Interruption of functional recovery by the NMDA glutamate antagonist MK801 after compression of the sensorimotor cortex: implications for treatment of tumors or other mass-related brain injuries

Glutamate antagonists have recently been shown to limit tumor growth, providing potential new therapeutic targets and strategies against brain tumors. Here, we demonstrate that the glutamate NMDA receptor antagonist MK801, after a delay, adversely reverses functional recovery in rats with compressive mass lesions of the sensorimotor cortex. Our data suggest that the controlled focal cortical compression model may be a valuable pre-clinical tool to screen compounds for the treatment of brain tumors. It may be possible to use this model to develop interventions that maintain anti-cancer effects but with diminished harm to bystander tissue and brain plasticity.     

L1 expressed by glioma cells promotes adhesion but not migration

L1 is an adhesion molecule of the immunoglobulin superfamily expressed by several types of cancer, including gliomas. It has been shown that L1 can act as chemoattractant to glioma cells, while the effects of L1 expressed by glioma cells themselves are unknown to date. We established a C6 rat glioma clone, conditionally expressing murine L1 under control of a tetracycline responsive promoter. In vitro experiments revealed increased adhesion on matrigel as well as increased intercellular adhesion in the presence of L1, whereas no L1-dependent effects on proliferation or migration on either matrigel or myelin were observed. In vivo experiments using transplantation into nude mouse striatum, where L1 expression by glioma cells was regulated by tetracycline via drinking water, did not show effects of L1 on tumor size or brain invasion. Our data suggest that L1 expressed on the surface of glioma cells increases cell-matrix and intercellular adhesion, but has no apparent effects on proliferation and invasion.     

CD24 promotes invasion of glioma cells in vivo.

Based on the hypothesis that adhesion molecules expressed on the surface of glioma cells mediate brain invasion, we examined the effect of CD24 on growth and migration of gliomas in vitro and in vivo. CD24, a glycosylphosphatidylinositol anchored, highly glycosylated adhesion molecule, is expressed in hematopoietic and neural cells. We found immunohistochemical expression of CD24 in human glioblastomas. We then established a clone from C6 rat glioblastoma cells, where mouse CD24 (also called heat stable antigen) is under control of a tetracycline-responsive promoter. In the presence of tetracycline (1 microg/ml) CD24 was downregulated by 20-fold. In vitro migration assays were performed on a basement membrane preparation (matrigel) and on myelin, the main substrates of in vivo glioma migration. While the cells were more motile on matrigel as compared with myelin, no relation between CD24 expression and motility was observed. We then transplanted the C6 clone into the striatum of nude mice and regulated CD24 expression via tetracycline in the drinking water (1 mg/ml). After 3 weeks, CD24 positive tumors of mice getting no tetracycline showed diffuse invasion of tumor cells in a brain area 10-fold larger than in CD24-suppressed tumors of mice receiving tetracycline. These data show that CD24 stimulates migration of gliomas in vivo and they suggest a role for this adhesion molecule in diffuse brain invasion of human gliomas.     

Intersectin1-s is involved in migration and invasion of human glioma cells

Malignant gliomas have a tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. Intersectin1 (ITSN1) as a molecular linker in the central nervous system is well known as an important regulator of endocytosis and exocytosis. ITSN1 has two isoforms: ITSN1-l and ITSN1-s. In this study, we show that siRNA-mediated down regulation of ITSN1-s inhibited migration and invasion of glioma cells. In addition, we demonstrate the possible mechanisms by which ITSN1-s functions in migration and invasion. Several key proteins, including cofilin, LIMK, PAK, FAK, integrin β1, and MMP-9, which are critical for cells migration and invasion, were probably involved in ITSN1-s signaling pathways. These results suggest that ITSN1-s contributes to glioma cells migration and invasion by regulating the formation of cytoskeleton, influencing adhesion and increasing expression of MMP-9. Our results indicate that ITSN1-s is a critical factor in gliomas invasion and identify that ITSN1-s is a new potentially antiinvasion target for therapeutic intervention in gliomas.     

