Kyung-Keun Kim

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.

COMP-Ang1, a chimeric form of Angiopoietin 1, enhances BMP2-induced osteoblast differentiation and bone formation

INTRODUCTIONnAngiogenesis is closely associated with bone formation, especially endochondral ossification. Angiopoietin 1 (Ang1) is a specific growth factor functioning to generate a stable and matured vasculature through the Tie2 receptor/PI3K/AKT pathway. Recently cartilage oligomeric matrix protein (COMP)-Ang1, an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor and AKT, was developed. This study was designed to examine the effects of angiogenic COMP-Ang1 on BMP2-induced osteoblast differentiation and bone formation.nnnMETHODSnExpression of endogenous Ang-1 and its binding receptor Tie 2 mRNA was examined in osteoblast-like cells and primary mouse calvarial cells by RT-PCR analysis, and was also monitored during osteoblast differentiation induced by BMP-2 and/or ascorbic acid and beta-glycerophosphate. Effects of COMP-Ang-1 on osteoblast differentiation and mineralization were evaluated by alkaline phosphatase (ALP) activity and osteocalcin (OC) production, and Alizarin red stain. For a molecular mechanism, Western blot and OG2 and 6xOSE promoter assays were done. For in vivo evaluation, adenoviral (Ad) vectors containing COMP-Ang-1 or BMP-2 gene were administered into thigh muscle of mice, and after 2 weeks bone formation was analyzed by micro-computed tomography and histology. Angiogenic event of COMP-Ang1 was confirmed by immunofluorescence analysis with anti-CD31 antibody.nnnRESULTSnExpression of Tie2 receptor was significantly increased in the course of osteoblast differentiation. Treatment or overexpression of COMP-Ang1 enhanced BMP2-induced ALP activity, OC production, and mineral deposition in a dose-dependent manner. In addition, COMP-Ang1 synergistically increased OG2 and 6xOSE promoter activities of BMP2, and sustained p38, Smad and AKT phosphorylation of BMP2. Notably, in vivo intramuscular injection of COMP-Ang1 dose-dependently enhanced BMP2-induced ectopic bone formation with increases in CD31 reactivity.nnnCONCLUSIONSnThese results suggest that COMP-Ang1 synergistically enhanced osteoblast differentiation and bone formation through potentiating BMP2 signaling pathways and angiogenesis. Combination of BMP2 and COMP-Ang1 should be clinically useful for therapeutic application to fracture and destructive bone diseases.

Brain tumor invasion model system using organotypic brain-slice culture as an alternative to in vivo model

Abstractn Purpose. The primary cause of local recurrence and therapeutic failure in the treatment of malignant gliomas is the invasion of tumor cells into the surrounding normal brain. While it is known that malignant gliomas infiltrate diffusely into regions of normal brain, it is frequently very difficult to unequivocally identify the solitary invading glioma cell in histopathological preparations, or in experimental glioma models. We have developed an experimental invasion assay system, which allows us to track the solitary invasive glioma cell, using human brain tissue obtained from routine craniotomies for seizures or trauma.n Methods. This tissue is cut into 1-mm thick slices and cultured in the upper chamber of Transwell culture dishes on top of a 0.4-µm pore size polyester membrane, which is fed on medium provided in the lower chamber. Glioma cells are stably transfected with vectors containing a green fluorescent protein (GFP) cDNA. Stable, high-level expression GFP transfectants were selected by direct visualization under fluorescence microscope. In addition, various tumor spheroids are stained with vital dye, DiI, to track the invading cells. GFP-expressing glioma cells or stained spheroids were then implanted on the center of the brain slice, and the degree of brain tumor invasion into the brain tissue was evaluated at different time points by optical sectioning using a confocal microscope.n Results. We observed that GFP-expressing glioma cells or stained spheroids could be readily tracked and followed with this model system. Individual tumor cells that exhibited green or red fluorescence could be identified and their migration path through the brain slices unequivocally followed.n Conclusion. This experimental invasion system may be of considerable utility in studying the process of brain tumor invasion and in evaluating its invasiveness in individual brain tumor because it not only provides a better representation of extracellular matrix molecules normally encountered by invading glioma cells, but also provides the fluorescent tag applied to the tumor cells.

