Candece L Gladson

Publications of Candece L Gladson

• Endothelial Expression of TNF Receptor-1 Generates a Proapoptotic Signal Inhibited by Integrin α6β1 in Glioblastoma.

  Authors: Ping Huang, M R Sandhya Rani, Manmeet S Ahluwalia, Eunnyung Bae, Richard A Prayson, Robert J Weil, Amy S Nowacki, Hirad Hedayat, Andrew E Sloan, Justin D Lathia, Jeremy N Rich, Russell Tipps, Candece L Gladson

  Cancer research. 03/2012; 72(6):1428-37.

  Activation of TNF receptor 1 (TNF-R1) can generate signals that promote either apoptosis or survival. In this study, we show that these signals can be determined by the character of the extracellularActivation of TNF receptor 1 (TNF-R1) can generate signals that promote either apoptosis or survival. In this study, we show that these signals can be determined by the character of the extracellular matrix in the tumor microenvironment. Specifically, through studies of glioblastoma, we showed that TNFα stimulation induced apoptosis of primary brain endothelial cells (EC) attached to collagen or fibronectin (which engage integrins α2β1/α3β1 and α5β1, respectively), but did not induce apoptosis of ECs attached to laminin (which engages integrins α6β1 and α3β1). TNF-R1 expression was significantly higher in ECs in glioblastoma (GBM) tumors compared with ECs in normal brain specimens. TNFα was also expressed in GBM tumor-associated ECs, which was associated with longer patient survival. ECs plated on anti-integrin α2 or α3 antibody were susceptible to TNFα-induced apoptosis, whereas those plated on anti-integrin α6 antibody were not. Moreover, the ECs plated on laminin, but not collagen, expressed cellular FLICE inhibitory protein (cFLIP) and TNFα stimulation of laminin-attached cells in which cFLIP had been downregulated resulted in the induction of apoptosis. In contrast, attachment to laminin did not induce cFLIP expression in GBM tumor stem cells. Together, our findings indicate that the laminin receptor integrin α6β1 promotes the survival of brain ECs by inhibiting prodeath signaling by TNF-R1, in part by inducing cFLIP expression. Cancer Res; 72(6); 1428-37. ©2012 AACR.

• Downregulation of FIP200 induces apoptosis of glioblastoma cells and microvascular endothelial cells by enhancing Pyk2 activity.

  Authors: Dongyan Wang, Mitchell A Olman, Jerry Stewart, Russell Tipps, Ping Huang, Paul W Sanders, Eric Toline, Richard A Prayson, Jeongwu Lee, Robert J Weil, Cheryl A Palmer, G Yancey Gillespie, Wei Michael Liu, Russell O Pieper, Jun-Lin Guan, Candece L Gladson

  PloS one. 01/2011; 6(5):e19629.

  The expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies ofThe expression of focal adhesion kinase family interacting protein of 200-kDa (FIP200) in normal brain is limited to some neurons and glial cells. On immunohistochemical analysis of biopsies of glioblastoma tumors, we detected FIP200 in the tumor cells, tumor-associated endothelial cells, and occasional glial cells. Human glioblastoma tumor cell lines and immortalized human astrocytes cultured in complete media also expressed FIP200 as did primary human brain microvessel endothelial cells (MvEC), which proliferate in culture and resemble reactive endothelial cells. Downregulation of endogenous expression of FIP200 using small interfering RNA resulted in induction of apoptosis in the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC. It has been shown by other investigators using cells from other tissues that FIP200 can interact directly with, and inhibit, proline-rich tyrosine kinase 2 (Pyk2) and focal adhesion kinase (FAK). In the human glioblastoma tumor cells, immortalized human astrocytes, and primary human brain MvEC, we found that downregulation of FIP200 increased the activity of Pyk2 without increasing its expression, but did not affect the activity or expression of FAK. Coimmunoprecipitation and colocalization studies indicated that the endogenous FIP200 was largely associated with Pyk2, rather than FAK, in the glioblastoma tumor cells and brain MvEC. Moreover, the pro-apoptotic effect of FIP200 downregulation was inhibited significantly by a TAT-Pyk2-fusion protein containing the Pyk2 autophosphorylation site in these cells. In summary, downregulation of endogenous FIP200 protein in glioblastoma tumor cells, astrocytes, and brain MvECs promotes apoptosis, most likely due to the removal of a direct interaction of FIP200 with Pyk2 that inhibits Pyk2 activation, suggesting that FIP200 expression may be required for the survival of all three cell types found in glioblastoma tumors.

