okadaic-acid and Prostatic-Neoplasms

Protein phosphatase 2A (PP2A) is a tumor suppressor complex that has recently been reported as a novel and highly relevant molecular target in prostate cancer (PCa). However, its potential therapeutic value remains to be fully clarified. We treated PC-3 and LNCaP cell lines with the PP2A activators forskolin and FTY720 alone or combined with the PP2A inhibitor okadaic acid. We examined PP2A activity, cell growth, prostasphere formation, levels of PP2A phosphorylation, CIP2A and SET expression, and AKT and ERK activation. Interestingly, both forskolin and FTY720 dephosphorylated and activated PP2A, impairing proliferation and prostasphere formation and inducing changes in AKT and ERK phosphorylation. Moreover, FTY720 led to reduced CIP2A levels. Treatment with okadaic acid impaired PP2A activation thus demonstrating the antitumoral PP2A-dependent mechanism of action of both forskolin and FTY720. Levels of PP2A phosphorylation together with SET and CIP2A protein expression were studied in 24 PCa patients and both were associated with high Gleason scores and presence of metastatic disease. Altogether, our results suggest that PP2A inhibition could be involved in PCa progression, and the use of PP2A-activating drugs might represent a novel alternative therapeutic strategy for treating PCa patients.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Colforsin; Fingolimod Hydrochloride; Humans; Male; Molecular Targeted Therapy; Okadaic Acid; Phosphorylation; Prostatic Neoplasms; Protein Phosphatase 2

Exon 2 of the Bcl-x gene undergoes alternative splicing in which the Bcl-xS splice variant promotes apoptosis in contrast to the anti-apoptotic splice variant Bcl-xL. In this study, the regulation of the alternative splicing of pre-mRNA of Bcl-x was examined in response to emetine. Treatment of different types of cancer cells with emetine dihydrochloride downregulated the level of Bcl-xL mRNA with a concomitant increase in the mRNA level of Bcl-xS in a dose- and time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), blocked emetine-induced alternative splicing in contrast to okadaic acid, a specific inhibitor of PP2A in cells, demonstrating a PP1-mediated mechanism. Our finding on the regulation of RNA splicing of members of the Bcl-2 family in response to emetine presents a potential target for cancer treatment.

Topics: Alternative Splicing; bcl-X Protein; Breast Neoplasms; Cell Line, Tumor; Cycloheximide; Down-Regulation; Emetine; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Marine Toxins; Okadaic Acid; Oxazoles; Prostatic Neoplasms; Protein Phosphatase 1; Protein Phosphatase 2; Up-Regulation; Uterine Cervical Neoplasms

Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKC alpha and the novel PKC delta isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574-7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645-655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKC alpha in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells and suggest that both p38 MAPK and Akt play critical roles as downstream effectors of PKC isozymes in this cellular model.

Topics: Androstadienes; Apoptosis; Blotting, Western; Cell Survival; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Prostatic Neoplasms; Protein Isoforms; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; RNA Interference; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors; Transfection; Tumor Cells, Cultured; Wortmannin

As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the alpha3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, alphav immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i. e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the alpha3beta1-mediated adhesion of PC-3 cells to collagen IV and the alpha2beta1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity.

Topics: Actins; Calcium-Binding Proteins; Calreticulin; Cell Adhesion; Cell Size; Collagen; Cytochalasin D; Cytoskeleton; Extracellular Matrix Proteins; Humans; Integrin alpha3beta1; Integrins; Jurkat Cells; Ligands; Male; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Prostatic Neoplasms; Protein Binding; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Receptors, Collagen; Ribonucleoproteins; Tumor Cells, Cultured; Vitronectin

Cells differ in the time required to execute cell death after receipt of a death signal. One reason may be the requirement for de novo synthesis of components of the death pathway. TSU-Pr1 prostate cancer cells treated with okadaic acid demonstrated activation of caspase-3, PARP cleavage, and nuclear fragmentation by 24 h and apoptosis by 72 h. Levels of procaspase-3 and procaspase-7, the precursor molecules of two effector caspases, were not depleted during apoptosis. Levels of procaspase-3 and -7 mRNA increased steadily in TSU-Pr1 cells up to 72 h after exposure to okadaic acid. Nuclear run-off experiments showed that the increase in mRNA was not due to transcriptional activation of caspase-3 and -7 mRNA. Antisense caspase-3 and caspase-7 oligodeoxynucleotides caused a depletion of procaspases-3 and -7 and a delay in apoptosis of TSU-Pr1 cells. Caspase antisense oligodeoxynucleotides inhibited apoptosis to a similar extent as peptide inhibitors of cysteine proteases. Synthesis of procaspases-3 and -7 was necessary to sustain programmed cell death in TSU-Pr1 prostate cancer cells.