Structure-based functional design of chemical ligands for AMPA-subtype glutamate receptors

Glutamate receptors (GluRs) function as transmembrane ion channels to regulate intracellular level of ions such as calcium in control of excitatory synaptic transmission of the central nervous system. Dysfunction of these glutamate receptors has been implicated in human brain neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's diseases. Despite such a significant role in both the biology and pathology of the central nervous system, detailed understanding of molecular mechanisms by which subtype- or subunit-specific glutamate receptors function in cells is still lacking. The recently determined three-dimensional crystal structure of the extracellular ligand-binding core of the prototypic AMPA-subtype GluR2, in complex with its agonist, provides a new opportunity for rational design of chemical ligands that could help elucidate the underlying mechanisms and also be useful in the therapy of the neurodegenerative diseases. Here we report our recent development in structure-based functional design of chemical ligands by using nuclear magnetic resonance (NMR) spectroscopy. The NMR structure-based method enables rapid identification of small molecular chemical ligands that bind to specific sites of the target protein. These chemical compounds can be optimized for selective binding to the target protein, and linked to produce chemical ligands with high-affinity and selectivity of the AMPA-subtype glutamate receptors.     

FRK通过FAK信号通路抑制脑胶质瘤细胞迁移与侵袭作用的研究

目的研究酪氨酸相关激酶(fyn-related kinase,FRK)是否通过粘着斑激酶(focal adhesion kinase,FAK)信号通路抑制脑胶质瘤细胞的迁移和侵袭。方法采用Western blot(WB)检测FRK质粒的转染效果,细胞划痕和Transwell侵袭实验检测过表达FRK及下调FAK对胶质瘤U251细胞迁移和侵袭能力的影响;WB检测过表达FRK对FAK、P-FAK蛋白水平的影响以及下调FAK对FRK蛋白水平的影响。结果FRK质粒转染成功。过表达FRK后胶质瘤U251细胞的迁移与侵袭能力下降。FAK siRNA干扰效率达到70%,下调FAK后胶质瘤U251细胞的迁移与侵袭能力下降。过表达FRK后P-FAK的蛋白水平明显下降,而FAK蛋白水平无明显变化,下调FAK对FRK的蛋白水平无明显影响。结论 FRK可能通过抑制FAK的磷酸化来调控胶质瘤的侵袭与迁移。     

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors signaling complexes in Bergmann glia

Glutamate, the major excitatory neurotransmitter, induces a wide array of signals from the membrane to the nucleus regulating gene expression. In Bergmann glia, Ca2+ -permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole- propionic acid (AMPA) receptors are involved in the short- and long-term interactions between these cells and the neurons that they surround. After activation, AMPA receptors become tyrosine phosphorylated and by these means form multiprotein signaling complexes. To characterize these events, cultured chick Bergmann glia cells as well as chick cerebellar slices were exposed to glutamate, and, by using a combination of immunoprecipitation assays coupled to Western blot analysis, we identified several signaling proteins that become associated with these receptors. A dose- and time-dependent association among AMPA receptors, the focal adhesion kinase pp125FAK, the phosphatidylinositol-3 kinase and paxillin was found. These results extend the concept of the transducisome to AMPA receptors and provide a framework in which a plausible control of the cytoskeletal network by glutamate is taking place, most possibly through AMPA receptors.     

Dissecting glioma invasion: interrelation of adhesion, migration and intercellular contacts determine the invasive phenotype.

