Shu-Guang Jin

The correlation and prognostic significance of MGMT promoter methylation and MGMT protein in glioblastomas.

OBJECTIVEThe aim of this study was to evaluate the correlation and prognostic significance of MGMT promoter methylation and protein expression in patients with glioblastoma. METHODSEighty-three patients with glioblastoma underwent surgery followed by radiotherapy and temozolomide chemotherapy between October 2000 and June 2008. To investigate the correlation between MGMT methylation and MGMT expression, methylation-specific polymerase chain reaction (MSP) and immunohistochemical staining was performed. To analyze the correlation between MGMT methylation and MGMT expression according to location, biopsies were obtained from 37 different sites within the tumors in 12 patients. Age, sex, Karnofsky Performance Scale status, extent of removal, chemotherapeutic methods, and MGMT promoter methylation and protein expression were analyzed as prognostic factors. RESULTSThe total median survival was 15.8 months (range, 12.6–19.1 months). The results of MSP were the same at various sites in 12 patients. A correlation between MSP and immunohistochemical staining was observed in 50% of the patients. In 73 patients, negative MGMT expression was detected in 70.5% of 44 patients with MGMT promoter methylation, and positive expression was observed in 55.2% of the 29 patients with unmethylated promoters. Multivariate analysis revealed that the extent of removal (P = 0.001) and the combination of MGMT promoter methylation and negative MGMT expression (median survival, 20.06 months; P = 0.006) were significantly associated with longer survival. CONCLUSIONWe report the feasibility of using MSP combined with immunohistochemical staining as a prognostic factor. The results of the present study suggest that MGMT promoter methylation in combination with negative MGMT expression might be a good prognostic factor in patients with glioblastoma.

Doxorubicin-incorporated nanoparticles composed of poly(ethylene glycol)-grafted carboxymethyl chitosan and antitumor activity against glioma cells in vitro.

In this study, methoxy poly(ethylene glycol)-grafted carboxymethyl chitosan (CMCPEG) was synthesized to make nanoparticles with doxorubicin (DOX) by ion complex formation. Since DOX has positive amine groups, it can interact with the carboxymethyl group of CMCPEG. The particle size of DOX-incorporated nanoparticles of CMCPEG was < 300 nm and nanoparticles had spherical shapes at morphological observation, indicating that DOX/CMCPEG mixtures can form spherical nanoparticles. In a drug release study, higher drug content induced an extended release of drug. Drug release was significantly changed by the release media pH. DOX release was faster at an acidic pH than a neutral or basic pH. The antitumor activity of DOX-incorporated nanoparticles in vitro was tested with DOX-resistant C6 glioma cells. Nanoparticles showed increased cytotoxicity compared to DOX alone. These results suggest that DOX was unable to penetrate into cells and did not effectively inhibit cell proliferation. In contrast, nanoparticles can penetrate into cells and effectively inhibit cell proliferation. Observation of cells under red fluorescence confirmed these results, i.e., nanoparticle-treated C6 cells, unlike DOX-treated cells, had strong red fluorescence. Since DOX has strong red fluorescence, DOX-incorporated nanoparticles entered into the tumor cells more than DOX alone. As a result, we suggest that DOX-incorporated nanoparticles of CMCPEG are superior candidates for antitumor drug delivery.

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 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. 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.

Preparation of polylactide-co-glycolide nanoparticles incorporating celecoxib and their antitumor activity against brain tumor cells.

