tretinoin and herbimycin

Transformed cells often express elevated levels of tyrosine-phosphorylated proteins. Inhibition of protein tyrosine kinases causes reversion of malignant cells to the normal phenotype. In the present study, we evaluated the possibility that the reversion of human endometrial adenocarcinoma RL95-2 cells to a stationary phenotype induced by retinoic acid was associated with inhibition of tyrosine phosphorylation of cellular proteins. We found that retinoic acid decreased the levels of tyrosine-phosphorylated proteins, as assessed by immunostaining and immunoprecipitations using specific anti-phosphotyrosine antibodies. In addition, the inhibitors of tyrosine kinases herbimycin A and tyrphostin mimicked retinoic acid, inducing F-actin reorganization and increasing the size of RL95-2 cells, as determined by measurement of cell perimeters. Because focal adhesions that connect actin filaments with the plasma membrane are major sites of tyrosine phosphorylation, we further investigated whether selected focal adhesion proteins were affected by retinoic acid. We found that retinoic acid altered the localization of focal adhesion kinase. All-trans retinoic acid was effective in reducing the levels of focal adhesion kinase and paxillin protein. Thirteen-cis retinoic acid increased the levels of vinculin protein in the cytosolic fraction of cells. These changes are consistent with actin reorganization and reversion toward a stationary phenotype induced by retinoic acid in endometrial adenocarcinoma RL95-2 cells. Our results indicate that the differentiating effects of retinoids on endometrial cells are associated with decreases in tyrosine phosphorylation and changes in the levels and distribution of focal adhesion proteins. These findings suggest that signaling pathways that involve tyrosine kinases are potential targets for drug design against endometrial cancer.

Topics: Actins; Adenocarcinoma; Analysis of Variance; Benzoquinones; Cell Adhesion Molecules; Endometrial Neoplasms; Enzyme Inhibitors; Female; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Isotretinoin; Kinetics; Lactams, Macrocyclic; Phosphoproteins; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tretinoin; Tumor Cells, Cultured

The effects of hymenialdisine (SK&F 108752) were evaluated on interleukin-1 (IL-1)-induced proteoglycan (PG) degradation, PG synthesis, nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) gene expression in bovine articular cartilage (BAC) and/or cartilage-derived chondrocytes. Cartilage disks from 0- to 3-month-old calves were treated with IL-1alpha or retinoic acid. PG release was determined by measuring glycosaminoglycan release, and nitrite production was measured as a readout for NO. Inhibition of iNOS gene expression was measured by Northern blot analysis in IL-1alpha-stimulated, cartilage-derived chondrocytes. To measure PG synthesis, chondrocytes were established in alginate beads and treated with hymenialdisine, and then [(35)S]sulfate incorporation into PGs was determined. Hymenialdisine inhibited IL-1alpha-stimulated PG breakdown in BAC in a dose-related manner with an IC(50) of approximately 0.6 microM. Herbimycin, a protein tyrosine kinase inhibitor, also inhibited PG breakdown, whereas RO 32-0432, a protein kinase C inhibitor, had no effect. Both hymenialdisine and herbimycin also were able to inhibit retinoic acid-stimulated PG release. IL-1alpha-stimulated NO production in BAC was inhibited by hymenialdisine and herbimycin at similar concentrations. The effect on iNOS gene expression was determined by Northern blot analysis in chondrocytes grown in monolayer, and inhibition by hymenialdisine was observed with an IC(50) of approximately 0.8 microM. In chondrocytes cultured in alginate beads, IL-1alpha inhibited PG synthesis, whereas hymenialdisine stimulated synthesis at low concentrations (0.6 and 1.25 microM), and higher doses (2.5 microM) were not stimulatory. Compounds with this profile may have utility in the treatment of osteoarthritis.

Topics: Animals; Azepines; Benzoquinones; Blotting, Northern; Cartilage, Articular; Cattle; Cells, Cultured; Chondrocytes; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Indicators and Reagents; Interleukin-1; Lactams, Macrocyclic; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Protein Kinase C; Protein-Tyrosine Kinases; Proteoglycans; Pyrroles; Quinones; Rifabutin; RNA; Sulfates; Tretinoin

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.

Topics: Base Sequence; Benzoquinones; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Dactinomycin; DNA, Complementary; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Genistein; Humans; Hydroquinones; Lactams, Macrocyclic; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; NF-kappa B; Okadaic Acid; Phenols; Proto-Oncogene Proteins c-raf; Quinones; Rifabutin; Signal Transduction; Tetradecanoylphorbol Acetate; Thromboplastin; Transcription Factor AP-1; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Midkine (MK), which induces chemotaxis of human neutrophils, was found to trigger mobilization of intracellular calcium of these cells. The maximum response was observed 150 sec after exposure to MK, suggesting a complex mechanism in the process. The calcium mobilization was inhibited by herbimycin A, Bordetella pertussis toxin and wortmannin, suggesting that a tyrosine kinase, a G protein-linked receptor and phosphatidyl inositol 3 kinase are involved in the process.

