ucn-1028-c and methyl-2-5-dihydroxycinnamate

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

We examined the effect of basic fibroblast growth factor (bFGF) on the activation of phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. bFGF stimulated both the formations of choline (EC50 was 30 ng/ml) and inositol phosphates (EC50 was 10 ng/ml). Calphostin C, an inhibitor of protein kinase C (PKC), had little effect on the bFGF-induced formation of choline. bFGF stimulated the formation of choline also in PKC down regulated cells. Genistein and methyl 2,5-dihydroxycinnamate, inhibitors of protein tyrosine kinases, significantly suppressed the bFGF-induced formation of choline. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, enhanced the bFGF-induced formation of choline. In vitro kinase assay for FGF receptors revealed that FGF receptor 1 and 2 were autophosphorylated after FGF stimulation. bFGF dose-dependently stimulated DNA synthesis of these cells. These results strongly suggest that bFGF activates phosphatidylcholine-hydrolyzing phospholipase D through the activation of tyrosine kinase, but independently of PKC activated by phosphoinositide hydrolysis in osteoblast-like cells.

Topics: Animals; Cinnamates; DNA; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fibroblast Growth Factor 2; Genistein; Inositol Phosphates; Isoflavones; Mice; Naphthalenes; Osteoblasts; Phosphatidylcholines; Phospholipase D; Phosphorylation; Phosphorylcholine; Precipitin Tests; Protein Kinase C; Protein-Tyrosine Kinases; Receptors, Fibroblast Growth Factor; Vanadates

Effects of various types of protein kinase inhibitor on the adhesion and spreading of BALB/c mouse 3T3 cells and on the phosphorylation and stability of focal adhesion kinase (FAK) in the cells were studied. Inhibitors of protein tyrosine kinases, methyl 2,5-dihydroxycinnamate and herbimycin A, inhibited tyrosine-phosphorylation of FAK and the adhesion of 3T3 cells to fibronectin. Among inhibitors of serine/threonine kinases tested, calphostin C, a specific inhibitor of protein kinase C, inhibited cell spreading rather than cell adhesion, and it induced the decrease of intracellular FAK within 30 min. Inhibitors of tyrosine kinase, A kinase, G kinase, and myosin light chain kinase did not induce such a rapid and specific decrease of FAK. When calphostin C (20 microM) was added to sub-confluent monolayer cultures, serine-phosphorylation of FAK was inhibited by 67% within 2 h, and decrease in the amount of FAK and rounding up of the cells began after 4 h. Label-chase experiments indicated that about 60% of 35S-labeled FAK degraded within 1-2 h after addition of calphostin C to monolayer cultures. These results indicated that serine-phosphorylation of FAK induced by protein kinase C was important in the regulation of metabolic stability of FAK.

Topics: 3T3 Cells; Animals; Benzoquinones; Cell Adhesion; Cell Adhesion Molecules; Cinnamates; Enzyme Inhibitors; Enzyme Stability; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Kinetics; Lactams, Macrocyclic; Mice; Mice, Inbred BALB C; Naphthalenes; Phosphorylation; Phosphoserine; Phosphotyrosine; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Rifabutin