ucn-1028-c and Melanoma

Protein kinase C (PKC) is a multigene family of serine/threonine protein kinases involved in cell signaling pathways of proliferation and motility. PKC interacts with Rho GTPases in the regulation of the actin cytoskeleton. The PKC-alpha isozyme binds the Rho GTPase cdc42, and both are coordinated with the Rac-phosphatidylinositol-3 kinase (PI3K) signaling pathway in melanoma cell invasion and migration on extracellular matrix proteins. To further define the role of PKC-alpha in melanoma cell migration, we tested the effect of PDBu and Ca dependent activation of PKC-alpha as well as treatment with the PKC-alpha inhibitors calphostin C and Go6976. Furthermore, we transfected siRNA targeted against PKC-alpha into human melanoma cells and performed time-lapse analysis of cell migration followed by western immunoblotting. We found that significant enhancement of cell migration at 0.5 h after PDBu treatment directly correlated with Ca dependent activation of PKC-alpha and was inhibited by the PKC-alpha inhibitor calphostin C. PKC-alpha siRNA transfection nearly abrogated PKC-alpha expression and significantly reduced melanoma cell migration compared with siRNA controls. These findings provide further evidence that PKC-alpha plays an important role in melanoma cell migration and may have implications in therapies designed to disrupt melanoma cell motility by alteration of PKC-alpha signaling.

Topics: Apoptosis; Carbazoles; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Movement; Extracellular Matrix; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Naphthalenes; Neoplasm Metastasis; Protein Isoforms; Protein Kinase C-alpha; RNA Interference; Time Factors

Internalization of the urokinase-type plasminogen activator (uPA) requires two receptors, the uPA receptor (uPAR) and the low density lipoprotein receptor-related protein (LRP)/alpha2-macroglobulin (alpha2M) receptor. Here, we address whether protein kinases are involved in the internalization of uPA by human melanoma cells. Initially, we found that the internalization of uPA was significantly inhibited by the serine/threonine protein kinase inhibitors staurosporine, K-252a and H-89, but not by the tyrosine kinase inhibitors, genistein and lavendustin A. Internalization of uPA was also inhibited by a pseudosubstrate peptide for cAMP-dependent protein kinase (PKA), but not by a pseudosubstrate peptide for protein kinase C. We confirmed a requirement for PKA-activity and implicated a specific isoform by using an antisense oligonucleotide against the regulatory subunit RI alpha of PKA which suppresses PKA-I activity. Exposure of cells to this oligonucleotide led to a specific, dose-dependent decrease in RI alpha protein and to a significant inhibition in the rate of uPA internalization. We further demonstrate that treatment of melanoma cells with either H-89 or PKA RI alpha antisense oligonucleotides also resulted in a decreased internalization of two other ligands of LRP, activated alpha2M and lactoferrin, indicating that PKA activity is associated with LRP. Finally, we demonstrate that PKA activity is also required for the internalization of transferrin, but not for the internalization of the epidermal growth factor or adenovirus 2, suggesting that in melanoma cells, PKA activity is not generally required for clathrin-mediated endocytosis, but is rather associated with specific internalization receptors.

Topics: Adenoviruses, Human; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Endocytosis; Enzyme Inhibitors; Epidermal Growth Factor; Genistein; Humans; Indole Alkaloids; Indoles; Isoflavones; Isoquinolines; Lactoferrin; Low Density Lipoprotein Receptor-Related Protein-1; Maleimides; Melanoma; Naphthalenes; Phenols; Protein Kinase C; Receptors, Immunologic; Signal Transduction; Staurosporine; Sulfonamides; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

We have examined the functional status of the VLA-4/alpha4beta1 integrin in a panel of human melanoma cell lines, focusing on the ability of cells expressing alpha4beta1 to mediate adhesion to the alpha4-specific ligands CS-1 peptide and VCAM-1. All melanoma cells expressing alpha4pbeta1 (8 of 10 lines examined) were capable of adhering to these specific ligands in adhesion assays, whereas 2 cell lines (HMB2 and VUP) which lacked surface alpha4 were unable to do so. Adherence of different melanoma cell lines to VCAM-1 was relatively uniform and not susceptible to upregulation with known integrin-activating factors, such as manganese ions, phorbol ester and activating monoclonal antibody (mAb) TS2/16. Cell adhesion to CS-1 peptide, however, varied according to cell surface receptor density and, in some cases, could be up-regulated by integrin-activating factors. Adhesion of SK23 cells to CS-1 peptide was increased by all 3 activating stimuli, whereas for all other melanoma cells an increase was obtained only by the use of TS2/16 mAb. Our data indicate not only an unusually low activation state of alpha4beta1 in SK23 cells but also heterogeneity in the activating capacity of the various stimuli. Moreover, a protein kinase C-dependent role in alpha4beta1 activity was suggested by adhesion assays carried out in the presence of the protein kinase C inhibitor calphostin C, which considerably reduced adhesion to CS-1 peptide.