• Targeting SRC in glioblastoma tumors and brain metastases: rationale and preclinical studies.

  Authors: Manmeet S Ahluwalia, John de Groot, Wei Michael Liu, Candece L Gladson

  Cancer letters. 10/2010; 298(2):139-49.

  Glioblastoma (GBM) is an extremely aggressive, infiltrative tumor with a poor prognosis. The regulatory approval of bevacizumab for recurrent GBM has confirmed that molecularly targeted agents haveGlioblastoma (GBM) is an extremely aggressive, infiltrative tumor with a poor prognosis. The regulatory approval of bevacizumab for recurrent GBM has confirmed that molecularly targeted agents have potential for GBM treatment. Preclinical data showing that SRC and SRC-family kinases (SFKs) mediate intracellular signaling pathways controlling key biologic/oncogenic processes provide a strong rationale for investigating SRC/SFK inhibitors, e.g., dasatinib, in GBM and clinical studies are underway. The activity of these agents against solid tumors suggests that they may also be useful in treating brain metastases. This article reviews the potential for using SRC/SFK inhibitors to treat GBM and brain metastases.

• Downregulation of FAK-related non-kinase mediates the migratory phenotype of human fibrotic lung fibroblasts.

  Authors: Guo-qiang Cai, Anni Zheng, Qingjiu Tang, Eric S White, Chu-Fang Chou, Candece L Gladson, Mitchell A Olman, Qiang Ding

  Experimental cell research. 05/2010; 316(9):1600-9.

  Fibroblast migration plays an important role in the normal wound healing process; however, dysregulated cell migration may contribute to the progressive formation of fibrotic lesions in the diseasedFibroblast migration plays an important role in the normal wound healing process; however, dysregulated cell migration may contribute to the progressive formation of fibrotic lesions in the diseased condition. To examine the role of focal-adhesion-kinase (FAK)-related non-kinase (FRNK) in regulation of fibrotic lung fibroblast migration, we examined cell migration, FRNK expression, and activation of focal adhesion kinase (FAK) and Rho GTPase (Rho and Rac) in primary lung fibroblasts derived from both idiopathic pulmonary fibrosis (IPF) patients and normal human controls. Fibrotic (IPF) lung fibroblasts have increased cell migration when compared to control human lung fibroblasts. FRNK expression is significantly reduced in IPF lung fibroblasts, while activation of FAK, Rho and Rac is increased in IPF lung fibroblasts. Endogenous FRNK expression is inversely correlated with FAK activation and cell migration rate in IPF lung fibroblasts. Forced exogenous FRNK expression abrogates the increased cell migration, and blocked the activation of FAK and Rho GTPase (Rho and Rac), in IPF lung fibroblasts. These data for the first time provide evidence that downregulation of endogenous FRNK plays a role in promoting cell migration through FAK and Rho GTPase in fibrotic IPF lung fibroblasts.

• Histidine-rich glycoprotein modulates the anti-angiogenic effects of vasculostatin.

  Authors: Philip A Klenotic, Ping Huang, Juan Palomo, Balveen Kaur, Erwin G Van Meir, Michael A Vogelbaum, Maria Febbraio, Candece L Gladson, Roy L Silverstein

  The American journal of pathology. 02/2010; 176(4):2039-50.

  Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein expressed on glial cells within the brain. Its expression is dramatically down-regulated in many glioblastomas, consistent with itsBrain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein expressed on glial cells within the brain. Its expression is dramatically down-regulated in many glioblastomas, consistent with its functional ability to inhibit angiogenesis and tumor growth in vivo. We have shown that the soluble anti-angiogenic domain of BAI1 (termed Vstat120) requires CD36, a cell surface glycoprotein expressed on microvascular endothelial cells (MVECs), for it to elicit an anti-angiogenic response. We now report that Vstat120 binding to CD36 on MVECs activates a caspase-mediated pro-apoptotic pathway, and this effect is abrogated by histidine-rich glycoprotein (HRGP). HRGP is a circulating glycoprotein previously shown to function as a CD36 decoy to promote angiogenesis in the presence of thrombospondin-1 or -2. Data here show that Vstat120 specifically binds HRGP. Under favorable MVEC growth conditions this interaction allows chemotactic-directed migration as well as endothelial tube formation to persist in in vitro cellular systems, and increased tumor growth in vivo as demonstrated in both subcutaneous and orthotopic brain tumor models, concomitant with an increase in tumor vascularity. Finally, we show that HRGP expression is increased in human brain cancers, with the protein heavily localized to the basement membrane of the tumors. These data help define a novel angiogenic axis that could be exploited for the treatment of human cancers and other diseases where excess angiogenesis occurs.

• Progress on antiangiogenic therapy for patients with malignant glioma.

  Authors: Manmeet S Ahluwalia, Candece L Gladson

  Journal of oncology. 01/2010; 2010:689018.

  Glioblastoma (GBM) is the most common primary brain tumor occurring in America. Despite recent advances in therapeutics, the prognosis for patients with newly diagnosed GBM remains dismal. As theseGlioblastoma (GBM) is the most common primary brain tumor occurring in America. Despite recent advances in therapeutics, the prognosis for patients with newly diagnosed GBM remains dismal. As these tumors characteristically show evidence of angiogenesis (neovascularization) there has been great interest in developing anti-angiogenic therapeutic strategies for the treatment of patients with this disease and some anti-angiogenic agents have now been used for the treatment of patients with malignant glioma tumors. Although the results of these clinical trials are promising in that they indicate an initial therapeutic response, the anti-angiogenic therapies tested to date have not changed the overall survival of patients with malignant glioma tumors. This is due, in large part, to the development of resistance to these therapies. Ongoing research into key features of the neovasculature in malignant glioma tumors, as well as the general angiogenesis process, is suggesting additional molecules that may be targeted and an improved response when both the neovasculature and the tumor cells are targeted. Prevention of the development of resistance may require the development of anti-angiogenic strategies that induce apoptosis or cell death of the neovasculature, as well as an improved understanding of the potential roles of circulating endothelial progenitor cells and vascular co-option by tumor cells, in the development of resistance.

• Plasminogen Kringle 5 Induces Apoptosis of Brain Microvessel Endothelial Cells: Sensitization by Radiation and Requirement for GRP78 and LRP1.

  Authors: Braden C McFarland, Jerry Stewart, Amal Hamza, Robert Nordal, Donald J Davidson, Jack Henkin, Candece L Gladson

  Cancer research. 07/2009;

  Recombinant plasminogen kringle 5 (rK5) has been shown to induce apoptosis of dermal microvessel endothelial cells (MvEC) in a manner that requires glucose-regulated protein 78 (GRP78). As we areRecombinant plasminogen kringle 5 (rK5) has been shown to induce apoptosis of dermal microvessel endothelial cells (MvEC) in a manner that requires glucose-regulated protein 78 (GRP78). As we are interested in antiangiogenic therapy for glioblastoma tumors, and the effectiveness of antiangiogenic therapy can be enhanced when combined with radiation, we investigated the proapoptotic effects of rK5 combined with radiation on brain MvEC. We found that rK5 treatment of brain MvEC induced apoptosis in a dose- and time-dependent manner and that prior irradiation significantly sensitized (500-fold) the cells to rK5-induced apoptosis. The rK5-induced apoptosis of both unirradiated and irradiated MvEC required expression of GRP78 and the low-density lipoprotein receptor-related protein 1 (LRP1), a scavenger receptor, based on down-regulation studies with small interfering RNA, and blocking studies with either a GRP78 antibody or a competitive inhibitor of ligand binding to LRP1. Furthermore, p38 mitogen-activated protein kinase was found to be a necessary downstream effector for rK5-induced apoptosis. These data suggest that irradiation sensitizes brain MvEC to the rK5-induced apoptosis and that this signal requires LRP1 internalization of GRP78 and the activation of p38 mitogen-activated protein kinase. Our findings suggest that prior irradiation would have a dose-sparing effect on rK5 antiangiogenic therapy for brain tumors and further suggest that the effects of rK5 would be tumor specific, as the expression of GRP78 protein is up-regulated on the brain MvEC in glioblastoma tumor biopsies compared with the normal brain. [Cancer Res 2009;69(13):5537-45].

