Invasion Of Malignant Glioma Cells Research Articles

3D Microfluidic System for Evaluating Inhibitory Effect of Chinese Herbal Medicine Oldenlandia diffusa on Human Malignant Glioma Invasion Combined with Network Pharmacology Analysis.

To investigate the anti-invasion efficacy of the ethanol extract of Oldenlandia diffusa Will. (EEOD) on a three-dimensional (3D) human malignant glioma (MG) cell invasion and perfusion model based on microfluidic chip culture and the possible mechanism of action of Oldenlandia diffusa Will. (OD). The comprehensive pharmacodynamic analysis method in this study was based on microfluidic chip 3D cell perfusion culture technology, and the action mechanism of Chinese medicine (CM) on human MG cells was investigated through network pharmacology analysis. First, the components of EEOD were analyzed by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Then, cell viability and apoptosis were assessed to determine the optimum concentration of EEOD for invasion experiments, and two-dimensional (2D) migration and invasion abilities of U87 and U251 MG cells were evaluated using scratch wound and Transwell assays. The possible mechanism underlying the effects of EEOD on glioma was analyzed through a network pharmacology approach. Thirty-five compounds of EEOD were detected by UPLC-Q-TOF/MS. EEOD suppressed the viability of MG cells, promoted their apoptosis, and inhibited their migratory and invasive potentials (all P<0.05). Network pharmacology analysis showed that OD inhibited the invasion of MG cells by directly regulating MAPK and Wnt pathways through MAPK, EGFR, MYC, GSK3B, and other targets. The anti-invasion effect of OD was also found to be related to the indirect regulation of microtubule cytoskeleton organization. ]EEOD could inhibit the invasion of human MG cells, and the anti-invasion mechanism of OD might be regulating MAPK and Wnt signaling pathways and microtubule cytoskeleton organization.

Long non-coding RNA LUCAT1 regulates the RAS pathway to promote the proliferation and invasion of malignant glioma cells through ABCB1

Long non-coding RNAs (lncRNAs) are closely related to the occurrence and development of tumors and have gradually become a hot topic in the field of glioma research in recent years. In this study, the role of lung cancer associated transcript 1 (lncRNA LUCAT 1) in glioma occurrence and development, as well as its possible regulatory mechanism, was explored. We utilized the gene chip technology in the preliminary experiment, and based on the experiment results, selected LUCAT1（NONHSAT102745）, which was significantly upregulated in glioma, and ATP-binding cassette Subfamily B member l (ABCB1), which was significantly down-regulated in co-expression analysis, for study. Next, the expression of LUCAT1 and ABCB1 in cells and tissues was immediately evaluated. Subsequently, the cells were transfected with scrambled siRNA, LUCAT1-siRNA/ABCB 1-siRNA, or overexpressed LUCAT1/ABCB1 plasmid + RAS signaling pathway inhibitor-farnesylthiosalicylic acid (FTS). By comparing with the normal combination negative control group, the cell proliferation and invasion ability were evaluated. Finally, subcutaneous tumor formation experiments in the nude mice confirmed the association between LUCAT1 and ABCB1 and RAS signaling pathways. The expression of LUCAT 1 was up-regulated with an increase in WHO grade, and the lncRNA-mRNA co-expression analysis showed that the expression of ABCB1 was low. LUCAT 1 gene knockout reduced the mRNA and protein levels of Ras signaling pathway related factors (Ras, Raf-1, p-AKT, and p-ERK) as regulating ABCB1 expression and inhibiting the ability of tumor in proliferation and invasion no matter in vitro or in vivo. For overexpressing of LUCAT 1, the opposite was true. After we knocked out ABCB1, the LUCAT1 expression was reversely regulated while the level of RAS signaling pathway related factors increased, and the ability of tumors in proliferation and invasion was enhanced. The abnormal LUCAT1 expression affected the biological behaviors of glioma cells, such as proliferation, invasion, etc. by regulating ABCB1 and promoting the activation of the RAS signaling pathway. This provided a new drug target and therapeutic approach for gene therapy of glioma, which is expected to significantly improve the prognosis of relevant patients.

