cytochalasin-d and Brain-Neoplasms

G-protein-coupled receptors can couple to different signal transduction pathways in different cell types (termed cell-specific signaling) and can activate different signaling pathways depending on the receptor conformation(s) stabilized by the activating ligand (functional selectivity). These concepts offer potential for developing pathway-specific drugs that increase efficacy and reduce side effects. Despite significant interest, functional selectivity has been difficult to exploit in drug discovery, in part due to the burden of multiple assays. Cellular impedance assays use an emerging technology that can qualitatively distinguish Gs, Gi/o, and Gq signaling in a single assay and is thereby suited for studying these pharmacological concepts. Cellular impedance confirmed cell-specific Gs and Gq coupling for the melanocortin-4 receptor and dual Gi and Gs signaling with the cannabinoid-1 (CB1) receptor. The balance of Gi versus Gs signaling depended on the cell line. In CB1-HEKs, Giand Gs-like responses combined to yield a novel impedance profile demonstrating the dynamic nature of these traces. Cellspecific signaling was observed with endogenous D1 receptor in U-2 cells and SK-N-MC cells, yet the pharmacological profile of partial and full agonists was similar in both cell lines. We conclude that the dynamic impedance profile encodes valuable relative signaling information and is sufficiently robust to help evaluate cell-specific signaling and functional selectivity.

Topics: alpha-MSH; Animals; Biological Assay; Brain Neoplasms; Cell Culture Techniques; Cell Line, Tumor; Cells, Cultured; CHO Cells; Cricetinae; Cricetulus; Cytochalasin D; Dopamine Agonists; Dose-Response Relationship, Drug; Electric Impedance; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Protein alpha Subunits, Gs; Humans; Inhibitory Concentration 50; Kidney; Neuroectodermal Tumors, Primitive, Peripheral; Osteosarcoma; Pertussis Toxin; Receptor, Muscarinic M1; Receptors, Dopamine D2; Receptors, Dopamine D5; Receptors, G-Protein-Coupled; Sensitivity and Specificity; Signal Transduction

Intracranial adoptive transfers of alloreactive cytotoxic T lymphocytes (aCTL) for brain tumor treatment were safe and showed promise in preclinical and early clinical trials. To better understand the endogenous immune responses that may ensue following cellular therapy with aCTL, we examined the ability of microglia to phagocytose aCTL-damaged and undamaged rat 9L gliosarcoma cells in vitro and in vivo. In vitro, 5.5+/-0.9% of microglial cells isolated from adult tumor-bearing rat brains phagocytosed aCTL-damaged 9L cells, whereas microglia did not bind to or ingest undamaged 9L cells. Addition of supernates from either 9L cell cultures or from aCTL+9L co-incubate cell cultures to microglia did not significantly alter their ability to bind to or phagocytose damaged glioma cells even though the latter contained T helper 1 and 2 cytokines. At 3 days following intracranial 9L cell infusion, 17.5+/-0.1% of the microglia phagocytosed CFSE-labeled aCTL-damaged 9L tumor cells within the adult rat brain, confirming the in vitro data. The results suggest that microglia within the tumor microenvironment of the adult rat glioma model selectively remove damaged, but not undamaged, glioma cells.

Topics: Animals; Animals, Newborn; Annexin A5; Brain; Brain Neoplasms; CD11b Antigen; Cell Line, Tumor; Cells, Cultured; Cytochalasin D; Cytotoxicity, Immunologic; Flow Cytometry; Gliosarcoma; Immunotherapy, Adoptive; Microglia; Nucleic Acid Synthesis Inhibitors; Phagocytosis; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; T-Lymphocytes, Cytotoxic; Time Factors; Ultraviolet Rays

