herbimycin and Disease-Models--Animal

Overexpression and activation of TPM3-ALK tyrosine kinase fusion protein is a causal oncogenic event in the development of Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic ALK-positive tumors. Thus, the development of ALK specific tyrosine kinase inhibitors is a current therapeutic challenge. Animal models are essential to assess, in vivo, the efficiency of ALK-oncogene inhibitors and to identify new and/or additional therapeutic targets in the ALK tumorigenesis pathway. Using the tetracycline system to allow conditional and concomitant TPM3-ALK and luciferase expression, we have developed a unique transplant model for bioluminescent TPM3-ALK-induced fibroblastic tumors in athymic nude mice. The reversible TPM3-ALK expression allowed us to demonstrate that this oncogene is essential for the tumor growth and its maintenance. In addition, we showed that this model could be used to precisely assess tumor growth inhibition upon ALK chemical inactivation. As proof of principle, we used the general tyrosine kinase inhibitor herbimycin A to inhibit ALK oncoprotein activity. As expected, herbimycin A treatment reduced tumor growth as assessed both by tumor volume measurement and bioluminescent imaging. We conclude that this transplant model for TPM3-ALK-induced tumors represents a valuable tool not only to accurately and rapidly evaluate in vivo ALK-targeted therapies but also to gain insight into the mechanism of ALK-positive tumor development.

Topics: Anaplastic Lymphoma Kinase; Animals; Antibiotics, Antineoplastic; Benzoquinones; Cell Transformation, Neoplastic; Disease Models, Animal; Drug Screening Assays, Antitumor; Genes, Reporter; Lactams, Macrocyclic; Luciferases; Luminescent Agents; Lymphoma, Large-Cell, Anaplastic; Mice; Mice, Nude; Neoplasm Transplantation; Oncogene Proteins, Fusion; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; Rifabutin; Tropomyosin

Pilocarpine-induced status epilepticus (SE) causes widespread tyrosine phosphorylation in the brain. It has been postulated that this intracellular signal may mediate potentially epileptogenic changes in the morphology and physiology of particular brain regions, including the hippocampus. The present study evaluated the effects of herbimycin A, a protein tyrosine kinase (PTK) inhibitor, over the acute (during which intense biochemical and electrophysiological activation occurs) and the chronic phase (characterized by spontaneous and recurrent epileptic seizures and the presence of synaptic reorganization, e.g., mossy fiber sprouting) of the pilocarpine model of epilepsy. The administration of a single dose of 1.74 nmol of herbimycin A (i.c.v., 5 microL) 5 min after the onset of SE did not change the acute behavioral manifestation of seizures despite significantly decreasing c-Fos immunoreactivity in different areas of the hippocampus and of the limbic cortex. Herbimycin-treated animals developed spontaneous recurrent seizures, as did control animals, with a similar latency for the appearance of the first seizure and similar seizure frequency. Neo-Timm staining revealed that all animals experiencing SE, regardless of whether or not injected with herbimycin, showed aberrant mossy fiber sprouting in the supragranular region of the dentate gyrus. Herbimycin did not obviously affect neuronal cell death as evaluated in Nissl-stained sections. These results indicate that the PTK blockade achieved with the current dose of herbimycin reduced the acute c-Fos expression but failed to alter the spontaneous seizure frequency or to attenuate the morphological modifications triggered by the SE.