Human giant larvae-1 promotes migration and invasion of malignant glioma cells by regulating N-cadherin.

Human giant larvae-1 (Hugl-1) is a human homologue of Drosophila tumor suppressor lethal (2)-giant larvae and has been reported to be involved in the development of human malignancies. Previous studies performed by our group demonstrated that Hugl-1 inhibits glioma cell proliferation in an intracranial model of nude mice. However, the exact molecular mechanisms underlying the participation of Hugl-1 in glioma invasion and migration, and in the depolarizing process remain largely unknown. Utilizing the U251-MG cells with stable expression of Hugl-1, the present study used wound healing, Transwell invasion and western blot assays to explore the role and specific mechanism of Hugl-1 in glioma invasion and migration. The results of the present study demonstrated that overexpression of Hugl-1 decreased cell-cell adhesion and increased cell-cell extracellular matrix adhesion. In addition, overexpression of Hugl-1 promoted pseudopodia formation, glioma cell migration and invasion. The molecular mechanism of action involved the negative regulation of N-cadherin protein levels by Hugl-1. Overexpression or knockdown of N-cadherin partially suppressed or enhanced the effects of Hugl-1 on glioma cell migration and invasion, respectively. Furthermore, Hugl-1 inhibited cell proliferation, while promoting cell migration, which suggests that it may serve a two-sided biological role in cellular processes. Taken together, these results suggest that Hugl-1 promotes the migration and invasion of malignant glioma cells by decreasing N-cadherin expression. Thus, Hugl-1 may be applied in the development of targeted and personalized treatment.

AKT Axis, miR-21, and RECK Play Pivotal Roles in Dihydroartemisinin Killing Malignant Glioma Cells.

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is known to play important roles in inhibiting proliferation rate, inducing apoptosis, as well as hindering the metastasis and invasion of glioma cells, but the underlying mechanisms are still unclear so far. In this study, methyl thiazolyl tetrazolium (MTT), colony-forming, wound healing, invasion, and apoptosis assays were performed to investigate the effect of DHA on malignant glioma cells. Results showed that DHA induced apoptosis of malignant glioma cells through Protein Kinase B (AKT) axis, induced death of malignant glioma cells by downregulating miR-21, and inhibited the invasion of malignant glioma cells corresponding with up-regulation of the reversion-inducing-cysteine-rich protein with kazal motifs (RECK). These results revealed that AKT axis, miR-21, and RECK play pivotal roles in DHA killing malignant glioma cells, suggesting that DHA is a potential agent for treating glioma.

Abstract 4099: Oligodendrocyte stimulates the invasion ability of glioblastoma cells

Abstract Background &amp; Aims: Glioblastoma (GBM, WHO Grade IV) is characterized by rapid cancer cell infiltration, and is one of the most lethal neoplasms among solid cancers. GBM might arise de novo, or following progression from lower grade gliomas. Recently, solid tumor progression has been recognized as the product of an evolving crosstalk between the cancer cells and its surrounding glial cells. The intensive invasion activity of GBM cells might be regulated by normal cells such as oligodendrocytes or fibroblasts. In this study, we evaluated the interaction between GBM cells and glial cells. Methods: Two GBM cell lines, T98G and U251, were used. Two oligodendrocyte cell lines, ODC1 and ODC2, were derived from surgical tissues of each patient with low-grade glioma (WHO Grade II). Two fibroblasts cell lines, GF1 and GF2, were derived from surgical tissues of each patient with GBM. The culture medium was composed of DMEM with 10% fetal calf serum. We examined the effects of glial cells on the invasion ability of GBM cells by wound-healing assay and invasion assay. Using IncuCyte invasion assay and matrigel invasion assay, the invasiveness of T98G and U251 cells was analyzed in the presence or absence of conditioned medium from oligodendrocyte cells or fibroblasts. The effect of the proliferation of T98G and U251 cells was examined by MTT assay or cell count. Results: ODC1 and ODC2 cells were positive for Olig2 staining but not for GFAP staining, suggesting that ODC1 and ODC2 were oligodendrocyte cells. GF1 and GF2 cells were spindle-shape and were negative for both Olig2 staining and GFAP staining, suggesting that GF1 and GF2 cells were fibroblasts. Oligodendrocyte cells, ODC1 and ODC2 cells, from grade II glioma significantly (p&amp;lt;0.01) increased the migration and invasion ability of GBM cells, T98G and U251 cells. In contrast, GF1 and GF2 fibroblasts from GBM did not affect on the migration and invasion ability of GBM cells. Both oligodendrocyte cells and fibroblasts did not stimulate the proliferation of T98G and U251 cells. Conclusion: Oligodendrocytes might up-regulate the invasiveness of malignant glioma cells. An invasion-stimulating factor(s) from oligodendrocyte cells might be partially associated with the progression from lowe-grade gliomas to GBM. Citation Format: Toshiyuki Kawashima. Oligodendrocyte stimulates the invasion ability of glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4099.