Alterations in cytoskeleton and subsequent cell shape changes exert specific effects on the expression of various genes. Our previous results suggested that malignant human gliomas express elevated levels of matrix metalloproteinases compared with normal brain tissue and low grade gliomas. To understand the role of cell shape changes on matrix metalloproteinase expression in human glioma cells, we treated SNB19 cells with cytochalasin-D, an inhibitor of actin polymerization, and colchicine-B, a tubulin inhibitor, in the presence of phorbol 12-myristate 13-acetate. Cytochalasin-D treatment of SNB19 cells resulted in the loss of phorbol 12-myristate 13-acetate-induced matrix metalloproteinase-9 (also known as gelatinase-B) expression and coincided with inhibition of actin polymerization, resulting in cell rounding. Moreover, compared with monolayers, cells grown as spheroids or cell aggregates failed to express matrix metalloproteinase-9 in the presence of phorbol 12-myristate 13-acetate. Matrix metalloproteinase-9 expression was also inhibited by calphostin-C, a protein kinase inhibitor, suggesting the involvement of protein kinase C in matrix metalloproteinase-9 expression. Phorbol 12-myristate 13-acetate-induced invasion of SNB19 cells through Matrigel was inhibited by cytochalasin-D and calphostin-C. These results suggest that the actin polymerization transduces signals that modulate the expression of matrix metalloproteinase-9 expression and the subsequent invasion of human glioma cells.

Topics: Actins; Biopolymers; Brain Neoplasms; Cell Adhesion Molecules; Collagenases; Cytochalasin D; Cytoskeleton; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Glioma; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; NF-kappa B; Protein Kinase C; Protein-Tyrosine Kinases; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Withangulatin A (WA), a newly discovered withanolide isolated from an antitumor Chinese herb, has been shown to be a vimentin intermediate filament-targeting drug by using immunofluorescence microscopy. Together with cytochalasin D and colchicine, these drugs were employed to investigate the importance of vimentin intermediate filaments, actin filaments, and microtubules in the development of acquired thermotolerance in 9L rat brain tumor cells treated at 45 degrees C for 15 min (priming heat-shock). Acquired thermotolerance was abrogated in cells incubated with WA before the priming heat-shock but it could be detected in cells treated with WA after the priming heat-shock. In contrast, cytochalasin D and colchicine do not interfere with the development of thermotolerance at all. The intracellular localizations of vimentin and the constitutive heat-shock protein70 (HSC70) in treated cells were examined by using immunofluorescence microscopy and detergent-extractability studies. In cells treated with WA before the priming heat-shock, vimentin IFs were tightly aggregated around the nucleus and unable to return to their normal organization after a recovery under normal growing conditions. In contrast, the IF network in cells treated with WA after the priming heat-shock was able to reorganize into filamentous form after a recovery period, a behavior similar to that of the cells treated with heat-shock only. HSC70 was found to be co-localized with vimentin during these changes. It is suggested that the integrity of intermediate filaments is important for the development of thermotolerance and that HSC70 may be involved in this process by stabilizing the intermediate filaments through direct or indirect binding.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Carrier Proteins; Cell Division; Colchicine; Cytochalasin D; Cytoskeleton; Hot Temperature; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Intermediate Filaments; Pregnenes; Rats; Tumor Cells, Cultured; Vimentin

Drebrins are developmentally regulated actin-binding proteins. In this study, we analyzed subcellular distribution of drebrin E in neuroblastoma cells (SH-SY5Y) in culture, especially in terms of its relationship to actin filaments. In undifferentiated cells, drebrin E was scattered as flocculus small dots along the stress fibers and also accumulated at adhesion plaques. In parallel with the neuronal differentiation following retinoic acid treatment, drebrin E was accumulated, accompanying filamentous (F) actin, in the submembranous cortical cytoplasm. Similar submembranous localization of drebrins was observed in primary cultured neurons. In the presence of drebrin E F-actin was more stable against cytochalasin D than F-actin lacking drebrin E. These results suggest that drebrin E plays a role in neuronal morphological differentiation by changing its subcellular localization with stabilized F-actin.

Topics: Actins; Brain Neoplasms; Cell Differentiation; Cytochalasin D; Cytoskeletal Proteins; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Immunohistochemistry; Neuroblastoma; Neurons; Neuropeptides; Subcellular Fractions; Tretinoin; Tumor Cells, Cultured