The invasive cellular behavior of malignant gliomas is determined by receptor mediated cell-substratum contacts and cell-cell interaction as well as cellular locomotion. This study attempts to break down the complex phenomena of the invasive process into their components of attachment to neighboring cells, aggregate formation, adhesion to matrix substratum, migration and invasion into three-dimensional cellular aggregates separately analyzed in different in vitro assay systems. Using a panel of 13 glioma cell lines, adhesion to non-specifically or merosin coated surfaces was correlated to monolayer cell migration and dissemination of tumor cells from aggregates plated on these substrates. The formation kinetics of aggregates were determined and compared to the ability of these cells to rapidly attach and form mechanically stable cell-cell contacts. The motility rates in the different assay systems as well as cell-cell attachment was correlated to invasion of re-aggregated tumor cells into fetal rat brain. A tight positive correlation was found for substrate adhesion and monolayer migration. In contrast, cell-substratum contacts had little influence on dissemination of cells out of three-dimensional aggregates and no association between monolayer migration and migration of cells out of aggregates was detected. The ability of glioma cells to rapidly form aggregates was associated with enhanced migration out of aggregates. The capacity to invade fetal rat brain aggregates was correlated with the capacity to form stable intercellular adhesion as measured in a cell-cell adhesion assay. Invasion in this system was not found to be associated with migration in monolayer or with migration out of tumor aggregates. This study highlights that current in vitro assays for invasion only represent isolated aspects of the multi-cascade process which is involved in tumor cell invasion.     

Experiment and observation on invasion of brain glioma in vivo.

Research on invasion and metastasis of glioma in vivo was performed by implanting C6 glioma cells transfected with enhanced green fluorescent protein (EGFP) gene into the brain of SD rats. Firstly, C6 glioma cells were transfected with a plasmid vector (pEGFP-N3) containing the EGFP gene. Stable EGFP-expressing clones were isolated and examination for these cells by flow cytometry and electron microscope was done. Secondly, EGFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections were examined using fluorescence microscopy after adjacent sections were examined by immunohistochemistry or routine hematoxylin and eosin staining for the visualization and detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of EGFP gene in vivo. The results showed that EGFP-transfected C6 glioma cells maintained stable high-level EGFP expression in the central nervous system during their growth in vivo. EGFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the visualization of distant micrometastasis and invasion on the single-cell level. Small locally invasive foci, including those immediately adjacent to the leading invasive edge of the tumor, were virtually undetectable by routine hematoxylin and eosin staining and immunohistochemistry. These results suggested that EGFP-transfected C6 cells can be visualized by fluorescence microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion and metastasis of brain glioma in vivo.     

Clinicoimmunological values of measurement of IgG-Fc receptor positive T cells in the patients with malignant brain tumors

It has recently been shown that human T-cell subpopulation can be identified functionally via surface receptors for Fc fragment of immunoglobulins. We detected peripheral blood T-cells bearing the Fc receptor for IgG molecules (T gamma cell) in patients with brain tumors who manifested a variety of immunological abnormalities. We also analyzed immunological values of T gamma cells which were thought to be suppressor and/or a part of killer T cells, comparing with other immunological parameters. The percentage of T gamma cells was significantly higher in patients with malignant brain tumors than in normal controls (6.54 +/- 1.6%). The values of T gamma cells in patients with glioma, metastatic brain tumor, benign brain tumor were 15.4 +/- 4.8%, 14.9 +/- 3.2%, and 8.1 +/- 3.7%, respectively. In the patients with glioma, the values increased in the uncontrolled group compared with the well-controlled group. Furthermore, the values were decreased by surgical removal of the tumor. On immunological study of T gamma cells in the patients with glioma, there was a definite correlation between the values of T gamma cells and ADCC activity. Furthermore, the T gamma cells in the patients showed higher ADCC activity than in normal controls. The values of T gamma cells also correlated with blastogenesis of the peripheral blood lymphocytes by PHA (r = -0.742, p less than 0.001) and Con A (r = -0.662, p less than 0.001). These results suggest that T gamma cells are playing two important roles in patients with glioma such as suppressor function and ADCC activity, and that these cells may change according to the tumor growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biology of angiogenesis and invasion in glioma