Background Celecoxib, a cyclo-oxygenase (COX)-2 inhibitor, has been reported to mediate growth inhibitory effects and to induce apoptosis in various cancer cell lines. In this study, we examined the potential effects of celecoxib on glioma cell proliferation, migration, and inhibition of COX-2 expression in vitro. Methods Celecoxib was incorporated into poly DL-lactide-co-glycolide (PLGA) nanoparticles for antitumor drug delivery. Results PLGA nanoparticles incorporating celecoxib had spherical shapes and their particle sizes were in the range of 50–200 nm. Drug-loading efficiency was not significantly changed according to the solvent used, except for acetone. Celecoxib was released from the PLGA nanoparticles for more than 2 days, and the higher the drug content, the longer the duration of drug release. PLGA nanoparticles incorporating celecoxib showed cytotoxicity against U87MG tumor cells similar to that of celecoxib administered alone. Furthermore, celecoxib did not affect the degree of migration of U87MG cells. PLGA nanoparticles incorporating celecoxib showed dose-dependent cytotoxicity similar to that of celecoxib alone in C6 rat glioma cells. Western blot assay of the C6 cells showed that neither celecoxib alone nor PLGA nanoparticles incorporating celecoxib affected COX-2 expression. Conclusion PLGA nanoparticles incorporating celecoxib had antitumor activity similar to that of celecoxib alone, even though these particles did not affect the degree of migration or COX-2 expression in the tumor cells.

Nogo-A expression in oligodendroglial tumors.

Nogo‐A belongs to the reticulon protein family and is expressed in the inner and outer loops of myelin sheaths of oligodendrocytes. We analyzed the patterns of Nogo‐A expression in human gliomas in an effort to identify a useful marker for the characterization of oligodendroglial tumors. We determined the expression of Nogo‐A in a panel of 58 astrocytic and oligodendroglial tumors using immunohistochemistry and compared the expression of Nogo‐A with Olig‐2, a recently identified marker for oligodendrogliomas. To localize Nogo‐A expression, immunofluorescent staining was performed using other glial markers (MAP‐2 and GFAP). We also confirmed the overexpression of the Nogo‐A protein in 53 astrocytic and oligodendroglial tumors using Western blot analysis. Based on immunohistochemical analysis, Nogo‐A and Olig‐2 had specificity in the detection of oligodendroglial tumors from astrocytic tumors (P = 0.001). The level of Nogo‐A staining was highly correlated with Olig‐2 (P = 0.001). The sensitivity and specificity of Nogo‐A for oligodendroglial tumors was 86.9% and 57.1%, respectively. Nogo‐A expression overlapped that of other oligodendroglial markers, but with different patterns of expression. Western blot analysis revealed that Nogo‐A is predominantly expressed in 85.7% of oligodendroglioma cells and 93.7% of anaplastic oligodendroglioma cells. Like other oligodendroglial markers, Nogo‐A is highly expressed in oligodendroglial tumors; however, it does not serve as a definite marker specific for oligodendroglial tumors.

Anticancer activity of PEGylated matrix metalloproteinase cleavable peptide-conjugated adriamycin against malignant glioma cells.

Although matrix metalloproteinases (MMPs) play a crucial role in the invasion and growth of malignant gliomas, their increased activity in tumor environment can be used as a specific target for chemotherapy. We investigated whether polymer-drug conjugates formed via MMP-cleavable peptide linkages could provide MMP-responsive tumor targeting and cytotoxicity for malignant glioma cells. One end of an MMP-cleavable peptide was attached to the end of methoxy polyethylene glycol (MPEG) while the other end was attached to adriamycin (ADR). The release of drugs in the presence of conditioned media of U87MG cells was investigated. The cytotoxicities of the MMP-cleavable MPEG-peptide-ADR (PPA) conjugates and non-cleavable MPEG-ADR (PA) conjugates were investigated using U87MG cells. The (1)H nuclear magnetic resonance (NMR) spectra confirmed the conjugation of the two ends of the peptide to the ends of MPEG and ADR, respectively. Gelatin zymography showed that MMP-2 was strongly expressed in the media of U87MG cells. The PA conjugate did not release ADR either in the phosphate buffered saline (PBS) or conditioned media of U87MG cells. The PPA conjugate released ADR in the presence of the conditioned media of U87MG cells, but not in PBS only. In the cytotoxicity test using U87MG cells, ADR and PPA conjugate showed similar anti-proliferative activities, while the cytotoxicity of PA conjugate was lower than that of ADR. Considering that the cytotoxicity of the PPA conjugate was similar to that of ADR, MMP-cleavable polymer-drug conjugates can be used as targeting carriers for the purpose of inhibiting the proliferation of malignant glioma cells.

Screening for motility-associated genes in malignant astrocytoma cell lines

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.

