herbimycin and Neuroblastoma

HIV-1 coat protein gp120 is able to kill neuronal cells in culture. Here we address the possible role of protein tyrosine kinases (PTKs) in gp120-induced neurotoxicity using the CHP100 human neuroblastoma cell line as experimental model. For this purpose, the effect of specific PTK inhibitors like genistein, herbimycin A and lavendustin A was evaluated on CHP100 cell death elicited by the viral protein. Here we report that genistein (1-10 microM) significantly reduced the cytotoxic effects induced by gp120 (10 pM). The same protective action was offered by a pre-treatment with herbimycin A (0.1-1 microM) or lavendustin A (1-10 microM). Conversely, daidzein (1-100 microM), a genistein analogue devoid of PTK inhibitory properties, failed to reduce CHP100 cell death caused by gp120. These findings suggest that PTKs can be involved in the signal transduction cascade by which the glycoprotein induces neurotoxicity.

Topics: Benzoquinones; Cell Death; Genistein; HIV Envelope Protein gp120; Humans; Isoflavones; Lactams, Macrocyclic; Neuroblastoma; Phenols; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured

Spontaneous epithelial (S) to neuroblast (N) conversion enhanced the capacity of SK-N-SH neuroblastoma (NB) cells to invade reconstituted basement membrane in vitro. This involved a switch to matrix metalloproteinase (MMP) activity, in particular MMP-9, and was associated with the induction of MMP-9 expression. N-type-specific MMP-9 expression was herbimycin A inhibitable tyrosine kinase (possibly c-src) dependent and was regulated transcriptionally through GT-box (-52), and nuclear factor kappaB (NFkappaB; -600) elements within the MMP-9 gene. GT-box function was associated with elevated levels of specific nuclear GT-box binding complexes in N-type cells. NFkappaB function was associated with specific p50- and p65-containing nuclear NFkappaB binding complex(es). No function could be attributed to the proximal AP-1 (-79) element, and minimal function was attributed to the SP-1 (-560), ets (-540), or distal AP-1 (-533) elements. This was despite elevated levels of specific junD/fra-1 containing proximal AP-1 element binding complex(es) in N-type cells. Our data highlight a pivotal role for the GT-box, in concert with the NFkappaB element, in the transcriptional up-regulation of MMP-9 expression during spontaneous S to N phenotype conversion by SK-N-SH cells involved in enhanced basement membrane invasivity.

Topics: Benzoquinones; Binding Sites; Cell Extracts; Cell Nucleus; Collagenases; CSK Tyrosine-Protein Kinase; Enzyme Inhibitors; Epithelial Cells; Humans; Lactams, Macrocyclic; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neuroblastoma; Neurons; NF-kappa B; Phenotype; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Quinones; Response Elements; Rifabutin; Sp1 Transcription Factor; src-Family Kinases; Transcription Factor AP-1; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation

Modulation of Ca2+ channel activity by protein kinases constitutes one of the major mechanisms regulating neuronal functions. Here, we explored the possible modulation of neuronal Ca2+ channels by protein tyrosine kinases (PTKs). To this end, the effects of PTK inhibitors on whole-cell Ba2+ currents (IBa) through voltage-gated Ca2+ channels were analysed in differentiated NG108-15 neuroblastoma x glioma hybrid cells. Genistein suppressed IBa in a concentration-dependent fashion (IC50 = 22 microM). Although daidzein, an analogue of genistein that is devoid of PTK inhibitory activity, also suppressed IBa, we estimated that specific PTK inhibition by genistein reduced IBa amplitude by 30%. In addition, lavendustin A (20 microM) and herbimycin A (20 microM), two other distinct PTK inhibitors, depressed IBa by 22% and 20%, respectively. Genistein suppressed N-type and T-type currents, sparing L-type current, and its effect was independent of G protein activation. The results suggest that the activity of neuronal Ca2+ channels can be modulated by PTKs, opening the possibility that some of the functions of PTKs in the nervous system are mediated by Ca2+ channel modulation.

Topics: Animals; Benzoquinones; Calcium Channel Blockers; Calcium Channels; Electric Conductivity; Enzyme Inhibitors; Genistein; Glioma; GTP-Binding Proteins; Hybrid Cells; Isoflavones; Kinetics; Lactams, Macrocyclic; Neuroblastoma; Nifedipine; omega-Conotoxin GVIA; Peptides; Phenols; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tumor Cells, Cultured

IMR32, a neuroblastoma cell line, and CADO LC6, a small cell lung cancer (SCLC) cell line, extended neurite-like processes when cultured on fibronectin (FN)-coated surfaces or cultured in a serum-free medium. Monoclonal antibodies against the integrin beta 1 subunit inhibited this process formation, suggesting that their morphological change is initiated by beta 1 integrin-dependent signal transduction to the cell interior. Anti-phosphorylation level of a 100-kDa protein, but not 125-kDa focal adhesion kindase, correlated well with the morphological change in both cell lines. This 100-kDa protein phosphorylation did not accompany FN-induced morphological changes in NIH 3T3 fibroblasts or A549 adenocarcinoma cells. These findings suggest that neuroblastoma and SCLC may share beta 1 integrin-mediated signaling events distinct from nonneuronal cells.