Treatment of adult brain tumors, in particular glioblastoma, remains a significant clinical challenge, despite modest advances in surgical technique, radiation, and chemotherapeutics. The formation of abnormal, dysfunctional tumor vasculature and glioma cell invasion along white matter tracts are believed to be major components of the inability to treat these tumors effectively. Recent insight into the fundamental processes governing glioma angiogenesis and invasion provide a renewed hope for development of novel strategies aimed at reducing the morbidity of this uniformly fatal disease. In this review, we discuss background biology of the blood brain barrier and its pertinence to blood vessel formation and tumor invasion. We will then focus our attention on the biology of glioma angiogenesis and invasion, and the key mediators of these processes. Last, we will briefly discuss recent and ongoing clinical trials targeting mediators of angiogenesis or invasion in glioma patients. The findings provide a renewed hope for those endeavoring to improve treatment of patients with glioma by providing a novel set of rational targets for translational drug discovery.     

Hydrodynamic cellular volume changes enable glioma cell invasion

Malignant gliomas are highly invasive brain tumors that currently lack effective treatment. Unlike other cancers, gliomas do not metastasize via the vasculature but invade surrounding brain solely along extracellular routes, primarily moving along the vasculature and nerve tracts. This study uses several model systems to visualize and quantitatively assess cell volume changes of human glioma cells invading within the brain's extracellular space of C.B.17 severe combined immunodeficient (scid) mice and tumor cells invading in a modified Boyden chamber using three-dimensional multiphoton and confocal time-lapse microscopy. Regardless of model system used to quantitatively assess volume changes, invading glioma cells maximally decreased their volume by 30-35%, a value that was independent of barrier and cell size. Through osmotic challenges, we demonstrate that the observed cellular volume changes during invasion represent the smallest achievable cell volume and require glioma cells to release all free unbound cytoplasmic water. Water osmotically follows the release of Cl(-) through ion channels and cotransporters and blockade of Cl(-) flux inhibits both volume changes and cell invasion. Hence, invading glioma cells use hydrodynamic volume changes to meet the spatial constraints imposed within the brain, using essentially all free, unbound cytoplasmic water to maximally alter their volume as they invade.     

Effects of Lamotrigine on brain nitrite and cGMP levels during focal cerebral ischemia in rats

Glutamate receptor antagonists are protective in animal models of focal cerebral ischemia. Lamotrigine (3,5-diamino-6-[2,3-dichlorophenyl]-1,2,4-triazine) is an anticonvulsant drug that blocks voltage-gated sodium channels and inhibits the ischemia-induced release of glutamate. Experiments in primary neuronal cultures implicate nitric oxide (NO) as a mediator of glutamatergic neurotoxicity acting via N-Methyl- d -Aspartate (NMDA) receptors. The effect of glutamate release inhibitor, Lamotrigine upon NO and cGMP production has been examined in focal cerebral ischemia in rats. Focal cerebral ischemia was produced by the permanent occlusion of right middle cerebral artery (MCA) in urethane anesthetized rats. A number of indicators of brain NO production (nitrite, cGMP) were determined in ipsilateral and contralateral cerebral cortex and cerebellum after 0, 10, 60 min of focal cerebral ischemia. The same parameters were measured in rats treated with Lamotrigine (20 mg/kg, i.p.) 30 min before or just after the occlusion of the right MCA.     

The brain tumor microenvironment

High-grade brain tumors are heterogeneous with respect to the composition of bona fide tumor cells and with respect to a range of intermingling parenchymal cells. Glioblastomas harbor multiple cell types, some with increased tumorigenicity and stem cell-like capacity. The stem-like cells maybe the cells of origin for tumor relapse. However, the tumor-associated parenchymal cells such as vascular cells,microglia, peripheral immune cells, and neural precursor cells also play a vital role in controlling the course of pathology.In this review, we describe the multiple interactions of bulk glioma cells and glioma stem cells with parenchymal cell populations and highlight the pathological impact as well as signaling pathways known for these types of cell-cell communication. The tumor-vasculature not only nourishes glioblastomas, but also provides a specialized niche for these stem-like cells. In addition, microglial cells,which can contribute up to 30% of a brain tumor mass,play a role in glioblastoma cell invasion. Moreover, non-neoplastic astrocytes can be converted into a reactive phenotype by the glioma microenvironment and can then secrete a number of factors which influences tumor biology. The young brain may have the capacity to inhibit gliomagenesis by the endogenous neural precursor cells, which secrete tumor suppressive factors. The factors, pathways, and interactions described in this review provide a new prospective on the cell biology of primary brain tumors, which may ultimately generate new treatment modalities. However, our picture of the multiple interactions between parenchymal and tumor cells is still incomplete.     