Collected literature on isoflavones and chronic diseases

AbstractIsoflavones are organic compounds, which have been linked to the health benefits and prevention of many diseases. Common isoflavones are genistein, daidzein, and glycitein. Genistein has been researched in regard to its effect on the reduction of menopausal symptoms and reduction of cardiovascular risk factors in osteopenic, post-menopausal women. Research on daidzein focuses on bone mineral density implications in post-menopausal women, therapeutic effects early in prostate cancer, and protection against DMBA-induced mammary carcinogenesis. The most recent research on daidzein has implications for its effect on cardiovascular risk reduction. Research on glycitein focuses on it bioavailability, as well as its role in angiogenesis and invasion of malignant glioma cells. The health benefits of these specific isoflavones are instrumental in the prevention and treatment of many diseases. This review of literature focuses on the effects of genistein, daidzein, and glycitein on health outcomes, such as ...

Sublethal dose of irradiation enhances invasion of malignant glioma cells through p53-MMP 2 pathway in U87MG mouse brain tumor model.

BackgroundGlioblastoma 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.MethodsThe 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.ResultsMMP-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.ConclusionRadiation-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0475-8) contains supplementary material, which is available to authorized users.

Suppression of TRPM7 inhibits proliferation, migration, and invasion of malignant human glioma cells.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a dismal prognosis. Despite intensive study on tumor biology, the underlying mechanisms of the unlimited proliferation and progressive local invasion are still poorly understood, and no effective treatment has been developed for GBM patients. We determine the role of TRPM7 channels in the growth, migration, and infiltration of malignant glioma cells. Using a combination of RT-PCR, Western blot, and patch-clamp techniques, we demonstrated the expression of functional TRPM7 channels of A172 cells, a human glioma cell line, as well as in human glioma tissues. Furthermore, we evaluated the role of TRPM7 in growth, migration, and infiltration of A172 cells with MTT and transwell migration and invasion assays. We showed the expression of functional TRPM7 channels in both A172 cells and human glioma tissues. Suppression of TRPM7 expression with TRPM7-siRNA dramatically reduced the proliferation, migration, and invasion of A172 cells. Pharmacological inhibition of TRPM7 channel with 2-aminoethoxydiphenyl borate (2-APB) showed a similar effect as TRPM7-siRNA. We demonstrate that human glioma cells express functional TRPM7 channel and that activation of this channel plays an important role in the proliferation, migration, and invasion of malignant glioma cells. TRPM7 channel may represent a novel and promising target for therapeutic intervention of malignant glioma.