Topics: Adenocarcinoma; Benzoquinones; Blotting, Western; Carcinoma, Small Cell; Cell Adhesion; Cell Adhesion Molecules; Cell Differentiation; Enzyme Inhibitors; Flow Cytometry; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin beta1; Lactams, Macrocyclic; Lung Neoplasms; Neuroblastoma; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Receptor, Insulin; Rifabutin; Signal Transduction; Tumor Cells, Cultured; Tyrosine

We investigated for the first time the effect of lipopolysaccharide and the signal transduction pathway on the biosynthesis of tetrahydrobiopterin [(6R-L-erythro-1',2'-dihydroxypropyl) -2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine], the cofactor for the enzymatic hydroxylation of the aromatic amino acids, in the murine neuroblastoma cell line N1E-115, which synthesizes tetrahydrobiopterin constitutively. Activation of N1E-115 cells with 1 microgram/ml lipopolysaccharide resulted in statistically significant increases in both intracellular tetrahydrobiopterin contents and the activity (Vmax) of GTP cyclohydrolase I, a rate-limiting enzyme in tetrahydrobiopterin de novo biosynthesis. Following simultaneous addition of the inhibitors of protein tyrosine kinases and GTP-binding proteins into serum-free culture media with lipopolysaccharide, we analyzed the transduction pathway of lipopolysaccharide signal toward the tetrahydrobiopterin biosynthetic system in N1E-115 cells. Our data indicate the following conclusions: (a) Protein tyrosine kinase systems are involved in mediating lipopoly-saccharide signal to tetrahydrobiopterin production, and (b) there may be a cross-talk between GTP-binding protein and the protein tyrosine kinase system in mediating lipopolysaccharide signal. These observations suggest that a neuronal cell such as N1E-115, which barely expresses CD14 on its cell surface, responds to lipopolysaccharide like macrophages and monocytes in the absence of soluble CD14.

Topics: Animals; Anti-Bacterial Agents; Antioxidants; Benzoquinones; Biopterins; Dose-Response Relationship, Drug; Enzyme Inhibitors; GTP Cyclohydrolase; Hydroquinones; Lactams, Macrocyclic; Lipopolysaccharides; Mice; Neuroblastoma; Quinones; Ribonucleosides; Rifabutin; Signal Transduction; Tumor Cells, Cultured

The relationships between the integrin-mediated activation of inward rectifyier K+ channels (KIR), the phosphorylation of pp125FAK and the rescue of neuritogenesis were studied in 41A3 mouse neuroblastoma cells. Neuritogenesis, elicited by adhesion to FN-enriched substrata, was reversibly impaired by pretreating these cells with the tyrosine kinase inhibitor Herbimycin A. This impairment mimicked that operated by Cs+ ions, which selectively inhibited the integrin-mediated activation of KIR channels. Various phosphotyrosine containing cellular proteins underwent a marked increase upon cell adhesion to FN-coated dishes. This increase was significantly reduced by Cs+ addition. Immunoprecipitation of pp125FAK revealed that the phosphorylation of this kinase and several associated proteins was significantly and reversibly inhibited by Cs+, indicating that integrin-mediated activation of KIR channels is a limiting step upstream to the phosphorylation of pp125FAK in the commitment to neuritogenesis.

Topics: Animals; Benzoquinones; Cell Adhesion Molecules; Cell Line; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Lactams, Macrocyclic; Membrane Potentials; Mice; Neoplasm Proteins; Neurites; Neuroblastoma; Patch-Clamp Techniques; Phosphoproteins; Phosphorylation; Phosphotyrosine; Potassium Channels; Potassium Channels, Inwardly Rectifying; Protein-Tyrosine Kinases; Quinones; Receptor, Insulin; Rifabutin; Tumor Cells, Cultured; Tyrosine

Neuropeptide Y (NPY) attenuated angiotensin II (AII)-or bradykinin (BK)-induced Ca2+ release from intracellular stores and inhibited forskolin-stimulated cAMP accumulation and omega-conotoxin-sensitive high K(+)-induced Ca2+ influx in the human neuroblastoma cell line SMS-KAN. All three NPY actions were mediated via Y2 receptors. Pretreatment with pertussis toxin completely abolished all of the NPY actions. Activation or down-regulation of protein kinase C had no effect on any NPY-mediated effect; herbimycin A, a tyrosine kinase inhibitor, only abolished the inhibitory effect of NPY on AII- or BK-induced Ca2+ mobilization. Herbimycin A also blocked platelet-derived growth factor-induced Ca2+ mobilization, which involves tyrosine kinase activation, and there was a good correlation in the concentration dependency between the two effects of herbimycin A, strongly suggesting that its ability to cancel the NPY effect is due to inhibition of tyrosine kinase activity. NPY attenuated AII- or BK-induced inositol 1,4,5-trisphosphate production, and herbimycin A reversed this NPY effect. These results provide the first evidence that Y2 receptors negatively couple to AII- or BK-induced phosphoinositide turnover leading to Ca2+ mobilization through pertussis toxin-sensitive GTP-binding protein(s). Inhibition of phospholipase C-beta activity by NPY seems to be mediated by activation of protein-tyrosine kinase or phosphotyrosine-containing protein(s).

Topics: Angiotensin II; Benzoquinones; Bradykinin; Calcium; Cyclic AMP; Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Lactams, Macrocyclic; Neuroblastoma; Neuropeptide Y; Pertussis Toxin; Potassium; Quinones; Receptors, Neuropeptide Y; Rifabutin; Second Messenger Systems; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Virulence Factors, Bordetella

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