• UROKINASE RECEPTOR MEDIATES LUNG FIBROBLAST ATTACHMENT AND MIGRATION TOWARDS PROVISIONAL MATRIX PROTEINS THROUGH INTERACTION WITH MULTIPLE INTEGRINS.

  Authors: Sha Zhu, Candece L Gladson, Kim E White, Qiang Ding, Jerry E Stewart Jr, Tong Huan Jin, Harold A Chapman Jr, Mitchell A Olman

  American journal of physiology. Lung cellular and molecular physiology. 06/2009;

  Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibit a dependence on urokinase receptor.Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibit a dependence on urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the u-PAR-integrin interactions, and determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR co-localizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin and collagens. Inhibitory antibody studies reveal that human lung fibroblasts utilize alphavbeta5 to attach to vitronectin, predominantly alpha5beta1 (and alphavbeta3) to attach to fibronectin, and alpha1beta1, alpha2beta1 and alpha3beta1 to attach to collagen. Blocking studies with alpha-integrin subunit decoy peptides and u-PAR neutralizing antibodies, indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple alpha integrins, but with a preference for alpha3. Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of u-PAR-integrin interactions can reduce fibroblast migration, and thereby potentially ameliorate fibroproliferative diseases of the lung. Key words: urokinase receptor, fibroblast, matrix.

• Thrombospondin-1-induced apoptosis of brain microvascular endothelial cells can be mediated by TNF-R1.

  Authors: Tanya A Rege, Jerry Stewart, Brian Dranka, Etty N Benveniste, Roy L Silverstein, Candece L Gladson

  Journal of cellular physiology. 09/2008;

  Thrombospondin-1 (TSP-1) treatment of dermal microvascular endothelial cells (MvEC) has been shown to upregulate Fas ligand (FasL) and to induce apoptosis by a mechanism that requires caspase-8Thrombospondin-1 (TSP-1) treatment of dermal microvascular endothelial cells (MvEC) has been shown to upregulate Fas ligand (FasL) and to induce apoptosis by a mechanism that requires caspase-8 activity. We have examined the potential anti-angiogenic effects of TSP-1 on primary human brain MvEC. The addition of TSP-1 to primary human brain MvEC cultured as monolayers on type 1 collagen, induced cell death and apoptosis (evidenced by caspase-3 cleavage) in a dose- (5-30 nM) and time-dependent (maximal at 17 h) manner. TSP-1 treatment for 17 h induced caspase-3 cleavage that required caspase-8 activity and the tumor necrosis factor receptor 1 (TNF-R1). We did not find a requirement for Fas, or the tumor necrosis-related apoptosis-inducing ligand receptors (TRAIL-R) 1 and 2. We confirmed the findings using caspase inhibitors, blocking antibodies and small interfering RNA (siRNA). Further analysis indicated that the TSP-1 induction of caspase-3 cleavage of primary human brain MvEC adherent to collagen required the synthesis of new message and protein, and that TSP-1 induced the expression of TNFalpha mRNA and protein. Consistent with these findings, when the primary human brain MvEC were propagated on collagen gels mAb anti-TNF-R1 reversed the inhibitory effect, in part, of TSP-1 on tube formation and branching. These data identify a novel mechanism whereby TSP-1 can inhibit angiogenesis-through induction of apoptosis in a process mediated by TNF-R1. J. Cell. Physiol. (c) 2008 Wiley-Liss, Inc.

• FAK-related non-kinase inhibits myofibroblast differentiation through differential MAPK activation in a FAK-dependent manner.