Topics: Apoptosis; Caspase 3; Caspase 7; Caspases; Enzyme Activation; Enzyme Precursors; Humans; Male; Okadaic Acid; Oligodeoxyribonucleotides, Antisense; Prostatic Neoplasms; Tumor Cells, Cultured

UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirement for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-alpha- and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis.

Topics: 3T3 Cells; Animals; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Cytoplasm; DNA Damage; Enzyme Activation; Glycogen Synthase Kinase 3; HL-60 Cells; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mitogen-Activated Protein Kinases; Okadaic Acid; Phorbol Esters; Prostatic Neoplasms; Signal Transduction; Time Factors; Tretinoin; Tumor Necrosis Factor-alpha; Ultraviolet Rays

We investigated the effect of protein kinase and phosphatase inhibitors on the growth of six human prostatic cancer cell lines: DU145, PC3, ND1, LNCaP, ALVA31 and JCA1. We studied okadaic acid and sodium orthovanadate as serine/threonine and tyrosine protein phosphatase inhibitors, respectively, and staurosporin and genistein as a serine/threonine and tyrosine protein kinase inhibitors, respectively. All inhibitors examined exhibited a dose-dependent growth inhibitory effect on prostatic cancer cell lines. Our data indicate that prostatic cancer cell lines express unique biochemical properties since the degree of growth inhibition varied greatly and was dependent on the specific cell line and inhibitor studied. In addition, we found that surface expression of endoglin (CD105) changed by treatment with all inhibitors in most of the cell lines. These data also indicate that endoglin appears to be involved both in protein phosphatase and kinase mediated phosphoprotein turnover.

Topics: Alkaloids; Antigens, CD; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Division; Endoglin; Enzyme Inhibitors; Ethers, Cyclic; Genistein; Humans; Hyaluronan Receptors; Intercellular Adhesion Molecule-1; Isoflavones; Male; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Receptors, Cell Surface; Staurosporine; Tumor Cells, Cultured; Vanadates; Vascular Cell Adhesion Molecule-1

This study examined the role of protein phosphorylation in TNF induction of apoptosis in several tumor cell lines by testing the effects of agents that either stimulate or inhibit protein phosphorylation. The serine-threonine phosphatase inhibitors, okadaic acid (OKA) and calyculin A (CLA), synergistically augmented TNF-induced apoptosis in several TNF-sensitive tumor cell lines including the U937 histiocytic lymphoma, the BT-20 mammary carcinoma, and the LNCap prostatic tumor cell line. Furthermore, the phosphatase inhibitors completely reversed the TNF resistance of a variant (U9-TR) derived from U937. CLA also inhibited phosphatase activity in cell-free extracts from both U937 and U9-TR at the same concentrations (0.4-2.0 nM) that it synergized with TNF. In contrast, TNF treatment of U937 cells did not result in inhibition of phosphatase activity mediated by protein phosphatase 1 (PP1) and PP2A in cell extracts. Since the phosphatase inhibitors are known to increase the overall levels of protein phosphorylation in cells, this suggested that TNF may act by stimulating protein kinase (PK) activity. This hypothesis was supported by the results of testing a panel of relatively specific protein kinase inhibitors. TNF activation of DNA fragmentation was blocked by a potent inhibitor of myosin light chain kinase (MLCK) but was unaffected by inhibitors of cAMP or cGMP-dependent PKs. We postulate that a defect in the activation of MLCK or possibly some other as yet unknown PK may be responsible for the TNF resistance of U9-TR. Furthermore, this resistance may be circumvented by promoting protein phosphorylation with the serine-threonine-dependent phosphatase inhibitors.

Topics: Alkaloids; Apoptosis; Breast Neoplasms; Carbazoles; DNA; Drug Synergism; Ethers, Cyclic; Humans; Indoles; Lymphoma, Large B-Cell, Diffuse; Male; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Prostatic Neoplasms; Protein Kinase Inhibitors; Recombinant Proteins; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha