Sanjeeva Mohanam

Downregulation of uPA inhibits migration and PI3k/Akt signaling in glioblastoma cells

The ability of glioma cells to migrate great distances from a primary tumor mass is the primary cause of tumor recurrence. The urokinase-type plasminogen activator (uPA) is a serine protease that can initiate proteolytic cascades, which result in remodeling of extracellular matrix and basement membrane, allowing cells to move across and through these barriers. The binding between uPA and its receptor uPAR also mediates several signaling events that seem to contribute to the evolution of a migratory phenotype. In this study, we determined how the downregulation of uPA affects the signaling pathways leading to cell migration. Stably transfecting human glioblastoma cells with antisense uPA decreased the amount of cell-bound uPA and disrupted actin cytoskeleton formation and cell migration. The phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathway has been suggested to mediate migration in various cancer cells. The antisense-uPA clones also had less phosphorylated PI3k and Akt than control cells, a finding associated with decreased cell migration, G2/M-phase arrest, and decreased clonogenic survival. Decreased activation of PI3k and the antiapoptotic factor Akt was not sufficient to induce apoptosis in the antisense-uPA clones, but staurosporine sensitized them to apoptosis to a greater extent than control cells. These results indicate that PI3k/Akt pathway is involved in the signaling cascade required to induce cell migration and that uPA has a direct role in regulating migration.

Down-regulation of cathepsin B expression impairs the invasive and tumorigenic potential of human glioblastoma cells

Increases in abundance of cathepsin B transcript and protein correlate with increases in tumor grade and alterations in subcellular localization and activity of cathepsin B. The enzyme is able to degrade the components of the extracellular matrix (ECM) and activate other proteases capable of degrading ECM. To investigate the role played by this protease in the invasion of brain tumor cells, we transfected SNB19 human glioblastoma cells with a plasmid containing cathepsin B cDNA in antisense orientation. Control cells were transfected with vector alone. Clones expressing antisense cathepsin B cDNA exhibited significant reductions in cathepsin B mRNA, enzyme activity and protein compared to controls. Matrigel Invasion assay showed that the antisense-transfected cells had a markedly diminished invasiveness compared with controls. When tumor spheroids containing antisense transfected SNB19 cells expressing reduced cathepsin B were co-cultured with fetal rat brain aggregates, invasion of fetal rat brain aggregates was significantly reduced. Green Fluorescent Protein (GFP) expressing parental cells and antisense transfectants were generated for detection in mouse brain tissue without any post-chemical treatment. Intracerebral injection of SNB19 stable antisense transfectants resulted in reduced tumor formation in nude mice. These results strongly support a role for cathepsin B in the invasiveness of human glioblastoma cells and suggest cathepsin B antisense may prove useful in cancer therapy.

Blockade of cathepsin B expression in human glioblastoma cells is associated with suppression of angiogenesis

The cysteine proteinase cathepsin B has been implicated in tumor progression by virtue of its increased mRNA and protein levels, as well as its localization at the invading front of the tumor. In this study, we examined whether blocking cathepsin B expression in human glioblastoma SNB19 cells affects angiogenesis. Stable transfectants of human glioblastoma cells with a plasmid containing antisense cathepsin B cDNA showed decreased migration rates in wound- and spheroid-migration assays. Analysis showed a reduction in VEGF protein and MMP-9 activity in the cathepsin B antisense cDNA-transfected cells. Regarding angiogenesis in vitro, we found that the conditioned medium of glioblastoma cells with downregulated cathepsin B expression reduced cell–cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary-like network formation. Furthermore, a marked reduction in microvasculature development was seen in an in vivo dorsal air sac assay of glioblastoma cells with downregulated cathepsin B expression. Taken together, these results provide evidence that inhibition of cathepsin B expression can suppress glioblastoma-induced neovascularization.

A novel function of tissue factor pathway inhibitor-2 (TFPI-2) in human glioma invasion.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G, and plasma kallikrein but not urokinase-type plasminogen activator, tissue plasminogen activator, or thrombin. Preliminary findings in our laboratory suggested that the expression of TFPI-2 is downregulated or lost during tumor progression in human gliomas. To investigate the role of TFPI-2 in the invasiveness of brain tumors, we stably transfected the human high-grade glioma cell line SNB19 and the human low-grade glioma cell line Hs683 with a vector capable of expressing a transcript complementary to the full-length TFPI-2 mRNA in either sense (0.7 kb) or antisense (1 kb) orientations. Parental cells and stably transfected cell lines were analysed for TFPI-2 protein by Western blotting and for TFPI-2 mRNA by Northern blotting. The levels of TFPI-2 protein and mRNA were higher in the sense clones (SNB19) and decreased in the antisense (Hs683) clones than in the corresponding parental and vector controls. In spheroid and matrigel invasion assays, the SNB19 parental cells were highly invasive, but the sense-transfected SNB-19 clones were much less invasive; the antisense-transfected Hs683 clones were more invasive than their parental and vector controls. After intracerebral injection in mice, the sense-transfected SNB19 clones were less able to form tumors than were their parental and vector controls, and the antisense-Hs683 clones but not the parental or vector controls formed small tumors. This is the first study to demonstrate that down- or upregulation of TFPI-2 plays a significant role in the invasive behavior of human gliomas.