  Authors: Qiang Ding, Candece L Gladson, Hongju Wu, Haurko Hayasaka, Mitchell A Olman

  The Journal of biological chemistry. 08/2008;

  Transforming growth factor beta-1 (TGF-ss1) induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinaseTransforming growth factor beta-1 (TGF-ss1) induces fibroblast transdifferentiation to myofibroblasts, a process that requires the involvement of integrin-mediated signaling and focal adhesion kinase (FAK). FAK-related non-kinase (FRNK) is known for its role in inhibiting integrin-mediated cell migration; however, its role in myofibroblast differentiation has not been defined. Here, we report that FRNK abrogates TGF-ss1-induced myofibroblast differentiation in vitro and in vivo. TGF-ss1 can induce a-smooth muscle actin (a-SMA) expression in the presence or absence of FAK; however, TGF-ss1-induced a-SMA expression is reduced (~73%) in FAK-deficient fibroblasts. Although both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation is required for maximal TGF-ss1-induced a-SMA expression, ERK is the major signaling intermediate in cells that express FAK. In contrast, p38 MAPK is the dominant mediator of TGF-ss1-induced a-SMA expression in FAK-deficient cells. FRNK overexpression blocks TGF-ss1-induced ERK or p38 MAPK activation in the presence, and surprisingly, in the absence of FAK. The loss of FRNK was tested in vivo during experimentally-induced pulmonary fibrosis in mice. FRNK-knockout mice have a greater increase in a-SMA-expressing cells in response to a pulmonary fibrotic stimulus in vivo, as compared to congenic wild-type mice. This is the first time that FRNK loss has been shown to modify the pathobiology in any animal disease model. Together, the data demonstrate that FRNK negatively regulates myofibroblast differentiation in vitro and in vivo. These data further suggest that modulation FRNK expression may be a novel avenue for therapeutic intervention in tissue fibrosis.

• Myofibroblast Differentiation Is Negatively Regulated by FAK-related Non-kinase.

  Authors: Qiang Ding, Candece L Gladson, Hongjui Wu, Mitchell A Olman

  Proceedings of the American Thoracic Society. 05/2008; 5(3):364.

• New molecular targets in angiogenic vessels of glioblastoma tumours.

  Authors: Joshua C Anderson, Braden C McFarland, Candece L Gladson

  Expert reviews in molecular medicine. 02/2008; 10:e23.

  Antiangiogenesis approaches have the potential to be particularly effective in the treatment of glioblastoma tumours. These tumours exhibit extremely high levels of neovascularisation, which mayAntiangiogenesis approaches have the potential to be particularly effective in the treatment of glioblastoma tumours. These tumours exhibit extremely high levels of neovascularisation, which may contribute to their extremely aggressive behaviour, not only by providing oxygenation and nutrition, but also by establishing a leaky vasculature that lacks a blood-brain barrier. This leaky vasculature enables migration of tumour cells, as well as the build up of fluid, which exacerbates tissue damage due to increased intracranial pressure. Here, we discuss the considerable progress that has been made in the identification of the pro- and antiangiogenic factors produced by glioblastoma tumours and the effects of these molecules in animal models of the disease. The safety and efficacy of some of these approaches have now been demonstrated in clinical trials. However, the ability of tumours to overcome these therapies and to re-establish angiogenesis requires further clinical research regarding potential multimodality therapies, as well as basic research into the regulation of angiogenesis by as yet unidentified factors. Optimisation of noninvasive procedures for monitoring of angiogenesis would greatly facilitate such research.

• ABT-510, a modified type 1 repeat peptide of thrombospondin, inhibits malignant glioma growth in vivo by inhibiting angiogenesis.

  Authors: Joshua C Anderson, J Robert Grammer, Wenquan Wang, L Burton Nabors, Jack Henkin, Jerry E Stewart, Candece L Gladson

  Cancer biology & therapy. 04/2007; 6(3):454-62.

  Anti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown toAnti-angiogenic therapies would be particularly beneficial in the treatment of malignant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin-1 (TSP-1) have been shown to inhibit angiogenesis in non-glioma tumor models and a modified TSR peptide, ABT-510, has now entered into Phase II clinical trials of its efficacy in non-glioma tumors. As microvascular endothelial cells (MvEC) exhibit heterogeneity, we evaluated the ability of the modified TSR peptide (NAcSarGlyValDallolleThrNvalleArgProNHE, ABT-510) to inhibit malignant glioma growth in vivo and to induce apoptosis of brain microvessel endothelial cells (MvEC) propagated in vitro. We found that daily administration of ABT-510 until euthanasia (days 7 to 19), significantly inhibited the growth of human malignant astrocytoma tumors established in the brain of athymic nude mice. The microvessel density was significantly lower and the number of apoptotic MvEC was significantly higher (3-fold) in the tumors of the ABT-510-treated animals. Similar results were found using a model in which the established tumor is an intracerebral malignant glioma propagated in a syngeneic mouse model. ABT-510 treatment of primary human brain MvEC propagated as a monolayer resulted in induction of apoptosis in a dose- and time-dependent manner through a caspase-8-dependent mechanism. It also inhibited tubular morphogenesis of MvEC propagated in collagen gels in a dose- and caspase-8 dependent manner through a mechanism that requires the TSP-1 receptor (CD36) on the MvEC. These findings indicate that ABT-510 should be evaluated as a therapeutic option for patients with malignant glioma.