MiR-145 Inhibits Migration and Invasion of Glioma Stem Cells by Targeting ABCG2

Despite advances in clinical therapies and technologies, the prognosis for patients with malignant glioma is poor. Our previous research demonstrated that glioma stem cells (GSCs) were crucial for glioma malignancy and accelerated tumor migration and invasion. The migration and invasion of malignant glioma cells into the surrounding normal brain tissues cause the poor outcome. miR-145, a miRNA found to be expressed in neurons, was recently found to have reduced expression in glioblastoma multiforme tumors. And miR-145 loss in glioma cells led to increased cell proliferation and invasion. However, its function on the migration and invasion of GSCs was still unknown. In this study, we aimed to identify the effects and mechanisms of miR-145 on the migration and invasion of GSCs. Our investigations revealed that miR-145 was low expressed in malignant glioma tissues and their corresponding GSCs. Knockdown of miR-145 in vitro could enhance the migration and invasion of GSCs, while up-regulation of miR-145 had the opposite effects. Further investigation of the potential mechanism demonstrated that the function of miR-145 in regulating the migration and invasion of GSCs is mediated by its targeting of ABCG2 mRNA. ABCG2 is an ATP-binding cassette transporter protein, which was identified to be overexpressed in GSCs and higher-grade glioma tissues. We found that miR-145 was negative correlated with ABCG2 levels in GSCs, and reduction in ABCG2 expression decreased the cell migration and invasion of GSCs. Further, a luciferase reporter proved that ABCG2 was a direct target of miR-145 in GSCs. Thus, these findings underscore the potential of miR-145 to regulate the migration and invasion of GSCs through targeting ABCG2.

NEDD4-1 Regulates Migration and Invasion of Glioma Cells through CNrasGEF Ubiquitination In Vitro

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) plays a great role in tumor cell growth, but its function and mechanism in cell invasive behavior are totally unknown. Here we report that NEDD4-1 regulates migration and invasion of malignant glioma cells via triggering ubiquitination of cyclic nucleotide Ras guanine nucleotide exchange factor (CNrasGEF) using cultured glioma cells. NEDD4-1 overexpression promoted cell migration and invasion, while its downregulation specifically inhibited them. However, NEDD4-1 did not affect the proliferation and apoptosis of glioma cells. NEDD4-1 physically interacted with CNrasGEF and promoted its poly-ubiquitination and degradation. Contrary to the effect of NEDD4-1, CNrasGEF downregulation promoted cell migration and invasion, while its overexpression inhibited them. Importantly, downregulation of CNrasGEF facilitated the effect of NEDD4-1-induced cell migration and invasion. Interestingly, aberrant up-regulated NEDD4-1 showed reverse correlation with CNrasGEF protein level but not with its mRNA level in glioma tissues. Combined with the in vitro results, the result of glioma tissues indicated post-translationally modification effect of NEDD4-1 on CNrasGEF. Our study suggests that NEDD4-1 regulates cell migration and invasion through ubiquitination of CNrasGEF in vitro.

Oleanolic Acid Suppresses Migration and Invasion of Malignant Glioma Cells by Inactivating MAPK/ERK Signaling Pathway

Mitogen-activated protein kinases/Extracellular signal-regulated kinase (MAPK/ERK) pathway is essential for migration and invasion of malignant glioma. It is efficient to inhibit migration and invasion of glioma cells by targeting this pathway. Oleanolic acid (OA) has been well demonstrated to suppress survival, growth and angiogenesis of glioma cells. However, it is still unknown if OA affects the migration and invasion of glioma cells. We utilized U-87 MG glioma cell lines and primary glioma cells from patients to study the effect of OA on migration and invasion of glioma cells with multidisciplinary approaches. In this study, we found that OA significantly decreased the ability of glioma cells to migrate and invade. Epithelial-mesenchymal transition (EMT) of glioma cells was also suppressed by OA treatment. Furthermore, MAPK/ERK pathway was greatly inhibited in glioma cells under OA treatment. MAPK/ERK reactivation induced by a recombinant lentiviral vector, Lv-MEK, was able to rescue the inhibitory effect of OA on migration and invasion of glioma cells. Taken together, we provided evidences that OA was a MAPK/ERK pathway-targeting anti-tumor agent. Although the concentrations we used exceeded its physiological level, OA may be used to prevent migration and invasion of glioma cells by developing its derivatives with enhanced bioactivity.