Downregulation of uPAR and Cathepsin B Induces Apoptosis via Regulation of Bcl-2 and Bax and Inhibition of the PI3K/Akt Pathway in Gliomas

BACKGROUND Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU)-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.

The hemopexin domain of MMP-9 inhibits angiogenesis and retards the growth of intracranial glioblastoma xenograft in nude mice

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‐G0/G1 content, fragmentation of DNA, caspase‐3, ‐8 and ‐9 activation and poly (ADP‐ribose) 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.

Modulation of invasive properties of human glioblastoma cells stably expressing amino-terminal fragment of urokinase-type plasminogen activator

The binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR) on the surface of tumor cells is involved in the activation of proteolytic cascades responsible for the invasiveness of those cells. The diffuse, extensive infiltration of glioblastomas into the surrounding normal brain tissue is believed to rely on modifications of the proteolysis of extracellular matrix components; blocking the interaction between uPA and uPAR might be a suitable approach for inhibiting glioma tumorigenesis. We assessed how expression of an amino-terminal fragment (ATF) of uPA that contains binding site to uPAR affects the invasiveness of SNB19 human glioblastoma cells. SNB19 cells were transfected with an expression plasmid (pcDNA3-ATF) containing a cDNA sequence of ATF-uPA. The resulting ATF-uPA-expressing clones showed markedly less cell adhesion, spreading, and clonogenicity than did control cells. Endogenous ATF expression also significantly decreased the invasive capacity of transfected glioblastoma cells in Matrigel and spheroid-rat brain cell aggregate models. ATF-uPA transfectants were also markedly less invasive than parental SNB19 cells after injection into the brains of nude mice, suggesting that competitive inhibition of the uPA-uPAR interaction on SNB19 cells by means of transfection with ATF cDNA could be a useful therapeutic strategy for inhibiting tumor progression.

Downregulation of uPA/uPAR inhibits intermittent hypoxia-induced epithelial-mesenchymal transition (EMT) in DAOY and D283 medulloblastoma cells

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.

Intermittent hypoxia regulates stem-like characteristics and differentiation of neuroblastoma cells.

Background Neuroblastomas 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 Findings Cell 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. Conclusions Our results suggest that intermittent hypoxia enhances stem-like characteristics and suppresses differentiation propensities in neuroblastoma cells.

Gadd45a sensitizes medulloblastoma cells to irradiation and suppresses MMP-9-mediated EMT.

Medulloblastomas are the most common malignant tumors of the central nervous system during childhood. Radiation-induced medulloblastoma tumor recurrences are aggressive and metastatic in nature. In the present study, we demonstrate that Gadd45a expression can sensitize medulloblastoma cells to radiotherapy. We have elucidated the role of Gadd45a in ionizing radiation (IR)-induced G2-M arrest and invasion and metastatic potential of the medulloblastoma cancer cell lines DAOY and D283. We demonstrate that Gadd45a is induced by IR and results in p53 phosphorylation. The role of IR-induced Gadd45a in G2-M arrest is demonstrated by fluorescence-activated cell sorting analysis in the cells treated with siRNA Gadd45a and Ov-exp Gadd45a. We show that Ov-exp Gadd45a aggravates G2-M blockage and also increases binding of Gadd45a to Cdc2 by immunocytochemistry analysis. Furthermore, we show the anti-tumorigenic role of Gadd45a to be mediated by the negative regulation of IR-induced cancer cell invasion and migration-associated proteins, such as matrix metallopeptidase (MMP)-9 and β-catenin. When compared with IR treatment alone, Ov-exp Gadd45a plus IR treatment resulted in decreased nuclear localization and increased membrane localization of β-catenin, and this was further confirmed by membrane distribution. We also show that Ov-exp Gadd45a resulted in downregulation of MMP-9 and suppression of epithelial-mesenchymal transition (EMT). Alternatively, inhibition of MMP-9 (pM) resulted in upregulation of Gadd45a and suppression of EMT. The anti-tumor effect of pM was correlated with increased expression of Gadd45a protein in nude mice intracranial tumors. Taken together, our studies demonstrate that upregulation of Gadd45a or suppression of MMP-9 (pM) with IR retards medulloblastoma tumor metastatic potential.