Hyang-Hwa Ryu

Preparation of poly(dl-lactide-co-glycolide) microspheres encapsulating all-trans retinoic acid

Poly(DL-lactide-co-glycolide) (PLGA) microspheres containing all-trans retinoic acid (atRA) were prepared by o/w solvent evaporation method and various preparation parameters, such as poly(vinyl alcohol) (PVA) concentration in aqueous solution, PVA MW, drug weight, solvent, polymer MW, and polymer weight, on the characteristics of microspheres and drug release were investigated. PVA concentration in water phase was a critical factor in making microspheres consistently with smooth surface and round shape. In our study, at least 2% (w/v) of PVA in aqueous solution was necessary for making microspheres with round shape. The particle size of microspheres ranged 10-100 microm. AtRA was slowly released from PLGA microspheres over 30 days. Sterilization of microspheres by ethylene oxide (EO) gas at 37 degrees C did not significantly affect the characteristics of drug release or its morphology. Cell growth inhibition of atRA was affected by preparation process of microspheres rather than the EO-gas sterilization process. These results indicate that PLGA microspheres containing atRA are acceptable for controlled release devices for use in the treatment of brain tumor.

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.

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.

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.

Increased expression of intracystic matrix metalloproteinases in brain tumors: relationship to the pathogenesis of brain tumor-associated cysts and peritumoral edema

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.

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.

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.

PDIA6 regulation of ADAM17 shedding activity and EGFR-mediated migration and invasion of glioblastoma cells

OBJECTIVE In patients with glioblastoma, local invasion of tumor cells causes recurrence and shortens survival. The goal of this study was to determine whether protein disulfide isomerase (PDI) A6 regulates migration and invasion of glioblastoma cells and the associated factors. METHODS U87MG cells were treated with either PDIA6 or ADAM17 small interfering RNA (siRNA) fragments or with both types of siRNA fragments, and expression was confirmed by reverse transcription-polymerase chain reaction and Western blot. Migration and invasion were assessed using a wound-healing assay, a Matrigel assay, and an organotypic culture system. After the U87MG cells were treated with siRNAs and epidermal growth factor receptor (EGFR) inhibitors, the expression of matrix metalloproteinase-2 (MMP-2), membrane Type 1-matrix metalloproteinase (MT1-MMP), integrin, phosphorylated focal adhesion kinase (pFAK), and phosphorylated EGFR (pEGFR) was detected by Western blotting and zymography. RESULTS U87MG cell migration and invasion increased significantly after inhibition of PDIA6. The MMP-2 activation ratio and ADAM17 activity (as a sheddase of the proligand) increased, and expression of pEGFR, pFAK, integrin α5β3, and MT1-MMP was induced, compared with control levels. Furthermore, heparin-binding epidermal growth factor (EGFR signaling ligand) was highly expressed in PDIA6-knockdown cells. After siPDIA6-transfected U87MG cells were treated with EGFR signaling inhibitors, expression of pFAK, MMP-2, and MT1-MMP decreased and invasion decreased significantly. Simultaneous double-knockdown of PDIA6 and ADAM17 reduced pEGFR and pFAK expression, compared with control levels. CONCLUSIONS The authors propose that inhibiting PDIA6 could transduce EGFR signaling by activating and inducing ADAM17 during migration and invasion of U87MG glioblastoma cells. The results of this study suggest that PDIA6 is an important component of EGFR-mediated migration and invasion of U87MG cells. This is the first report of the effects of PDIA6 on migration and invasion in glioblastoma.

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.