Topics: Anti-Allergic Agents; Antigens, Surface; Biomarkers, Tumor; Cell Adhesion; Enzyme Inhibitors; Flow Cytometry; Humans; Integrin alpha4beta1; Integrins; Melanoma; Membrane Proteins; Microfilament Proteins; Naphthalenes; Precipitin Tests; Protein Kinase C; Receptors, Lymphocyte Homing; Rhabdomyosarcoma; Tumor Cells, Cultured; Vascular Cell Adhesion Molecule-1

Differentiation therapy is an attractive option for malignant melanoma, as traditional forms of chemotherapy seem to have little effect on this type of tumour. Among the several pathways for the experimental induction of differentiation of melanoma, we have focused on signal transduction mediated by protein kinases. We have examined the effects of calphostin C (a protein kinase C inhibitor), genistein and methyl 2,5-dihydroxycinnamate (tyrosine kinase inhibitors), and exogenous phosphotyrosine (an activator of protein tyrosine phosphatases) on the growth, morphology and differentiation of malignant melanomas in vitro. All four compounds tested were able to inhibit cell proliferation, but only genistein and methyl 2,5-dihydroxycinnamate were able to induce morphological changes, yielding a more dendritic or a rounder phenotype, respectively. The latter two drugs were also able to induce specific cell-cycle alterations, in contrast to calphostin C and phosphotyrosine. Melanin content was increased greatly in phophotyrosine treated cells and, to a smaller extent, in cells treated with genistein. RNA expression of specific genes encoding cytolytic T-cell antigens was not altered by the two tyrosine kinase inhibitors, in spite of the phenotypic changes observed. Together, these results suggest that tyrosine kinases are involved in cell cycle, growth, and differentiation pathways in malignant melanomas; however, these pathways may not be co-dependent. The results also suggest that these pathways may be sensitive to specific tyrosine kinase inhibitors or activators of protein tyrosine phosphatases.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Cell Cycle; Cell Differentiation; Cinnamates; Enzyme Inhibitors; Eye Neoplasms; Gene Expression Regulation, Neoplastic; Genistein; Humans; Melanoma; Naphthalenes; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; Skin Neoplasms; Tumor Cells, Cultured

Phorbol 12-myristate 13-acetate (PMA) is a tumour promotor that acts as a potent protein kinase C (PKC) activator that has significant effects on tumour cell attachment and spreading. We tested whether these effects of PMA may be observed in human melanoma cells, and whether a specific response to extracellular matrix proteins may be mediated by shifts in the expression of beta 1 integrins. We used cell attachment assays, video time lapse cell spreading assays, flow cytometry, function blocking monoclonal antibodies (MAbs) and PKC inhibitor Calphostin C to address these questions. We established that PMA induces a rapid and temporary enhancement of cell attachment and spreading which was not accompanied by a significant change in the expression of beta 1 integrins. Spreading of melanoma cells that were not stimulated with PMA could be significantly blocked with a function blocking MAb (clone P4C10) against the common beta 1 integrin subunit. The spreading and attachment of the PMA treated cells was also significantly reduced, but less so, after MAb treatment. The PMA enhanced cell attachment and spreading could be effectively blocked by RGD sequences and PKC inhibitor. Taken together, our data indicate that PMA induces a rapid and temporary ECM-dependent enhancement of melanoma cell attachment and spreading, and that the response to ECM components appears not to be due to significant shifts in beta 1 integrin expression, but rather to activation of beta 1 integrins.

Topics: Amino Acid Sequence; Cell Adhesion; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Humans; Integrins; Melanoma; Molecular Sequence Data; Naphthalenes; Oligopeptides; Polycyclic Compounds; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured