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Background: Radiosurgery has been recognized as a reasonable treatment for metastatic brain tumors. Increasing the radiosensitivity and synergistic effects are possible ways to improve the therapeutic efficacy of specific regions of tumors. c-Jun-N-terminal kinase (JNK) signaling regulates H2AX phosphorylation to repair radiation-induced DNA breakage. We previously showed that blocking JNK signaling influenced radiosensitivity in vitro and in an in vivo mouse tumor model. Drugs can be incorporated into nanoparticles to produce a slow-release effect. This study assessed JNK radiosensitivity following the slow release of the JNK inhibitor SP600125 from a poly (DL-lactide-co-glycolide) (LGEsese) block copolymer in a brain tumor model., Materials and Methods: A LGEsese block copolymer was synthesized to fabricate SP600125-incorporated nanoparticles by nanoprecipitation and dialysis methods. The chemical structure of the LGEsese block copolymer was confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy. The physicochemical and morphological properties were observed by transmission electron microscopy (TEM) imaging and measured with particle size analyzer. The blood-brain barrier (BBB) permeability to the JNK inhibitor was estimated by BBBflammaTM 440-dye-labeled SP600125. The effects of the JNK inhibitor were investigated using SP600125-incorporated nanoparticles and by optical bioluminescence, magnetic resonance imaging (MRI), and a survival assay in a mouse brain tumor model for Lewis lung cancer (LLC)-Fluc cells. DNA damage was estimated by histone γ H2AX expression and apoptosis was assessed by the immunohistochemical examination of cleaved caspase 3., Results: The SP600125-incorporated nanoparticles of the LGEsese block copolymer were spherical and released SP600125 continuously for 24h. The use of BBBflammaTM 440-dye-labeled SP600125 demonstrated the ability of SP600125 to cross the BBB. The blockade of JNK signaling with SP600125-incorporated nanoparticles significantly delayed mouse brain tumor growth and prolonged mouse survival after radiotherapy. γ H2AX, which mediates DNA repair protein, was reduced and the apoptotic protein cleaved-caspase 3 was increased by the combination of radiation and SP600125-incorporated nanoparticles.

Nogo or reticulon-4 (RTN4), also known as neurite outgrowth inhibitor, is a member of the reticulon family of genes. Nogo-A, one of the three isoforms, is enriched in the central nervous system (CNS). The extracellular domain of Nogo-A, Nogo-66, has neurite growth inhibitory activity that is specific for neurons and is mediated by the Nogo receptor. However, most of its functions are not known yet. We investigated whether Nogo-A modulates the migration and invasion of a glioblastoma cell line, as well as the factors that have an effect on Nogo-A. The expression of Nogo-A was evaluated using western blotting and immunohistochemistry in human brain tumor specimens. U87MG cells were transfected with a sense-Nogo-A cDNA construct (U87-Nogo-A cells expressing Nogo-A) and an empty vector (U87MG-E cells not expressing Nogo-A). The migration and invasion abilities of these cells were investigated using simple scratch and Matrigel invasion assays. Morphologic and cytoskeletal changes were documented by confocal microscopy. The proliferation rate was estimated using doubling time assay. The effects of Nogo-A on Rho activity and phosphorylated cofilin were determined by a Rho activity assay and western blotting. Among primary brain tumors, Nogo-A expression was found in a higher percentage of oligodendrogliomas (90.0%) compared with the percentage in the glioblastomas (68.4%). In addition, the percentage in mixed gliomas was 42.9%, while it was not expressed in pituitary adenomas or schwannomas. The migration and invasion abilities of the U87-Nogo-A cells were decreased compared with the control. In the U87-Nogo-A cell line, Rho activity and phosphorylated cofilin expression were also decreased and morphology became more flat in comparison with the U87MG-E cell line. Nogo-A may inhibit the migration and invasion of human malignant glioma cells via the downregulation of RhoA-cofilin signaling.

Purpose: Pilocytic astrocytoma (PA) is a World Health Organization grade I neoplasm that generally follows a benign course. However, in some patients, PA exhibits an aggressive clinical course. Here, we examined the clinical course of pediatric and adult PAs with progression at a single institution., Methods: Between 1995 and 2013, 39 patients with PA were treated. Nineteen were pediatric patients (mean age, 12 years; range, 1-17 years) with a male-to-female patient ratio of 10:9, while 20 were adults (mean age, 36.4 years; range, 19-65 years) with a male-to-female ratio of 9:11. We analyzed and compared tumor location, extent of tumor resection, adjuvant treatment, and clinical course in all patients., Results: In the 19 pediatric patients, tumors were located in the cerebellar vermis, cerebellar hemisphere, optic pathways plus hypothalamus, hypothalamus, brainstem, and the temporal lobe in 6 (31.6%), 5 (26.3%), 3 (15.8%), 2 (10.5%), and 2 (10.5%) patients and 1 (5.3%) patient, respectively. The mass was totally, subtotally, or partially resected in 11 (57.9%), 2 (10.5%), and 4 (21.1%) patients, respectively; biopsies were performed in 2 (10.5%) patients. Immediate postoperative adjuvant treatment was carried out in 6 patients. Tumor progression was detected in 3 patients at 3.0, 4.6, and 5.2 years after treatment, respectively, without significant symptoms. In the 20 adult patients, tumors were located in the cerebellar hemisphere, cerebellar vermis, hypothalamus, brainstem, cerebral hemisphere, and lateral ventricle in 5 (25%), 4 (20%), 3 (15%), 3 (15%), 3 (15%), and 2 (10%) patients, respectively. The mass was totally, subtotally, or partially resected in 11 (55%) and 6 (30%) patients and 1 (5%) patient, respectively; biopsies were performed in 2 patients. Immediate adjuvant treatment was carried out in 2 patients. Progression was detected in 3 patients at 0.3, 0.9, and 2.5 years after treatment, respectively, with progressive neurologic symptoms. There was one case of disease-related mortality during follow-up among the adult patients., Conclusion: Most of the PA cases evaluated in this study were benign. However, tumor progression in adult PAs followed a more aggressive clinical course than those in pediatric PAs.

Background: Glioblastoma is a highly lethal neoplasm that frequently recurs locally after radiotherapy, and most of these recurrences originate from near the irradiated target field. In the present study, we identified the effects of radiation on glioma invasion and p53, TIMP-2, and MMP-2 expression through in vitro and in vivo experiments., Methods: The U87MG (wt p53) and U251 (mt p53) human malignant glioma cell lines were prepared, and the U2OS (wt 53) and Saos2 (del p53) osteosarcoma cell lines were used as p53 positive and negative controls. The four cell lines and p53 knock-downed U87MG cells received radiation (2-6 Gy) and were analyzed for expression of p53 and TIMP-2 by Western blot, and MMP-2 activity was detected by zymography. In addition, the effects of irradiation on directional invasion of malignant glioma were evaluated by implanting nude mice with bioluminescent u87-Fluc in vivo followed by MMP-2, p53, and TIMP-2 immunohisto-chemistry and in situ zymography., Results: MMP-2 activity and p53 expression increased in proportional to the radiation dose in cell lines with wt p53, but not in the cell lines with del or mt p53. TIMP-2 expression did not increase in U87MG cells. MMP-2 activity decreased in p53 knock-downed U87MG cells but increased in the control group. Furthermore, radiation enhanced MMP-2 activity and increased tumor margin invasiveness in vivo. Tumor cells invaded by radiation overexpressed MMP-2 and p53 and revealed high gelatinolytic activity compared with those of non-radiated tumor cells., Conclusion: Radiation-induced upregulation of p53 modulated MMP-2 activity, and the imbalance between MMP-2 and TIMP-2 may have an important role in glioblastoma invasion by degrading the extracellular matrix. Bioluminescent "U87-Fluc"was useful for observing tumor formation without sacrifice after implanting tumor cells in the mouse brain. These findings suggest that the radiotherapy involved field for malignant glioma needs to be reconsidered, and that future trials should investigate concurrent pharmacologic therapies that inhibit invasion associated with radiotherapy.

KITENIN (KAI1 COOH-terminal interacting tetraspanin) promotes tumor invasion and metastasis in various cancers. This study assessed the association between KITENIN expression and advanced glioma grade in patients. In vitro assays revealed that KITENIN knockdown inhibited the invasion and migration of glioma cells, whereas KITENIN overexpression promoted their invasion and migration. In orthotopic mouse tumor models, mice transplanted with KITENIN-transfected glioma cells had significantly shorter survival than mice transplanted with mock-transfected cells. Patients with low KITENIN expression showed a significantly longer progression-free survival than patients with high KITENIN expression. KITENIN induced the expression of the epithelial-mesenchymal transition (EMT) markers (N-cadherin, ZEB1, ZEB2, SNAIL and SLUG) as well as the glioma stemness markers (CD133, ALDH1 and EPH-B1). Taken together, these findings showed that high levels of KITENIN increased glioma invasiveness and progression, associated with the up-regulation of EMT and stemness markers.