Topics: Analysis of Variance; Animals; Benzoquinones; Cell Count; Cell Death; Disease Models, Animal; Drug Administration Schedule; Drug Interactions; Enzyme Inhibitors; Epilepsy, Temporal Lobe; Immunohistochemistry; Lactams, Macrocyclic; Male; Muscarinic Agonists; Pilocarpine; Proto-Oncogene Proteins c-fos; Quinones; Rats; Rats, Wistar; Rifabutin

The pathogenesis of retinopathy of prematurity involves dysregulated angiogenesis resulting in pre-retinal growth of new vessels. Inhibition of tyrosine kinase-dependent pro-angiogenic signals may provide a rational therapeutic approach to the reduction of pre-retinal neovascularization. Vascular endothelial growth factor stimulates endothelial cell mitogenesis, differentiation and migration, by binding and activating the receptor tyrosine kinases vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2. One of the vascular endothelial growth factor receptor substrates implicated in vascular endothelial growth factor signal transduction is c-Src. The ability of herbimycin A, a c-Src-selective tyrosine kinase inhibitor, to inhibit vascular endothelial growth factor-induced bovine retinal microvascular endothelial cell proliferation and tube formation was investigated. The ability of the compound to inhibit pathologic angiogenesis was tested in a rat model of retinopathy of prematurity. Exposure of neonatal rats to oxygen concentrations cycling between 10 and 50% induced severe pre-retinal neovascularization in all rats. Some of the eyes of these variable oxygen-exposed rats were herbimycin A-injected or vehicle-injected 1 or 3 days post-oxygen exposure while some eyes were non-injected. All rats were sacrificed for assessment 6 days post-exposure. Herbimycin A inhibited both vascular endothelial growth factor-induced bovine retinal microvascular endothelial cell proliferation and capillary tube formation in a dose-dependent manner. Injection of herbimycin A into oxygen-treated rats 1 day post-oxygen exposure produced a 63% decrease in pre-retinal neovascularization relative to vehicle (P = 0.0029). There was a 41% decrease in pre-retinal neovascularization in herbimycin-injected eyes relative to vehicle-injected eyes 3 days post-oxygen (P = 0.031). Pre-retinal neovascularization was reduced in vehicle-injected eyes relative to non-injected eyes at both injection times. There were no significant differences in retinal vascular area between any of the experimental groups. Based on the results of this study, herbimycin A inhibits endothelial cell proliferation and tube formation at non-toxic concentrations and reduces pre-retinal neovascularization in a rat model of retinopathy of prematurity. Reduction of angiogenesis by the inhibition of tyrosine kinase activity may be a viable route to the development of effective che

Topics: Animals; Benzoquinones; Cell Division; Cells, Cultured; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Humans; Infant, Newborn; Lactams, Macrocyclic; Protein-Tyrosine Kinases; Quinones; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinal Vessels; Retinopathy of Prematurity; Rifabutin; Vascular Endothelial Growth Factor A

Expression of heat shock proteins (HSPs) as a heat stress response is associated with acquisition of thermotolerance. Herbimycin A is a tyrosine kinase inhibitor that has been shown to induce HSPs. The present study aims to investigate the effects of herbimycin A on thermotolerance in rats subjected to heat stress exposure. Herbimycin A induced hsp70 to peak levels 12 h post-injection in rats without heat stress. No change in hsp70 levels was observed in the vehicle- and saline-treated rats. In rats exposed to heat stress at 45 degrees C for 25 min, 12 h post-treatment, lower peak temperatures were attained in herbimycin A-treated group as compared to the vehicle- and saline-treated groups. Terminal transferase-mediated d-UTP nick end labeling (TUNEL) showed that a significant decrease in apoptosis of hepatocytes in herbimycin A-treated rats as compared to the vehicle- and saline-treated rats. Caspase-3 activation was also lower in herbimycin A-treated rats, compared to the vehicle- and saline-treated rats. The present study has demonstrated that herbimycin A is effective for development of thermotolerance and therefore protects rats from heat stress.

Topics: Animals; Apoptosis; Benzoquinones; Blotting, Western; Caspase 3; Caspases; Disease Models, Animal; Enzyme Activation; Heat Stress Disorders; HSP70 Heat-Shock Proteins; In Situ Nick-End Labeling; Lactams, Macrocyclic; Liver; Male; Quinones; Rats; Rats, Sprague-Dawley; Rifabutin