大鼠C6脑胶质瘤模型的病理特征与MRI的观察

目的建立SD大鼠C6胶质瘤模型并对其病理特征及MRI进行观察。方法50只SD大鼠随机分成5组，每组10只，c6胶质瘤细胞悬液立体定向接种于大鼠的右侧尾状核，接种后观察大鼠的生活状态、生存期；分别于接种后不同时段进行MRI观察肿瘤生长特性及肿瘤体积的测量；取不同时段组大鼠脑标本行脑组织HE染色、透射电镜(transmission electron microscope，TEM)、脑组织含水量测量(与10只正常大鼠对照)、胶质纤维酸性蛋白(GFAP)免疫组化检查。结果立体定向颅内接种成功率97．5％，未见远处及颅外转移，肿瘤在一定时期内牛长较快，脑水肿随肿瘤的生长明显加重，生存期观察组中7只荷瘤鼠死亡，3只肿瘤自发部分消退。结论立体定向建立大鼠C6胶质瘤模型成功率高，接种后颅内肿瘤呈浸润性生长，与人脑胶质瘤具有相似性，由于生存期观察组中有部分荷瘤鼠出现肿瘤自发部分消退，故应用该模型评价治疗效果时应慎重；TIWI增强扫描可清晰显示肿瘤影像，且能更早发现肿瘤；MRI联合病理可较好反映肿瘤生长方式及发展过程。     

Imaging glioma cell invasion in vivo reveals mechanisms of dissemination and peritumoral angiogenesis

Infiltration of cancer cells into normal tissue is a hallmark of malignant gliomas and compromises treatment options. A lack of appropriate models limits the study of this invasion in vivo, which makes it difficult to fully understand its anatomy and the role of dynamic interactions with structures of the normal brain. We developed a novel methodology by utilizing multiphoton laser scanning microscopy (MPLSM) to image the movement of glioma cells deep within the normal brain of live mice in real time. This allowed us to track the invasion of individual RFP‐expressing GL261 cells in relation to perfused vasculature or GFP‐labeled endothelial cells repetitively over days, up to a depth of 0.5 mm. Glioma cells moved faster and more efficiently when the abluminal site of a blood vessel was utilized for invasion. Cells that invaded perivascularly were frequently found next to (a) multiple capillary structures where microvessels run parallel to each other, (b) capillary loops or glomeruloid‐like bodies, and (c) dilated capillaries. Dynamic MPLSM for more than 48 h revealed that single invasive glioma cells induced intussusceptive microvascular growth and capillary loop formation, specifically at the microvascular site with which they had contact. As the main tumor grew by cooption of existing brain vessels, these peritumoral vascular changes may create a beneficial environment for glioma growth. In conclusion, our study revealed new mechanisms of peritumoral angiogenesis and invasion in gliomas, providing an explanation for their interdependence. © 2009 Wiley‐Liss, Inc.     

