BackgroundNeuroblastomas are the most common extracranial solid tumors in children. Neuroblastomas are derived from immature cells of the sympathetic nervous system and are characterized by clinical and biological heterogeneity. Hypoxia has been linked to tumor progression and increased malignancy. Intermittent hypoxia or repeated episodes of hypoxia followed by re-oxygenation is a common phenomenon in solid tumors including neuroblastoma and it has a significant influence on the outcome of therapies. The present study focuses on how intermittent hypoxia modulates the stem-like properties and differentiation in neuroblastoma cells.Methods and FindingsCell survival was assessed by clonogenic assay and cell differentiation was determined by morphological characterization. Hypoxia-inducible genes were analyzed by real-time PCR and Western blotting. Immunofluorescence, real-time PCR and Western blotting were utilized to study stem cell markers. Analysis of neural crest / sympathetic nervous system (SNS) markers and neuronal differentiation markers were done by real-time PCR and Western blotting, respectively. Intermittent hypoxia stimulated the levels of HIF-1α and HIF-2 α proteins and enhanced stem-like properties of neuroblastoma cells. In intermittent hypoxia-conditioned cells, downregulation of SNS marker genes and upregulation of genes expressed in the neural crest were observed. Intermittent hypoxia suppressed the retinoic acid-induced differentiation of neuroblastoma cells.ConclusionsOur results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.
Matrix Metalloproteinase-9 (MMP-9) consists of a prodomain, catalytic domain with 3 fibronectin-like type II modules and C-terminal hemopexin-like (PEX) domain. These domains play distinct roles in terms of proteolytic activity, substrate binding and interaction with inhibitors and receptors. To assess the potential of the MMP-9-PEX domain to interfere with tumor progression, we stably transfected human glioblastoma cells with an expression vector containing a cDNA sequence of the MMP-9-PEX. The selected clones exhibited decreased MMP-9 activity and reduced invasive capacity. We assessed how secretion of MMP-9-PEX by glioblastoma cells affects angiogenic capabilities of human microvascular endothelial cells (HMECs) in vitro. MMP-9-PEX conditioned medium treatment caused a reduction in migration of HMECs and inhibited capillary-like structure formation in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level. The suppression of HMECs survival by conditioned medium from MMP-9-PEX stable transfectants was associated with apoptosis induction characterized by an increase in cells with a sub-G 0 /G 1 content, fragmentation of DNA, caspase-3, -8 and -9 activation and poly (ADPribose) polymerase (PARP) cleavage. A significant tumor growth inhibition was observed in intracranial implants of MMP-9-PEX stable transfectants in nude mice with attenuation of CD31 and MMP-9 protein expression. These results demonstrate that MMP-9-PEX inhibits angiogenic features of endothelial cells and retards intracranial glioblastoma growth. ' 2008 Wiley-Liss, Inc.Key words: MMP-9; glioblastoma; angiogenesis; tumor growth inhibition Glioblastomas are the most common and malignant central nervous system tumors.1 Current therapeutic modalities include combinations of surgery, radiotherapy and chemotherapy; however, these therapies remain ineffective and patients with glioblastoma multiform have a median survival time of less than 1 year. [2][3][4] Glioblastomas are very aggressive, characterized by a high proliferation rate, extensive angiogenesis and marked tumor cell invasion into normal brain parenchyma. 4,5 The process of neovascularization involves a complex series of sequential events targeted in the activation of matrix degrading enzymes and motility.6,7 Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) play an important role in the degradation of extracellular matrix components and are involved in a multitude of physiological and pathological processes.8-10 MMP-2 and MMP-9 are the 2 most abundant matrix metalloproteinases (MMPs) found in gliomas. 11-13Like most MMPs, MMP-2 and MMP-9 contain an N-terminal prodomain, catalytic zinc-binding domain and the C-terminal hemopexinlike (PEX) domain 9,14 but differ from other MMPs by containing 3 fibronectin type II repeats within the catalytic domain. 15The PEX domain of various MMPs has been reported to have biological activities independent of the full-length enzyme. [16][17][18] MMP-2-PEX has a role in ...
The hypoxic microenvironment of solid tumors is associated with malignant progression and it renders tumors more resistant to cancer therapies. Endothelial cell damage may occur following hypoxic conditions and lead to dysfunction; however, endothelial cells in tumors survive hypoxic conditions providing nutrients and oxygen to facilitate tumor growth. In this study, we investigated the effects of tumor-conditioned medium on hypoxia-induced changes in endothelial cell growth, migration and survival. Tumor conditioned medium collected from U87 human glioblastoma cells were applied to endothelial cultures in normoxia or hypoxia conditions. Hypoxia caused a reduction in clonogenic cell survival response and an increase of the sub-G 1 phase of the cell cycle in endothelial cells. Cell migration was measured by spheroid and wound-induced migration assays and hypoxia compared with normoxia significantly increased the number of migrating endothelial cells. Nuclear staining with Hoechst 33258 and caspase-9 and-3 activation in endothelial cells show that hypoxia-induced apoptosis involves caspase-dependent mechanism. Exposure to hypoxia caused an increase in gene expression of VEGF and VEGFR2 and activities of MMP-2 and MMP-9. Furthermore, hypoxia induced an increase in capillary-like structure formation in endothelial cells seeded into Matrigel. Tumor conditioned medium enhanced survival and rescued endothelial cells from apoptosis induced by hypoxia. These molecular changes in endothelial cells could, in part, contribute to the angiogenic response that occurs during hypoxia-induced angiogenesis in glial tumors.
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