• New concepts regarding focal adhesion kinase promotion of cell migration and proliferation.

  Authors: Braden D Cox, Meera Natarajan, Michelle R Stettner, Candece L Gladson

  Journal of cellular biochemistry. 10/2006; 99(1):35-52.

  Focal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that plays a key role in the regulation of proliferation and migration of normal and tumor cells. FAK associates withFocal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that plays a key role in the regulation of proliferation and migration of normal and tumor cells. FAK associates with integrin receptors and recruits other molecules to the site of this interaction thus forming a signaling complex that transmits signals from the extracellular matrix to the cell cytoskeleton. Crk-associated substrate (CAS) family members appear to play a pivotal role in FAK regulation of cell migration. Cellular Src bound to FAK phosphorylates CAS proteins leading to the recruitment of a Crk family adaptor molecule and activation of a small GTPase and c-Jun N-terminal kinase (JNK) promoting membrane protrusion and cell migration. The relocalization of CAS and signaling through specific CAS family members appears to determine the outcome of this pathway. FAK also plays an important role in regulating cell cycle progression through transcriptional control of the cyclin D1 promoter by the Ets B and Kruppel-like factor 8 (KLF8) transcription factors. FAK regulation of cell cycle progression in tumor cells requires Erk activity, cyclin D1 transcription, and the cyclin-dependent kinase (cdk) inhibitor p27Kip1. The ability of FAK to integrate integrin and growth factor signals resulting in synergistic promotion of cell migration and proliferation, and its potential regulation by nuclear factor kappa B (NFkappaB) and p53 and a ubiquitously expressed inhibitory protein, suggest that it is remarkable in its capacity to integrate multiple extracellular and intracellular stimuli.

• A novel technique to quantify glioma tumor invasion using serial microscopy sections.

  Authors: N Shastry Akella, Qiang Ding, Ingrid Menegazzo, Wenquan Wang, G Yancey Gillespie, J Robert Grammer, Candece L Gladson, L Burton Nabors

  Journal of neuroscience methods. 07/2006; 153(2):183-9.

  Here we present a new technique to quantitatively characterize malignant glioma invasion in a syngeneic mouse model. The GL261 mouse malignant glioma cell line was injected intracerebrally into theHere we present a new technique to quantitatively characterize malignant glioma invasion in a syngeneic mouse model. The GL261 mouse malignant glioma cell line was injected intracerebrally into the C57B1/6 black mouse and allowed to propagate for 10 or 17 days, followed by euthanasia of the animal, harvesting of the brain, fixation, and serial sectioning. Histologic examination was performed and the primary tumor mass and discontinuous sites of tumor invasion were traced on digital images of serial microscopy sections, followed by analysis of the invasion characteristics using a custom-written MATLAB program. We found a significant increase in the number of discontinuous tumor invasion sites and in the distance of these sites from the tumor centroid in mice that were euthanized at 17 days post-tumor cell injection, as compared to mice euthanized at 10 days. Furthermore, a scatter plot analyses indicated that the invasion site data could be grouped based on the characteristics of area and distance from the tumor centroid to reveal significant differences between the two experimental groups of mice. This quantitative method will allow a future in vivo analysis of invasion characteristics in glioma cells expressing altered levels or function of invasion genes, and of new therapy targeting invading glioma cells.

• Syndecan-2 is expressed in the microvasculature of gliomas and regulates angiogenic processes in microvascular endothelial cells.

  Authors: Constance Y Fears, Candece L Gladson, Anne Woods

  The Journal of biological chemistry. 06/2006; 281(21):14533-6.

