Abstract Normal progression of the cell cycle requires sequential expression of cyclins. Rapid induction of cyclin D1 and its associated binding with cyclin-dependent kinases, in the presence or absence of mitogenic signals, is considered a rate-limiting step during cell cycle progression through the G1 phase. In the present study, human umbilical cord blood stem cells (hUCBSC) co-cultured with glioblastoma cells (U251 and 5310) not only induced G0-G1 phase arrest, but also reduced the number of cells at the S and G2-M phases of the cell cycle. Western and FACS analyses of cell cycle regulatory proteins showed decreased expression levels upon treatment with hUCBSC. Co-localization experiments under in vivo conditions in nude mice brain xenografts with cyclin D1 and CD81 antibodies demonstrated decreased expression of cyclin D1 in the presence of hUCBSC. Treatment with hUCBSC decreased expression of the cyclin D1 and CDK4 complex as well as cyclin D1 and CDK6 complexes. However, there was no significant change in the CDK4 and CDK6 complex. Our results indicate that hUCBSC can attenuate uncontrolled cell cycle progression of glioma cells by downregulating the expression levels of cyclin D1 and its partner kinases CDK4 and CDK6 at the cell cycle level. Our data elucidate a model to regulate glioma cell cycle progression in which hUCBSC act to control cyclin D1 induction and its partner kinases, CDK4 and CDK6, by mediating cell cycle arrest via G0-G1 arrest. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3319. doi:10.1158/1538-7445.AM2011-3319
The urokinase-type plasminogen activator (uPA) and uPA receptor (UPAR) play important roles in the proteolytic cascade involved in the invasiveness of gliomas and other invasive tumors. High-level expression of uPAR has been correlated with high-grade glioma cell lines and tumors We report here that down-regulating uPAR levels by antisense strategy using an adenovirus construct (Ad-uPAR) inhibited glioma invasion in Matrigel and spheroid in vitro models. sc. (U87-MG) and intracranial (SNB19) injections of Ad-uPAR-infected glioma cells did not produce tumors in nude mice. However, injection of the Ad-uPAR construct into previously established so U87-MG tumors in nude mice caused regression of those tumors. Our results support the therapeutic potential of targeting the uPA-uPAR system for the treatment of gliomas and other cancers.
Abstract Neuroblastoma is the most common extracranial pediatric solid tumor that is derived from the developing sympathetic nervous system and results from the improper differentiation of neural crest cells. Neuroblastomas show a tremendous clinical heterogeneity ranging from benign ganglioneuromas to highly aggressive immature tumors. Hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. Intermittent hypoxia (IH) which is characterized by cyclic periods of hypoxia and reoxygenation, occurs in tumor cells that are dependent on tumor blood vessels having intermittent perfusion fluctuations in blood flow. The occurrence of IH episodes varies significantly in rapidly growing malignant tumors. Bone is one of the target organs of metastasis in advanced neuroblastoma. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by the osteoclast. The present study focuses on how IH-conditioned neuroblastoma cells modulate the differentiation of osteoclasts. IH-conditioned cells were derived by exposing tumor cells to 10 repeated cycles of hypoxia followed by reoxygenation. In order to assess the role of HIF-1α overexpression in osteolysis, we stably transfected human neuroblastoma cells with an expression vector containing a HIF-1α cDNA. Additionally, cells were stably transfected with a plasmid expressing HIF-1α shRNA or luciferase shRNA and selected in neomycin. The expression of HIF-1α protein was analyzed by western blotting in IH-conditioned and stable transfectants of neuroblastoma cells. Changes in gene expression were determined by real-time PCR for IL-8, MCP-1α, PTHrP, CXCR-4 and RANKL and the expression of osteoclastogenic factors was found increased in IH-conditioned and HIF-1α protein overexpressing cells. Conditioned medium collected from IH-conditioned and HIF-1α cDNA overexpressing neuroblastoma cells has shown enhanced osteoclast formation capabilities in the tartrate-resistant acid phosphatase (TRAP) assay. IH-conditioned and HIF-1α protein overexpressing neuroblastoma cells secrete increased amounts of VEGF protein, which has been found to promote osteoclast activity. Calcium-sensing receptor (CaR) plays a pivotal role in osteoclast differentiation. We found enhanced expression of CaR in RAW 264.7 (osteoclast precursors) cells treated with conditioned medium collected from IH-conditioned and HIF-1α protein overexpressing neuroblastoma cells compared with the control. Thus, IH was found to enhance osteolytic capabilities of neuroblastoma cells in vitro in part through HIF-1α protein stabilization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 408. doi:1538-7445.AM2012-408
