Metallothinein 1E Enhances Glioma Invasion through Modulation Matrix Metalloproteinases-2 and 9 in U87MG Mouse Brain Tumor Model

Malignant glioma cells invading surrounding normal brain are inoperable and resistant to radio- and chemotherapy, and eventually lead to tumor regrowth. Identification of genes related to motility is important for understanding the molecular biological behavior of invasive gliomas. According to our previous studies, Metallothionein 1E (MT1E) was identified to enhance migration of human malignant glioma cells. The purpose of this study was to confirm that MT1E could modulate glioma invasion in vivo. Firstly we established 2 cell lines; MTS23, overexpressed by MT1E complementary DNA construct and pV12 as control. The expression of matrix metalloproteinases (MMP)-2, -9 and a disintegrin and metalloproteinase 17 were increased in MTS23 compared with pV12. Furthermore it was confirmed that MT1E could modulate MMPs secretion and translocation of NFkB p50 and B-cell lymphoma-3 through small interfering ribonucleic acid knocked U87MG cells. Then MTS23 and pV12 were injected into intracranial region of 5 week old male nude mouse. After 4 weeks, for brain tissues of these two groups, histological analysis, and immunohistochemical stain of MMP-2, 9 and Nestin were performed. As results, the group injected with MTS23 showed irregular margin and tumor cells infiltrating the surrounding normal brain, while that of pV12 (control) had round and clear margin. And regrowth of tumor cells in MTS23 group was observed in another site apart from tumor cell inoculation. MT1E could enhance tumor proliferation and invasion of malignant glioma through regulation of activation and expression of MMPs.

Bacitracin Inhibits the Migration of U87-MG Glioma Cells via Interferences of the Integrin Outside-in Signaling Pathway

OBJECTIVE: Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. METHODS: U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. RESULTS: Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). CONCLUSION: Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.

Proteomic Analysis between U87MG and U343MG-A Cell Lines: Searching for Candidate Proteins for Glioma Invasion

BACKGROUND: To investigate the molecular basis for invasion of malignant gliomas, proteomic analysis approach was carried out using two human glioma cell lines, U87MG and U343MG-A that demonstrate different motility and invasiveness in in vitro experiments. METHODS: High-resolution two-dimensional gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry analysis were performed. RESULTS: Nine distinct protein spots that were recognized with significant alteration between the two cell lines. Five of these protein spots were up-regulated in U87MG and four were up-regulated in U343MG-A. CONCLUSION: Among these proteins, cathepsin D was shown to be one of the important proteins which are related with glioma invasion. However, further studies are necessary to reveal the exact role and mechanism of cathepsin D in glioma invasion.

Characterization of Invading Glioma Cells Using Molecular Analysis of Leading-Edge Tissue

OBJECTIVE: We have introduced a method of characterization of invading glioma cells by using molecular analysis of marginal invading tumor cells and molecular profiles of glioma tumor margin. METHODS: Each of tumor core and marginal tissues was obtained in 22 glioma patients. Tumor core cells and marginal cells from each glial tumor were collected by laser capture microdissection or intraoperative microdissection under the operating microscope. Expression of MMP-2, MMP-9, CD44 and RHAMM mRNA by invading glioma cells compared with tumor core was confirmed by realtime-PCR of twenty-four glioma specimens. Clinical data also were reviewed for invasion and recurrence pattern of the gliomas radiologically and invasive rim pattern microscopically. RESULTS: Overall results of the molecular analysis showed that relative overexpression of MMP-2, MMP-9 and RHAMM were noted at the invasive edge of human glioma specimens comparing to the tumor core but CD44 was highly expressed in the tumor core comparing to the margin. High marginal expression of MMP-2 and MMP-9 were noted in poorly ill-defined margin on the pathological finding. High marginal expression of CD44 and MMP-2 were demonstrated in the midline cross group on the radiological review, and that of RHAMM and MMP-2 were showed in the aggressive recurrence group. High expression of MMP-2 seems to be involved in the various invasion-related phenomenons. CONCLUSION: Up-regulation of MMP-2, MMP-9, CD44 and RHAMM was noted in invasive edge of gliomas according to the various clinical situations.

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

OBJECTIVE: 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. METHODS: 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 micrometer were regarded as positive ones and counted. Statistical analysis of migration ability and invasion properties of each cell lines was performed using t-test. RESULTS: 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. CONCLUSION: 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.