  Angiogenesis is the formation of new blood vessels from the existing vasculature and is necessary for tumor growth. Syndecan-2 (S2) is highly expressed in the microvasculature of mouse gliomas. WhenAngiogenesis is the formation of new blood vessels from the existing vasculature and is necessary for tumor growth. Syndecan-2 (S2) is highly expressed in the microvasculature of mouse gliomas. When S2 expression was down-regulated in mouse brain microvascular endothelial cells (MvEC), this inhibited cell motility and reduced the formation of capillary tube-like structures in vitro. Pro-angiogenic growth factors and enzymes up-regulated during glioma tumorigenesis stimulated shedding of the S2 ectodomain from endothelial cells in vitro. The effect of shed S2 on angiogenic processes was investigated by incorporating recombinant S2 ectodomain (S2ED) into in vitro angiogenesis assays. S2ED promoted membrane protrusion, migration, capillary tube formation, and cell-cell interactions. We therefore propose that S2 is necessary for angiogenesis of MvEC, proangiogenic factors expressed during glioma progression regulate S2 shedding, and shed S2 ectodomain may increase endothelial cell angiogenic processes.

• Low-density lipoprotein receptor-related protein contributes to the antiangiogenic activity of thrombospondin-2 in a murine glioma model.

  Authors: Constance Y Fears, J Robert Grammer, Jerry E Stewart, Douglas S Annis, Deane F Mosher, Paul Bornstein, Candece L Gladson

  Cancer research. 11/2005; 65(20):9338-46.

  Host antiangiogenesis factors defend against tumor growth. The matricellular protein, thrombospondin-2 (TSP-2), has been shown to act as an antiangiogenesis factor in a carcinogen-induced model ofHost antiangiogenesis factors defend against tumor growth. The matricellular protein, thrombospondin-2 (TSP-2), has been shown to act as an antiangiogenesis factor in a carcinogen-induced model of skin cancer. Here, using an in vivo malignant glioma model in which the characteristics of the tumors formed after intracerebral implantation of GL261 mouse glioma cells are assessed, we found that tumor growth and microvessel density were significantly enhanced in tumors propagated in TSP-2(-/-) mice. Mechanistically, matrix metalloproteinase (MMP)-2 has been associated with neoangiogenesis and it has been proposed that the levels of available MMP-2 may be down-regulated by formation of a complex with TSP-2 that is internalized by low-density lipoprotein receptor-related protein 1 (LRP1). We found elevated expression of MMP-2 and MMP-9 in tumors propagated in TSP-2(-/-) mice, with a preferential localization in the microvasculature. In wild-type mice, MMP-2 was coexpressed with TSP-2 in the tumor microvasculature. In vitro, addition of recombinant (rec) TSP-2 to mouse brain microvessel endothelial cells reduced MMP-2 levels and invasion through mechanisms that could be inhibited by a competitive inhibitor of ligand binding to LRP1 or by siLRP1. Thus, the antiangiogenic activity of TSP-2 is capable of inhibiting the growth of gliomas in part by reducing the levels of MMP-2 in the tumor microvasculature. This mechanism is mediated by LRP1.

• The role of the extracellular matrix in angiogenesis in malignant glioma tumors.

  Authors: Dongyan Wang, Joshua C Anderson, Candece L Gladson

  Brain pathology (Zurich, Switzerland). 11/2005; 15(4):318-26.

  Angiogenesis is a promising target for the development of effective strategies for the treatment of malignant brain tumors in that it has the potential to starve large tumors and prevent the regrowthAngiogenesis is a promising target for the development of effective strategies for the treatment of malignant brain tumors in that it has the potential to starve large tumors and prevent the regrowth of residual margins. Two critical steps in angiogenesis, the proliferation of activated endothelial cells and their migration into the perivascular space (sprouting), require adherence of the endothelial cells to the extracellular matrix (ECM). Thus, the availability of the appropriate ligands within the ECM contributes to the regulation of angiogenesis. In addition, several components of the ECM can act through other mechanisms to further promote angiogenesis or inhibit it. Current evidence suggests that the regulation of angiogenesis is a dynamic process in which the endothelial cells can promote angiogenesis by secreting proteases that remodel the ECM, tumor cells can further promote angiogenesis by secreting ECM components and actively remodeling their environment, and stromal cells may respond to angiogenesis associated with tumors and inflammatory reactions by secreting inhibitory molecules. Here, we provide a critical review of the protein and proteoglycan components of the ECM that have been implicated in angiogenesis with an emphasis on their role in promoting or inhibiting angiogenesis in brain tumors.