Tumor-Associated Microglia/Macrophages Enhance the Invasion of Glioma Stem-like Cells via TGF-β1 Signaling Pathway

The invasion of malignant glioma cells into the surrounding normal brain tissues is crucial for causing the poor outcome of this tumor type. Recent studies suggest that glioma stem-like cells (GSLCs) mediate tumor invasion. However, it is not clear whether microenvironment factors, such as tumor-associated microglia/macrophages (TAM/Ms), also play important roles in promoting GSLC invasion. In this study, we found that in primary human gliomas and orthotopical transplanted syngeneic glioma, the number of TAM/Ms at the invasive front was correlated with the presence of CD133(+) GSLCs, and these TAM/Ms produced high levels of TGF-β1. CD133(+) GSLCs isolated from murine transplanted gliomas exhibited higher invasive potential after being cocultured with TAM/Ms, and the invasiveness was inhibited by neutralization of TGF-β1. We also found that human glioma-derived CD133(+) GSLCs became more invasive upon treatment with TGF-β1. In addition, compared with CD133(-) committed tumor cells, CD133(+) GSLCs expressed higher levels of type II TGF-β receptor (TGFBR2) mRNA and protein, and downregulation of TGFBR2 with short hairpin RNA inhibited the invasiveness of GSLCs. Mechanism studies revealed that TGF-β1 released by TAM/Ms promoted the expression of MMP-9 by GSLCs, and TGFBR2 knockdown reduced the invasiveness of these cells in vivo. These results demonstrate that TAM/Ms enhance the invasiveness of CD133(+) GSLCs via the release of TGF-β1, which increases the production of MMP-9 by GSLCs. Therefore, the TGF-β1 signaling pathway is a potential therapeutic target for limiting the invasiveness of GSLCs.

Triptolide inhibits proliferation and invasion of malignant glioma cells

Malignant glioma is the most devastating and aggressive tumor in brain, characterized by rapid proliferation and diffuse invasion. Chemotherapy and radiotherapy are the pivotal strategies after surgery; however, high drug resistance of malignant glioma and the blood-brain barrier usually render chemotherapy drugs ineffective. Here, we find that triptolide, a small molecule with high lipid solubility, is capable of inhibiting proliferation and invasion of malignant glioma cells effectively. In both investigated malignant glioma cell lines, triptolide repressed cell proliferation via inducing cell cycle arrest in G0/G1 phase, associated with downregulation of G0/G1 cell cycle regulators cyclin D1, CDK4, and CDK6 followed by reduced phosphorylation of retinoblastoma protein (Rb). In addition, triptolide induced morphological change of C6 cells through downregulation of protein expression of MAP-2 and inhibition of activities of GTPases Cdc42 and Rac1/2/3, thus significantly suppressing migratory and invasive capacity. Moreover, in an in vivo tumor model, triptolide delayed growth of malignant glioma xenografts. These findings suggest an important inhibitory action of triptolide on proliferation and invasion of malignant glioma, and encourage triptolide as a candidate for glioma therapy.

Identification of MMP-9 specific microRNA expression profile as potential targets of anti-invasion therapy in glioblastoma multiforme

The poor prognosis of glioblastoma multiforme (GBM) is largely attributed to their highly invasive nature and MMP-9 plays a pivotal role in regulating invasiveness of malignant glioma cells. MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to regulate a wide range of biological processes via targeting messenger RNA. Previous reports have shown many oncogenes regulate survival and invasion via targeting MMP-9 in GBM. But no literature indicates that miRNAs regulate glioma cell invasion through targeting MMP-9. Here, we show MMP-9 overexpression conferred a poor prognosis in 163 GBM patients. Furthermore, MMP-9 specific miRNA expression profile (14 positively and 31 negatively correlated miRNAs with MMP-9) was established via miRNA microarrays in 60 GBM samples. Among them, two miRNAs: miR-885-5p and miR-491-5p, were chosen for functional validation for their high positive correlation with MMP-9 expression. And upregulation of miR-885-5p and miR-491-5p were demonstrated to reduce the levels of MMP-9 expression and inhibit cellular invasion in U251 and U87 glioma cells. Furthermore, we found that miR-491-5p suppressed glioma cell invasion via targeting MMP-9 directly. To our knowledge, this is the first study to identify the MMP-9 specific microRNA signature which may provide potential targets for anti-invasion therapy in GBM.