The brain tumor microenvironment

High-grade brain tumors are heterogeneous with respect to the composition of bona fide tumors cells and with respect to a range of intermingling parenchymal cells. Glioblastomas harbor multiple cell types, some with increased tumorigenicity and stem cell-like capacity. The stem-like cells may be the cells of origin for tumor relapse. However, the tumor-associated parenchymal cells-such as vascular cells, microglia, peripheral immune cells, and neural precursor cells-also play a vital role in controlling the course of pathology. In this review, we describe the multiple interactions of bulk glioma cells and glioma stem cells with parenchymal cell populations and highlight the pathological impact and signaling pathways known for these types of cell-cell communication. The tumor-vasculature not only nourishes glioblastomas, but also provides a specialized niche for these stem-like cells. In addition, microglial cells, which can contribute up to 30% of a brain tumor mass, play a role in glioblastoma cell invasion. Moreover, non-neoplastic astrocytes can be converted into a reactive phenotype by the glioma microenvironment and can then secrete a number of factors which influences tumor biology. The young brain may have the capacity to inhibit gliomagenesis by the endogenous neural stem and progenitor cells, which secrete tumor suppressive factors. The factors, pathways, and interactions described in this review provide a new prospective on the cell biology of primary brain tumors, which may ultimately generate new treatment modalities. However, our picture of the multiple interactions between parenchymal and tumor cells is still incomplete. ? 2011 Wiley-Liss, Inc.     

The neurotransmitter glutamate and human T cells: glutamate receptors and glutamate-induced direct and potent effects on normal human T cells,cancerous human leukemia and lymphoma T cells,and autoimmune human T cells

Glutamate is the most important excitatory neurotransmitter of the nervous system, critically needed for the brain’s development and function. Glutamate has also a signaling role in peripheral organs. Herein, we discuss glutamate receptors (GluRs) and glutamate-induced direct effects on human T cells. T cells are the most important cells of the adaptive immune system, crucially needed for eradication of all infectious organisms and cancer. Normal, cancer and autoimmune human T cells express functional ionotropic and metabotropic GluRs. Different GluR subtypes are expressed in different T cell subtypes, and in resting vs. activated T cells. Glutamate by itself, at low physiological 10^?8M to 10^?5M concentrations and via its several types of GluRs, activates many key T cell functions in normal human T cells, among them adhesion, migration, proliferation, intracellular Ca²⁺ fluxes, outward K⁺ currents and more. Glutamate also protects activated T cells from antigen-induced apoptotic cell death. By doing all that, glutamate can improve substantially the function and survival of resting and activated human T cells. Yet, glutamate’s direct effects on T cells depend dramatically on its concentration and might be inhibitory at excess pathological 10^?3M glutamate concentrations. The effects of glutamate on T cells also depend on the specific GluRs types expressed on the target T cells, the T cell’s type and subtype, the T cell’s resting or activated state, and the presence or absence of other simultaneous stimuli besides glutamate. Glutamate also seems to play an active role in T cell diseases. For example, glutamate at several concentrations induces or enhances significantly very important functions of human T-leukemia and T-lymphoma cells, among them adhesion to the extracellular matrix, migration, in vivo engraftment into solid organs, and the production and secretion of the cancer-associated matrix metalloproteinase MMP-9 and its inducer CD147. Glutamate induces all these effects via activation of GluRs highly expressed in human T-leukemia and T-lymphoma cells. Glutamate also affects T cell-mediated autoimmune diseases. With regards to multiple sclerosis (MS), GluR3 is highly expressed in T cells of MS patients, and upregulated significantly during relapse and when there is neurological evidence of disease activity. Moreover, glutamate or AMPA (10^?8M to 10^?5M) enhances the proliferation of autoreactive T cells of MS patients in response to myelin proteins. Thus, glutamate may play an active role in MS. Glutamate and its receptors also seem to be involved in autoimmune rheumatoid arthritis and systemic lupus erythematosus. Finally, T cells can produce and release glutamate that in turn affects other cells, and during the contact between T cells and dendritic cells, the latter cells release glutamate that has potent effects on the T cells. Together, these evidences show that glutamate has very potent effects on normal, and also on cancer and autoimmune pathological T cells. Moreover, these evidences suggest that glutamate and glutamate-receptor agonists might be used for inducing and boosting beneficial T cell functions, for example, T cell activity against cancer and infectious organisms, and that glutamate-receptor antagonists might be used for preventing glutamate-induced activating effects on detrimental autoimmune and cancerous T cells.