rottlerin and safingol

Recently, we demonstrated that activation of the protein kinase C (PKC) signalling pathway promoted morphological differentiation of GT1 hypothalamic neurones via an increase in beta-catenin, a cell-cell adhesion molecule, indicating a possible involvement of PKC in cellular motility. In this study, we explored the differential roles of PKC isoforms in GT1 cell migration. First, we transiently transfected GT1 cells with enhanced green fluorescence protein (EGFP)-tagged actin to monitor the dynamic rearrangement of filamentous-actin (F-actin) in living cells. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, markedly promoted lamellipodia formation, while safingol (a PKC alpha-selective inhibitor) blocked the TPA-induced lamellipodial actin structure. Both wound-healing and Boyden migration assays showed that TPA treatment promoted neuronal migration of GT1 cells; however, cotreatment of TPA with safingol or rottlerin (a PKC delta-selective inhibitor) clearly blocked this TPA effect, indicating that both PKC alpha and PKC delta may be positive regulators of neuronal migration. By contrast, PKC gamma-EGFP-expressing GT1 cells exhibited decreased cellular motility and weak staining for actin stress fibres, suggesting that PKC gamma may act as a negative mediator of cell migration in these neurones. Among the PKC downstream signal molecules, p130Cas, a mediator of cell migration, and its kinase, focal adhesion kinase (FAK), increased following TPA treatment; phosphorylation of p130Cas was induced in a PKC alpha-dependent manner. Together, these results demonstrate that PKC alpha promotes GT1 neuronal migration by activating focal adhesion complex proteins such as p130Cas and FAK.

Topics: Acetophenones; Actins; Benzopyrans; Cell Line, Transformed; Cell Movement; Enzyme Activation; Enzyme Inhibitors; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression; Green Fluorescent Proteins; Hypothalamus; Isoenzymes; Luminescent Proteins; Neurons; Phosphoproteins; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Protein-Tyrosine Kinases; Proteins; Recombinant Fusion Proteins; Retinoblastoma-Like Protein p130; Sphingosine; Tetradecanoylphorbol Acetate; Transfection

The growth and proliferation of multiple myeloma (MM) cells are influenced by various cytokines produced by bone marrow stromal cells. As cytokine interaction between malignant plasma cells and neighbouring stromal cells is important in the pathogenesis of MM, the understanding of intracellular signalling events elicited by this interaction is of central importance. Recent reports have shown that protein kinase C (PKC) is directly involved in modulating apoptosis in different cells types, including those of haematopoietic neoplasms. In the present study, we analysed the expression patterns of PKC isoforms in the myeloma cell lines U266, RPMI-8226 and K620. This analysis demonstrated common expression of PKC-delta, PKC-iota, PKC- micro and PKC-zeta in all three myeloma cell lines. PKC-delta expression in plasma cells from 11 patients with MM was also shown by immunohistochemistry, utilizing a monoclonal mouse anti-human PKC-delta antibody. U266 cells treated with the broad PKC inhibitor safingol (l-threo-dihydrosphingosine) or the PKC-delta-specific inhibitor rottlerin (3'-[(8-Cinnamoyl-5,7-dihydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)methyl]-2',4',6'-trihydroxy-5'-methylacetophenone) showed decreased PKC-delta in the particulate fraction and resulted in significant apoptosis. Primary myeloma cells also showed apoptosis after treatment with the PKC inhibitors, as detected by both flow cytometric and morphological evaluation. Our results indicate that PKC-delta is commonly expressed in myeloma cells and plays an important role in plasma cell survival.

Topics: Acetophenones; Apoptosis; Benzopyrans; Blotting, Western; Down-Regulation; Enzyme Inhibitors; Humans; Multiple Myeloma; Protein Kinase C; Protein Kinase C-delta; Sphingosine; Tumor Cells, Cultured

The protein kinase C (PKC) is a family of serine/threonine kinases that are key regulatory enzymes involved in growth, differentiation, cytoskeletal reorganization, tumor promotion, and migration. We investigated the functional involvement of PKC isotypes and of E-cadherin in the regulation of the locomotion of six human colon-adenocarcinoma cell lines. The different levels of the PKC alpha and the E-cadherin expression have predictable implications in the spontaneous locomotory activity. With the use of PKC alpha--specific inhibitors (safingol, Go6976) as well as the PKC delta--specific inhibitor rottlerin, we showed that only PKC alpha plays a major role in the regulation of tumor cell migration. The results were verified by knocking out the translation of PKC isozymes with the use of an antisense oligonucleotide strategy. After stimulation with phorbol ester we observed a translocation and a colocalization of the activated PKC alpha at the plasma membrane to the surrounding extracellular matrix. Furthermore, we investigated the functional involvement of E-cadherin in the locomotion with the use of a blocking antibody. A high level of PKC alpha expression together with a low E-cadherin expression was strongly related to a high migratory activity of the colon carcinoma cells. This correlation was independent of the differentiation grade of the tumor cell lines.

Topics: Acetophenones; Benzopyrans; Biological Transport; Cadherins; Carbazoles; Cell Membrane; Cell Movement; Colonic Neoplasms; Enzyme Activation; Enzyme Inhibitors; Humans; Indoles; Isoenzymes; Oligonucleotides, Antisense; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Sphingosine; Tumor Cells, Cultured

Trivalent arsenic (arsenite) is a human carcinogen. However, the molecular mechanism of arsenite-induced carcinogenesis is still not well understood. In this study, we found that arsenite induced translocation of PKCepsilon, PKCdelta, and PKCalpha from cytosol to membranes. Rottlerin, a selective inhibitor for PKCdelta, and safingol, a specific inhibitor for PKCalpha, both markedly inhibited arsenite-induced AP-1 activity. These inhibitory effects by rottlerin and safingol appeared to be dose dependent. Arsenite-induced phosphorylation of Erks was inhibited by rottlerin, while safingol inhibited arsenite-induced phosphorylation of JNKs and p38 kinases. Dominant negative mutant transfectant of PKCepsilon markedly blocked arsenite-induced AP-1 activity and the phosphorylation of Erks, JNKs, and p38 kinases. These data demonstrate that PKCdelta, PKCepsilon, and PKCalpha mediate arsenite-induced AP-1 activation in JB6 cells through different MAP kinase (Erks, JNKs, and p38 kinases) pathways.

Topics: Acetophenones; Animals; Arsenites; Benzopyrans; Carcinogens; Cell Membrane; Cells, Cultured; Cytosol; Enzyme Activation; Enzyme Activators; Enzyme Inhibitors; Isoenzymes; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinases; Mutation; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Protein Kinase C-epsilon; Skin; Sphingosine; Transcription Factor AP-1; Transfection

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCepsilon and PKCdelta, but not PKCalpha, from cytosol to membrane. Dominant negative mutant (DNM) PKCepsilon or PKCdelta inhibited UVB-induced translocation of PKCepsilon and PKCdelta, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCepsilon and PKCdelta, whereas the DNM of PKCalpha was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCdelta, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCalpha, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 microM) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 microM), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCdelta and PKCepsilon, but not PKCalpha, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs.

Topics: Acetophenones; Animals; Apoptosis; Benzopyrans; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cell Membrane; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Imidazoles; Indoles; Isoenzymes; Maleimides; Mice; Mutation; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Protein Kinase C-epsilon; Pyridines; Signal Transduction; Sphingosine; Ultraviolet Rays