Inhibition of tissue factor/protease-activated receptor-2 signaling limits proliferation, migration and invasion of malignant glioma cells

Tissue factor (TF) is upregulated in several malignant diseases, including gliomas. Here, we demonstrate pronounced differences in the expression of TF and its interactors factor VII and protease-activated receptor 2 (PAR-2) in nine human glioma cell lines (U87, U251, U343, U373, MZ-18, MZ-54, MZ-256, MZ-304, Hs 683) as detected by RT-PCR and Western blot analysis. Inhibition of TF signaling by a neutralizing monoclonal antibody (mAb TF9-10H10) led to significantly reduced proliferation in high-grade astroglial (MZ-18 and MZ-304) and oligodendroglial (Hs 683) cell lines abundantly expressing TF, but not in U373 cells expressing low amounts of TF. Scratch migration assays and Boyden chamber assays indicated that mAb TF9-10H10 and lentiviral knockdown of TF significantly reduced cell migration and invasion of MZ-18, MZ-304 and Hs 683 cells, both under normoxic and hypoxic conditions. Of note, all three cell lines displayed increased cell migration and invasion under hypoxic conditions (1% O2), which was associated with enhanced expression of TF and increased phosphorylation of p44/42 mitogen-activated protein kinase (ERK1/2). Silencing of TF blocked activation of the ERK pathway, induction of TF expression and the potentiating effect of hypoxia on cell migration and invasion. RNA interference against PAR-2 abrogated the autocrine effects of TF on cell proliferation, migration and invasion, indicating that TF signals via PAR-2 in glioma cells. Our results suggest an important role for the TF/FVIIa/PAR-2/ERK axis in tumor growth and invasion of glioma and suggest that TF may be a suitable target for the development of novel therapies against high-grade glioma.

The Pyk2 FERM domain as a target to inhibit glioma migration

The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH(2)-terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the beta5C-alpha1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro. Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression.

The Effect of Hyaluronic Acid on the Invasiveness of Malignant Glioma Cells : Comparison of Invasion Potential at Hyaluronic Acid Hydrogel and Matrigel

Hyaluronidase (HAse), a degrading enzyme of hyaluronic acid (HA), is highly expressed in patients with malignant glioma. The purpose of this study was to verify whether HAse is related to the invasion of glioma cells. We also investigated if glioma cells with higher mobility in 2-dimensioal (2-D) method have also higher mobility at 3-dimensional (3-D) environment. Malignant glioma cell lines (U87MG, U251MG, U343MG-A, and U373MG) were used, and their HAse expressions were evaluated by HA zymography. The migration ability was evaluated by simple scratch technique. The invasiveness of each cell lines was evaluated by Matrigel invasion assay and HA hydrogel invasion assay. In HA hydrogel invasion assay, colonies larger than 150 microm were regarded as positive ones and counted. Statistical analysis of migration ability and invasion properties of each cell lines was performed using t-test. In scratch test to examine migration ability of each cell lines, U87MG cells were most motile than others, and U343MG-A least motile. The HAse was expressed in U251MG and U343MG-A cell lines. However, U87MG and U373MG cell lines did not express HAse activity. In Matrigel invasion assay, the cell lines expressing HAse (U251MG and U343MG-A) were more invasive in the presence of HA than HAse deficient cell lines (U87MG and U373MG). In HA hydrogel invasion assay, the HAse-expressing cell lines formed colonies more invasively than HAse-deficient ones. Malignant Glioma cells expressing HAse were more invasive than HAse-deficient ones in 3-dimensional environment. Therefore, it might be suggested that invasion of malignant gliomas is suppressed by inhibition of HAse expression or HA secretion. Additionally, the ability of 2-D migration and 3-D invasion might not be always coincident to each other in malignant glioma cells.