GRIM-19 Expression and Function in Human Gliomas

OBJECTIVE We determined whether the expression of GRIM-19 is correlated with pathologic types and malignant grades in gliomas, and determined the function of GRIM-19 in human gliomas. METHODS Tumor tissues were isolated and frozen at -80 just after surgery. The tissues consisted of normal brain tissue (4), astrocytomas (2), anaplastic astrocytomas (2), oligodendrogliomas (13), anaplastic oligodendrogliomas (11), and glioblastomas (16). To profile tumor-related genes, we applied RNA differential display using a Genefishing DEG kit, and validated the tumor-related genes by reverse transcription polymerase chain reaction (RT-PCR). A human glioblastoma cell line (U343MG-A) was used for the GRIM-19 functional studies. The morphologic and cytoskeletal changes were examined via light and confocal microscopy. The migratory and invasive abilities were investigated by the simple scratch technique and Matrigel assay. The antiproliferative activity was determined by thiazolyl blue Tetrazolium bromide (MTT) assay and FACS analysis. RESULTS Based on RT-PCR analysis, the expression of GRIM-19 was higher in astrocytic tumors than oligodendroglial tumors. The expression of GRIM-19 was higher in high-grade tumors than low-grade tumors or normal brain tissue; glioblastomas showed the highest expression. After transfection of GRIM-19 into U343MG-A, the morphology of the sense-transfection cells became larger and more spindly. The antisense-transfection cells became smaller and rounder compared with wild type U343MG-A. The MTT assay showed that the sense-transfection cells were more sensitive to the combination of interferon-beta and retinoic acid than U343MG-A cells or antisense-transfection cells; the anti-proliferative activity was related to apoptosis. CONCLUSION GRIM-19 may be one of the gene profiles which regulate cell death via apoptosis in human gliomas.

Solitary intracranial subdural osteoma: Intraoperative findings and primary anastomosis of an involved cortical vein

We report a patient with a intracranial subdural osteoma with a large cortical vein passing through the subdural calcified mass. A 60-year-old man presented with an approximately 3-year history of persistent headache. Computerized tomography (CT) scanning showed a homogeneous high-density nodule attached to the inner surface of the right frontal skull. Intraoperatively, the hard mass was found to be located in the intradural subarachnoid space. A large cortical vein passed through the subdural mass and was anastomosed in an end-to-end fashion after the excision of the segment involved by the tumor. The histopathologic examination showed lamellated bony trabeculae lined by osteoblasts and the underlying dura was uninvolved by the tumor cells.

Possible role of matrix metalloproteinases (MMPs) in hyperostosis of intracranial meningiomas

ObjectAlthough bone invasion and hyperostosis are common phenomena in patients with intracranial meningiomas, the basic pathomechanism is not fully understood. Based on an immunohistochemical study of surgically resected samples with hyperostosis, we postulate a possible mechanism of hyperostosis in patients with intracranial meningiomas.Materials and methodsForty-six meningiomas were evaluated in this study. Twenty-six meningiomas associated with hyperostosis specimens served as the study group, and 20 meningiomas without any bony changes served as controls. An immunohistochemical staining technique was used to detect the expression of matrix metalloproteinase (MMP)-2, -9, and -13, membrane type (MT)1-MMP, estrogen receptor (ER), and progesterone receptor (PR) in the main tumor and hyperostotic portions of the studied samples.ResultsIn the non-hyperostosis group, expression of MMP-13, MT1-MMP, and ER was significantly less than in the main tumor portion of hyperostotic meningiomas, while there was no difference in the expression of MMP-2 and -9 and PR in the main tumor between the two groups. In the hyperostosis group, the immunoreactivity of MMP-2 in the hyperostotic portion revealed a higher pattern of expression than the main tumor (p < 0.002). The expression of MMP-9, MT1-MMP, ER, and PR had relatively positive immunoreactivity in the main tumor portion (P < 0.05).ConclusionsIncreased expression of MMP-13 and MT1-MMP in the tumor portion of hyperostosis of meningiomas might contribute to the initiation of osteolysis. Activated MMP-2 in hyperostotic lesions may change the physiological metabolism of the skull bone, thus playing an important role in hyperostosis formation.