Tumor vasculature provides the infrastructure by which malignant tissue can be nourished; therefore, targeting angiogenesis may be an effective means of treating cancer. We showed previously that SNB19 glioblastoma cells modulate bovine retinal endothelial cells in cocultures to form capillary-like network structures, that matrix metalloproteinase-9 (MMP-9) expression is critical for endothelial morphogenesis, and that MMP-9 expression in glioblastoma cells is regulated by extracellular signal-regulated kinase-1 (ERK-1). In the present study, we investigated whether interfering with the activation of this mitogen-activated protein (MAP) kinase would repress MMP-9 synthesis and inhibit capillary formation.Cocultures of bovine retinal endothelial and SNB19 cells were analyzed for MMP-9 secretion, and phospho- and total ERK levels. These cocultures were treated with PD98059, a specific inhibitor of MAP/ERK kinase 1, or transfected with dominant-negative ERK-1 mutant containing expression vector. Alterations in capillary-like structure formation, and actin cytoskeleton and secretion of vascular endothelial growth factor (VEGF), MMP-9, and tissue inhibitor of metalloproteinase-1 were determined by immunofluorescence, gelatin zymography, and Western blotting.We found that inhibition of the ERK-1/2 pathway with PD98059 abrogated glial cell-mediated capillary formation by the endothelial cells and reduced the levels of MMP-9 in the coculture. Strikingly, the abrogation of MAP kinase signaling by a dominant-negative ERK-1 mutant inhibited glial-induced capillary network formation by reducing VEGF levels and MMP-9 activity and increasing the levels of tissue inhibitor of metalloproteinase-1. Inhibition of ERK activity also disrupted the formation of the actin cytoskeleton, a prerequisite for endothelial cell migration.The mechanism underlying activation of ERK is involved in reorganization of the actin cytoskeleton, and induction of VEGF and MMP-9, thereby stimulating endothelial cell morphogenesis. These studies clearly provide experimental evidence that ERK inhibition diminishes glial-induced endothelial-cell morphogenesis; therefore, interfering with ERK signaling may be a viable approach to target angiogenesis.
Urokinase-type plasminogen activator receptors (uPARs) play an important role in tumor invasion by localizing degradative enzymes at the invasive zone. In the present study, we examined the presence and distribution of uPARs in human gliomas in vivo. The amounts of uPARs were measured by radioreceptor assays and Northern blotting and were significantly higher in anaplastic astrocytomas and glioblastomas than they were in normal brain tissues and low-grade gliomas. In situ hybridization was performed to investigate the cellular source of uPAR mRNA in various types of astrocytomas and normal brain tissues. uPAR mRNA was localized in astrocytoma cells and endothelial cells within tumor tissue, especially near sites of vascular proliferation and at the leading edges of tumors. uPAR mRNA was also expressed in tumor cells near necrotic areas. Expression was barely detectable in low-grade astrocytomas and normal brain tissues. These results suggest that expression of uPAR in the invading astrocytoma cells may play a significant role in the invasive behaviors of glioblastomas.
Hypoxia is known to induce overexpression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) and thus overexpression promotes uPAR-mediated survival signaling in various cancers. Moreover, hypoxia/ overexpression of uPAR in cancer cells promote the epithelial-mesenchymal transition (EMT) and thereby invasiveness and metastasis. In this study, we show that intermittent hypoxia has a more pronounced effect than chronic hypoxia and contributes to EMT, invasion and migration in medulloblastoma cells. Intermittent hypoxia induced expression of mesenchymal markers (i.e., SNAIL, Vimentin and N-cadherin) and reduced expression of epithelial markers (i.e., Zo-1, E-cadherin) in medulloblastoma cells. Further, intermittent hypoxia also leads to enhancement in cell invasion, migration and angiogenesis in medulloblastoma cells. Intermittent hypoxia also inhibited expression of pro-anti-apoptotic proteins (Bax and Bad), and induced expression of anti-pro-apoptotic proteins (Bcl2 and Bcl-xL), and activation of ERK in medulloblastoma cells. Transcriptional inactivation of either uPA or uPAR inhibits the intermittent hypoxia-induced invasion and migration, and expression of Vimentin. uPA/ uPAR downregulation also induces E-cadherin expression and inhibits activation of ERK. Thus, transcriptional inactivation of either uPA or uPAR enhances the apoptotic response in medulloblastoma cells exposed to intermittent hypoxia. This study provides evidence of the anti-tumor efficacy of down-regulation of uPA or uPAR in medulloblastoma tumors to target hypoxia-induced cell EMT, invasion and migration, to achieve better therapeutic outcomes in the treatment of malignant medulloblastoma.
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