The Role of Fascin in the Migration and Invasiveness of Malignant Glioma Cells

Malignant glioma is the most common primary brain tumor, and its ability to invade the surrounding brain parenchyma is a leading cause of tumor recurrence and treatment failure. Whereas the molecular mechanisms of glioma invasion are incompletely understood, there is growing evidence that cytoskeletal-matrix interactions contribute to this process. Fascin, an actin-bundling protein, induces parallel actin bundles in cell protrusions and increases cell motility in multiple human malignancies. The role of fascin in glioma invasion remains unclear. We demonstrate that fascin is expressed in a panel of human malignant glioma cell lines, and downregulation of fascin expression in glioma cell lines by small interfering RNA (siRNA) is associated with decreased cellular attachment to extracellular matrix (ECM) and reduced migration. Using immunofluorescence analysis, we show that fascin depletion results in a reduced number of filopodia as well as altered glioma cell shape. In vitro invasiveness of U251, U87, and SNB19 glioma cells was inhibited by fascin siRNA treatment by 52.2%, 40.3%, and 23.8% respectively. Finally, we show a decreased invasiveness of U251-GFP cells by fascin knockdown in an ex vivo rat brain slice model system. This is the first study to demonstrate a role for fascin in glioma cell morphology, motility, and invasiveness.

Ezrin expression in tissue microarray of primary and recurrent gliomas.

Malignant progression, infiltrative growth pattern and recurrencies after surgery are characteristic features of diffuse gliomas. Ezrin is a membrane-cytoskeleton linker protein expressed in several types of neoplasms. In experimental models, increased ezrin expression correlates with invasion of malignant glioma cells. We studied ezrin expression and its correlation with patient survival in 229 primary and recurrent astrocytomas (WHO grades II-IV), oligodendrogliomas (II-III) and oligoastrocytomas (II-III) of 113 patients. Ezrin expression as evaluated by immunohistochemistry and immunoblotting was detected in all studied glioma types. Staining intensity and number of immunoreactive cells correlated with increasing malignancy of astrocytomas and oligoastrocytomas (P = 0.001). Ezrin expression was strongest in astrocytomas (P = 0.006). Also oligodendrogliomas were positive for ezrin. High ezrin expression in primary gliomas correlated with shorter time to recurrence (P < 0.05) and poor overall survival (P < 0.05) of the patients. Ezrin expression increased during progression of the tumours (P < 0.05). However, ezrin was not an independent prognostic factor. The results of this study show that ezrin expression is associated with progression of gliomas and correlates with histological cell type and WHO tumour grade.

Connexin 43 enhances the adhesivity and mediates the invasion of malignant glioma cells.

A hallmark of astrocytic tumors is their infiltrative nature. Although their aggressive and typically widespread dispersal in the adult brain differs fundamentally from that of other brain tumors, little is known about their cellular basis. Astrocytic tumors express the gap junction protein connexin 43 (Cx43), and we show here that Cx43 expression induced the morphological transformation of glioma cells into an epithelial phenotype. In a short-term aggregation assay, Cx43 expression was associated with a several-fold increase in the competence of glioma cells to aggregate. Antibodies directed against the extracellular domain of Cx43 restored the connexin-deficient phenotype, as manifested by a dose-dependent reduction in aggregation. Apart from their role in gap junction formation, connexins may therefore be considered a distinct class of membrane proteins with adhesive properties. Moreover, implanted Cx43-expressing glioma cells established functional gap junction channels with host astrocytes and dispersed through a substantially greater volume of brain parenchyma than mock- and mutant Cx43-transfected sister cells. Cx43 expression therefore may modulate not only the adhesion of astrocytes to one another, but the spread of glial tumor cells throughout astrocytic syncytia. These observations widen our concept of the potential interactions between tumor cells and their surroundings and suggest that both connexin proteins and their derived gap junctions are critical determinants of the invasiveness of central gliomas.