Metallothionein 1E (MT1E) has been found to be highly expressed in motile cell lines. We investigated whether MT1E actually modulates the migration and invasion of human glioma cell lines and the types of factors that have an effect on MT1E. RNA differential display was performed using Genefishing™ technology in the human glioma cell lines U343MG-A, U87MG and U87MG-10'; the results were validated by RT-PCR and northern blot analysis, in order to detect possible genetic changes as the determining factors for migration ability in malignant glioma. MT1E was identified in U87MG, a highly motile cell line. The migration and invasion abilities of human glioma cell lines, and MT1E transfectants were investigated using simple scratch testing and Matrigel invasion assays. Morphological and cytoskeletal (actin, vimentin) changes were documented by light and confocal microscopy. The expression of MT1E in four glioma cell lines was assessed by RT-PCR and western blotting. In addition, the effects of MT1E on the activity of the NF-κB p50/p65 transcription factor, MMP-2 and -9 were examined by western blotting and zymography. The endogenous MT1E expression in the human glioma cell lines was statistically correlated with their migratory abilities and invasion. The U87-MT-AS cells became more round and had decreased stress fibers, compared with the U87MG cells. Endogenous MT1E expression in the four human glioma cell lines was directly correlated with migration. Two antisense MT1E-transfected cell lines showed decreased NF-κB p50 translocation into the nucleus, which led to decreased activity of MMP-9 in conditioned media. It may be postulated that MT1E can enhance the migration and invasion of human glioma cells by inducing MMP-9 inactivation via the upregulation of NF-κB p50.

Object: Galectin-1 is highly expressed in motile cell lines. The authors investigated whether galectin-1 actually modulates the migration and invasion of human glioblastoma multiforme (GBM) cell lines, and whether its expression with respect to invasion and prognosis is attributable to certain glioma subgroups., Methods: In the human GBM cell lines U343MG-A, U87MG, and U87MG-10', the RNA differential display was evaluated using Genefishing technology. The results were validated by reverse transcription polymerase chain reaction and Northern blot analysis to detect possible genetic changes as the determining factors for the motility of the malignant glioma. The migration and invasion abilities were investigated in human GBM cell lines and galectin-1 transfectant using an in vitro brain slice invasion model and a simple scratch technique. The morphological and cytoskeletal (such as the development of actin and vimentin) changes were examined under light and confocal microscopy. Galectin-1 expression was assessed on immunohistochemical tests and Western blot analysis., Results: Endogenous galectin-1 expression in the human GBM cell lines was statistically correlated with migratory abilities and invasiveness. The U87-G-AS cells became more round than the U87MG cells and lacked lamellipodia. On immunohistochemical staining, galectin-1 expression was increased in higher-grade glioma subgroups (p = 0.027)., Conclusions: Diffuse gliomas demonstrated higher expression levels than pilocytic astrocytoma in the Western blot. Galectin-1 appears to modulate migration and invasion in human glioma cell lines and may play a role in tumor progression and invasiveness in human gliomas.

Although several types of brain tumors are commonly associated with cyst formation, the pathogenesis of tumor-associated cysts (TAC) is unknown. We investigated the matrix metalloproteinase (MMP) expression of cyst fluids to elucidate the pathogenesis of TAC in brain tumors. We also examined the relationship between the severity of peritumoral edema and the expression of intracystic MMP. We collected 40 cyst fluid samples from 34 patients with TAC and studied the expression of MMP-2 and -9 in the cyst fluid using gelatin zymography. Radiological studies were used to estimate the severity of the peritumoral edema and to determine the presence of TAC. Although gelatin zymography of the cyst fluid showed high levels of MMPs, there was no correlation between the expression of MMPs in the cyst fluid and that in the tumor tissue. The level of MMP expression in the cyst fluid did not reflect the pathologic grade of the individual tumors. However, the total and activated MMP-9 levels were significantly associated with the severity of the peritumoral edema (p<0.05). These results suggest that MMPs may be partly involved in the pathogenesis of TAC and peritumoral edema in brain tumors.

The most characteristic feature of a malignant astrocytoma is its early and extensive infiltration into adjacent parenchymal structures. We focused on detecting the possible expression changes as the determining factors for malignant astrocytoma's motile ability. We confirmed that four of 39 genes showed different expression on DD-PCR by RT-PCR and Northern blot analysis. These findings suggest that the genes identified may be important for determining high motility in astrocytoma cell lines. These findings may help us understand the molecular invasion mechanism in astrocytomas.

Background: Despite the well-documented importance of Nm23 in the control of metastasis, there is currently a paucity of data regarding the role of this gene family in the control of glioma invasion., Materials and Methods: Nm23-H1 expression in gliomas was assessed via immunohistochemistry, Western blot, RT-PCR and Northern blot analyses. The migration and invasion ability were also investigated in primary glioma culture cells, human glioma cell lines and nm23-H1 transfectant, using an in vitro brain slice invasion model and a simple scratch technique., Results: Although no significant correlations were detected between nm23-H1 expression and pathological grade, the endogenous nm23-H1 expression in gliomas was found to be inversely correlated with their migratory abilities. Additionally, the nm23-H1 transfectant resulted in a reduction of approximately 45% of the migratory ability and suppressed the invasiveness of the parental cell line., Conclusion: Our overall findings suggest that nm23-H1 may play an important role in the suppression of glioma invasion and migration.