Topics: Androstadienes; Benzoquinones; Calcium; Carrier Proteins; Chemotaxis, Leukocyte; Cytokines; Flow Cytometry; Heparin; Humans; Intracellular Fluid; Lactams, Macrocyclic; Midkine; Neutrophils; Quinones; Rifabutin; Signal Transduction; Tretinoin; Wortmannin

In this study, it was shown that the proportion of guanosine triphosphate (GTP)-bound active Ras increased in TPA (12-o-tetradecanoyl phorbol-13-acetate)-induced monocytic differentiation of HL-60 cells. The increase of active Ras was observed at 24 hours after TPA stimulation and attained to threefold (15%) over the proportion in nontreated HL-60 cells. Herbimycin A, an inhibitor of tyrosine kinase, prevented the activation of Ras, as well as the induction of monocytic differentiation. In parallel with the activation of Ras, the proteins with molecular weights of 52, 56, 62, and 190 kD were tyrosine-phosphorylated and formed a complex with GTPase-activating protein (GAP) for Ras. In addition to the 116-kD GAP (type I GAP), the 100-kD GAP (type II GAP) molecule was markedly induced at 24 hours after TPA stimulation of HL-60 cells. These phenomena sustained for a further 24 hours during monocytic differentiation. However, they were not observed during retinoic acid-induced granulocytic differentiation of the cells. The HL-60 transfectants, which expressed a dominant inhibitory Ha-ras Asn17, showed a low level of tyrosine-phosphorylated GAP-associated proteins and did not undergo full differentiation in response to TPA. Taken together, these data indicate that the activation of Ras and GAP complex formation mutually correlate and function downstream of protein-tyrosine kinases in the signaling pathway for monocytic differentiation of HL-60 cells.

Topics: Benzoquinones; Cell Differentiation; Dexamethasone; Enzyme Activation; Granulocytes; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Lactams, Macrocyclic; Monocytes; Mutation; Oncogene Protein p21(ras); Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proteins; Quinones; ras GTPase-Activating Proteins; Rifabutin; Tetradecanoylphorbol Acetate; Transfection; Tretinoin; Tumor Cells, Cultured; Tyrosine

Four retinoids, i.e. retinol (vitamin A), retinoic acid, retinyl acetate and synthetic chalcone carboxylic acid (Ch 55), were examined for their effects on embryonic angiogenesis using 4.5-day chorioallantoic membranes of chick embryo. The effects of these retinoids were compared with that of antibiotic herbimycin A, which was the most powerful inhibitor of the angiogenesis reported previously. The four retinoids strongly inhibited embryonic angiogenesis; the order of inhibitory activity was Ch 55 greater than retinoic acid greater than herbimycin A greater than retinyl acetate based on the dose required for the half-maximal inhibitory effect. The present results suggest that retinoids are effective inhibitors of angiogenesis, and can be applied for the management of certain diseases accompanied by aberrant angiogenesis, particularly that which occurs during progressive growth of solid tumors.

Topics: Animals; Benzoquinones; Chalcone; Chalcones; Chick Embryo; Diterpenes; Dose-Response Relationship, Drug; In Vitro Techniques; Lactams, Macrocyclic; Neovascularization, Pathologic; Quinones; Retinoids; Retinyl Esters; Rifabutin; Tretinoin; Vitamin A

We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines. Growth curve analyses with SK-N-SH suggested that herb-A greatly reduced the number of initially growing cells, whereas RA slightly enhanced initial cell growth. Morphological changes were determined with the use of rhodaminephalloidin staining of actin. Retinoic acid caused an increase in the fraction of neuroblast cell in SK-N-SH, and conversely of nonneuronal cells in SH-SY5Y and Kelly cell lines. Both drugs also caused partial differentiation towards a neuronal phenotype, and herb-A induced selective lysis of nonneuronal cells of SK-N-SH. Because of their discrepant effects, RA (10 microM) and herb-A (236 nM) were tested in combination at a concentration that had only moderate effects when given alone. The combination further reduced cell growth and colony formation and dramatically enhanced differentiation towards a neuronal morphology. The Kelly cell line with amplified N-myc genome, which correlates with clinical progression of neuroblastoma, was also sensitive to RA plus herb-A. These results recommend the combination of RA and herb-A for differentiation therapy of neuroblastoma.

Topics: Anti-Bacterial Agents; Benzoquinones; Cell Differentiation; Drug Combinations; Humans; Lactams, Macrocyclic; Neuroblastoma; Neurons; Quinones; Receptors, Opioid; Rifabutin; Tretinoin; Tumor Cells, Cultured; Tumor Stem Cell Assay