• Inhibition of cystine uptake disrupts the growth of primary brain tumors.

  Authors: Wook Joon Chung, Susan A Lyons, Gina M Nelson, Hashir Hamza, Candece L Gladson, G Yancey Gillespie, Harald Sontheimer

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 09/2005; 25(31):7101-10.

  Glial cells play an important role in sequestering neuronally released glutamate via Na+-dependent transporters. Surprisingly, these transporters are not operational in glial-derived tumorsGlial cells play an important role in sequestering neuronally released glutamate via Na+-dependent transporters. Surprisingly, these transporters are not operational in glial-derived tumors (gliomas). Instead, gliomas release glutamate, causing excitotoxic death of neurons in the vicinity of the tumor. We now show that glutamate release from glioma cells is an obligatory by-product of cellular cystine uptake via system xc-, an electroneutral cystine-glutamate exchanger. Cystine is an essential precursor for the biosynthesis of glutathione, a major redox regulatory molecule that protects cells from endogenously produced reactive oxygen species (ROS). Glioma cells, but not neurons or astrocytes, rely primarily on cystine uptake via system xc- for their glutathione synthesis. Inhibition of system xc- causes a rapid depletion of glutathione, and the resulting loss of ROS defense causes caspase-mediated apoptosis. Glioma cells can be rescued if glutathione status is experimentally restored or if glutathione is substituted by alternate cellular antioxidants, confirming that ROS are indeed mediators of cell death. We describe two potent drugs that permit pharmacological inhibition of system xc-. One of these drugs, sulfasalazine, is clinically used to treat inflammatory bowel disease and rheumatoid arthritis. Sulfasalazine was able to reduce glutathione levels in tumor tissue and slow tumor growth in vivo in a commonly used intracranial xenograft animal model for human gliomas when administered by intraperitoneal injection. These data suggest that inhibition of cystine uptake into glioma cells through the pharmacological inhibition of system xc- may be a viable therapeutic strategy with a Food and Drug Administration-approved drug already in hand.

• Lyn kinase activity is the predominant cellular SRC kinase activity in glioblastoma tumor cells.

  Authors: Michelle R Stettner, Wenquan Wang, L Burton Nabors, Suman Bharara, Daniel C Flynn, J Robert Grammer, G Yancey Gillespie, Candece L Gladson

  Cancer research. 07/2005; 65(13):5535-43.

  Cellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. AsCellular Src activity modulates cell migration, proliferation, and differentiation, and recent reports suggest that individual members of the Src family may play specific roles in these processes. As we have found that Lyn, but not Fyn, activity promotes migration of glioblastoma cells in response to the cooperative signal generated by platelet-derived growth factor receptor beta and integrin alpha(v)beta3, we compared the activity and expression of Lyn and Fyn in glioblastoma (grade IV) tumor biopsy samples with that in anaplastic astrocytoma (grade III) tumors, nonneoplastic brain, and normal autopsy brain samples. Lyn kinase activity was significantly elevated in glioblastoma tumor samples. Notably, the Lyn kinase activity accounted for >90% of pan-Src kinase activity in glioblastoma samples but only approximately 30% of pan-Src kinase activity in the other groups. The levels of phosphorylation of the autophosphorylation site were consistent with significantly higher Lyn activity in glioblastoma tumor tissue than nonneoplastic brain. Although the normalized levels of Lyn protein and the relative levels of Lyn message were significantly higher in glioblastoma samples than nonneoplastic brain, the normalized levels of Lyn protein did not correlate with Lyn activity in the glioblastoma samples. There was no significant difference in the normalized levels of c-Src and Fyn protein and message in the glioblastoma and nonneoplastic brain. Immunostaining revealed that Lyn is located primarily in the glioblastoma cells in the tumor biopsies. These data indicate that Lyn kinase activity is significantly elevated in glioblastoma tumors and suggest that it is the Lyn activity that promotes the malignant phenotype in these tumors.