ABSTRACT
We previously showed that inhibition of the platelet-derived growth factor receptor (PDGFR) blocks the survival and migration of medulloblastoma cells. Identification of in vitro PDGFR-targeting pharmacologic agents that are suitable for preclinical testing in medulloblastoma models in vivo will be critical for efficiently translating these agents to clinical investigation in children with medulloblastoma. In this study, we investigated whether the multi-tyrosine kinase inhibitor sunitinib, effectively inhibits PDGFR signaling required for medulloblastoma cell migration. Daoy and D556 human medulloblastoma cells pre-treated for 1 h with 0.2 µM sunitinib demonstrated induction of PTEN expression and significant inhibition of PDGFR signaling activity and transactivation of EGFR, in a RAS-independent manner, in response to PDGF-BB stimulation. Sunitinib pre-treatment markedly reduced medulloblastoma cell migration in response to both PDGF-BB and 10% serum at 4 and 24 h after treatment. Pre-treatment with sunitinib for 1 h also resulted in detachment and decreased viability of D556, but not Daoy, cells and only after 48 h following treatment. However, sunitinib did not induce apoptosis in either cell line at any time point, indicating that the anti-migratory effects of sunitinib were not due to impeding cell survival. Sunitinib similarly inhibited PDGFR signaling and migration of primary murine Smo/Smo medulloblastoma cells, suggesting that the Smo/Smo mouse is an appropriate model for preclinical testing of sunitinib. These results indicate that sunitinib may be an important pharmacologic agent for the treatment of invasive medulloblastoma, particularly given evidence of its ability to cross the blood-brain barrier to target tumor cells, and thus warrants further in vivo testing for confirmation of efficacy.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Movement/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Indoles/pharmacology , PTEN Phosphohydrolase/metabolism , Pyrroles/pharmacology , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction/drug effects , Angiogenesis Inducing Agents/pharmacology , Animals , Becaplermin , Cell Count/methods , Cell Line, Tumor , Cells, Cultured , Dose-Response Relationship, Drug , Epidermal Growth Factor/pharmacology , Humans , Medulloblastoma/pathology , Mice , PTEN Phosphohydrolase/genetics , Platelet-Derived Growth Factor/pharmacology , Proto-Oncogene Proteins c-sis , Receptors, Platelet-Derived Growth Factor/genetics , Sunitinib , Time FactorsABSTRACT
Platelet-derived growth factor (PDGF) receptor (PDGFR) expression correlates with metastatic medulloblastoma. PDGF stimulation of medulloblastoma cells phosphorylates extracellular signal-regulated kinase (ERK) and promotes migration. We sought to determine whether blocking PDGFR activity effectively inhibits signaling required for medulloblastoma cell migration and invasion. DAOY and D556 human medulloblastoma cells were treated with imatinib mesylate (Gleevec), a PDGFR tyrosine kinase inhibitor, or transfected with small interfering RNA (siRNA) to PDGFRB to test the effects of blocking PDGFR phosphorylation and expression, respectively. PDGFR cell signaling, migration, invasion, survival, and proliferation following PDGF-BB stimulation, with and without PDGFR inhibition, were measured. PDGF-BB treatment of cells increased PDGFRB, Akt and ERK phosphorylation, and transactivated epidermal growth factor receptor (EGFR), which correlated with enhanced migration, survival, and proliferation. Imatinib (1 µmol/L) treatment of DAOY and D556 cells inhibited PDGF-BB- and serum-mediated migration and invasion at 24 and 48 h, respectively, and concomitantly inhibited PDGF-BB activation of PDGFRB, Akt, and ERK but increased PTEN expression and activity. Imatinib treatment also induced DAOY cell apoptosis at 72 h and inhibited DAOY and D556 cell proliferation at 48 h. siRNA silencing of PDGFRB similarly inhibited signaling, migration, and survival and both siRNA and imatinib treatment inhibited PDGF-BB-mediated EGFR transactivation, indicating that the effects of imatinib treatment are specific to PDGFRB target inhibition. These results indicate that PDGFRB tyrosine kinase activity is critical for migration and invasion of medulloblastoma cells possibly by transactivating EGFR; thus, imatinib may represent an important novel therapeutic agent for the treatment of medulloblastoma.
Subject(s)
Antineoplastic Agents/pharmacology , ErbB Receptors/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/drug effects , Medulloblastoma/pathology , Piperazines/pharmacology , Pyrimidines/pharmacology , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Apoptosis/drug effects , Benzamides , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , ErbB Receptors/metabolism , Humans , Imatinib Mesylate , Neoplasm Invasiveness , PTEN Phosphohydrolase/agonists , PTEN Phosphohydrolase/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction/drug effectsABSTRACT
Ongoing neurogenesis in the adult mammalian dentate gyrus and olfactory bulb is generally accepted, but its existence in other adult brain regions is highly controversial. We labeled newly born cells in adult rats with the S-phase marker bromodeoxyuridine (BrdU) and used neuronal markers to characterize new cells at different time points after cell division. In the neocortex and striatum, we found BrdU-labeled cells that expressed each of the eight neuronal markers. Their size as well as staining for gamma-aminobutyric acid (GABA), glutamic acid decarboxylase 67, calretinin and/or calbindin, suggest that new neurons in both regions are GABAergic interneurons. BrdU and doublecortin-immunoreactive (BrdU+/DCX+) cells were seen within the striatum, suggesting migration of immature neurons from the subventricular zone. Surprisingly, no DCX+ cells were found within the neocortex. NG2 immunoreactivity in some new neocortical neurons suggested that they may instead be generated from the NG2+ precursors that reside within the cortex itself.
