorylase Phosphatase; Potassium Chloride; Rats; Tumor Cells, Cultured

Phosphorylation/dephosphorylation of intracellular proteins are important steps in the regulation of cell growth. Okadaic acid, an inhibitor of the serine/threonine protein phosphatases 1 and 2A, is a potent tumor promoter. This effect may be through the inhibition of dephosphorylation (termed "hyperphosphorylation") and subsequent inactivation of tumor-suppressor proteins. We examined whether okadaic acid regulates growth of human pancreatic cancer cells (MIA PaCa-2 and Panc-1) or alters the phosphorylation of the retinoblastoma tumor-suppressor protein. Growth studies, nuclear labeling analyses, and Western blotting for retinoblastoma protein were performed. Okadaic acid stimulated cell growth and induced hyperphosphorylation of the retinoblastoma protein. The growth-stimulatory effect of okadaic acid on these human pancreatic cancer cells may be mediated by inactivation of the growth suppressive effect of the retinoblastoma protein by hyperphosphorylation. These studies suggest that the growth of these human pancreatic cancer cells is still regulated by tumor-suppressor proteins.

Topics: Blotting, Western; Cell Division; Enzyme Inhibitors; Humans; Okadaic Acid; Pancreatic Neoplasms; Phosphorylation; Retinoblastoma Protein; Tumor Cells, Cultured

This study investigates the occurrence and regulation of serine/threonine protein phosphatases (PPases) in insulin-secreting RINm5F insulinoma cells. PPases types 1 and 2A were identified in crude RINm5F cell homogenates by both enzymatic assay and Western blot analysis. We then characterized and compared the inhibitory actions of several compounds isolated from cyanobacteria, marine dinoflagellates and marine sponges, (viz. okadaic acid, microcystin-LR, calyculin-A and nodularin) cation-independent PPase activities in RINm5F cell homogenates. It was found that okadaic acid was the least potent inhibitor (IC50 approximately 10(-9) M, IC100 approximately 10(-6) M), while the other compounds exhibited IC50 values of approximately 5 x 10(-10) M and IC100 approximately 5 x 10(-9) M. The findings indicate that the inhibitory substances employed in this study may be used pharmacologically to investigate the role of serine/threonine PPases in RINm5F cell insulin secretion, a process that is likely to be regulated to a major extent by protein phosphorylation.

Topics: Animals; Ethers, Cyclic; In Vitro Techniques; Insulinoma; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Pancreatic Neoplasms; Peptides, Cyclic; Phosphoprotein Phosphatases; Rabbits; Rats; Tumor Cells, Cultured