Possible Pathophysiological Role of Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloproteinases (MMPs) in Metastatic Brain Tumor-associated Intracerebral Hemorrhage

SummaryBackgroundIntratumoral hemorrhage, as one of the cerebrovascular complications in various tumor-related conditions, occurs mainly in malignant brain tumors. Recent studies have shown that the overexpression of vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) may play a role for the loss of vascular integrity and the subsequent hemorrhage in several instances, in addition to their well-known properties in tumor development and metastasis.MethodsTo investigate the potential role of VEGF and MMP in hemorrhagic complication of metastatic brain tumor, we estimated the expression of VEGF, MMP-2 & -9 by immunohistochemical studies in pathological specimens of metastatic brain tumors obtained from 16 patients, 7 in hemorrhagic and 9 in non-hemorrhagic group. We also examined the expression of collagen type IV, CD34, Factor VIII in order to evaluate the status of tumor vasculature.ResultsPatients in hemorrhagic group showed a higher VEGF expression with neovascularization than those in non-hemorrhagic group. The basement membranes of newly formed vessels were disrupted in cases with high expression in both MMP-2 and -9. These results indicate that rapid growing nascent blood vessels, responding vigorously to VEGF, are concentrated around the hemorrhagic tumors. Besides, these results suggest a possibility that the basement membranes of these nascent vessels could be disrupted proteolytically by MMP.ConclusionWe conclude that overexpression of VEGF and MMP may play a role in metastatic brain tumor-associated hemorrhage. Presumably, the underlying pathophysiological mechanisms are through rapid growth and breakdown of vessels around the tumors caused by overexpression of VEGF and MMP of tumor cells.

Effect of Topical Cyclosporin A on Herpetic Stromal Keratitis in a Mouse Model

Purpose: To study the effect of topical cyclosporin A on herpetic stromal keratitis (HSK) in a mouse model. Methods: The corneas of BALB/c mice were infected with herpes simplex type 1 virus. The mice were divided into 4 groups according to topical treatment methods: vehicle solution, 0.01% cyclosporin A, 0.1% cyclosporin A, and 1% cyclosporin A. The severity of stromal keratitis was graded clinically at 3, 7, 10, and 14 days after treatment. Histologic and immunohistochemical examinations were performed to analyze infiltrating inflammatory cells and T lymphocytes at 14 days after treatment. Flow cytometry was performed to count CD4+ T cells at 14 days after treatment. Results: On day 3 after treatment, there were no significant differences in mean severity scores of HSK between the vehicle and cyclosporin A-treated groups. On days 7, 10, and 14, the severity scores in the groups treated with 0.1% or 1% cyclosporin A decreased significantly compared with the vehicle group (P < 0.05). However, the scores in the group treated with 0.01% cyclosporin A did not change up to 14 days after treatment. By histologic and immunohistochemical analysis, a significant decrease in the total number of inflammatory cells and T lymphocytes was detected in the groups treated with 0.1% or 1% cyclosporin A. Flow cytometry also showed similar findings. Conclusions: Topical cyclosporin A effectively reduces stromal haze and inflammation in experimental HSK, and cyclosporin A eyedrops with a >0.1% concentration can be used for the treatment of HSK.

Knockdown of RON Inhibits AP-1 Activity and Induces Apoptosis and Cell Cycle Arrest Through the Modulation of Akt/FoxO Signaling in Human Colorectal Cancer Cells

Background/AimsAltered Recepteur d’Origine nantais (RON) expression transduces signals inducting invasive growth phenotype that includes cell proliferation, migration, matrix invasion, and protection of apoptosis in human cancer cells. The aims of the current study were to evaluate whether RON affects tumor cell behavior and cellular signaling pathways including activator protein-1 (AP-1) and Akt/forkhead box O (FoxO) in human colorectal cancer cells.MethodsTo study the biological role of RON on tumor cell behavior and cellular signaling pathways in human colorectal cancer, we used small interfering RNA (siRNA) to knockdown RON gene expression in human colorectal cancer cell line, DKO-1.ResultsKnockdown of RON diminished migration, invasion, and proliferation of human colorectal cancer cells. Knockdown of RON decreased AP-1 transcriptional activity and expression of AP-1 target genes. Knockdown of RON activated cleaved caspase-3, -7, -9, and PARP, and down-regulated the expression of Mcl-1, survivin and XIAP, leading to induction of apoptosis. Knockdown of RON induced cell cycle arrest in the G2/M phase of cancer cells by an increase of p27 and a decrease of cyclin D3. Knockdown of RON inhibited the phosphorylation of Akt/FoxO signaling proteins such as Ser473 and Thr308 of Akt and FoxO1/3a.ConclusionsThese results indicate that knockdown of RON inhibits AP-1 activity and induces apoptosis and cell cycle arrest through the modulation of Akt/FoxO signaling in human colorectal cancer cells.

Expression of KITENIN in human colorectal cancer and its relation to tumor behavior and progression

KAI1 COOH‐terminal interacting tetraspanin (KITENIN) contributes to tumor invasion and metastasis in various cancers. The aim of current study was to evaluate whether KITENIN affects tumor cell invasion and prognosis in human colorectal cancers. We investigated the biologic role of KITENIN on tumor cell invasion by using small interfering RNA in Caco2, DLD1, and SW480. We evaluated the expression of KITENIN and activator protein‐1 (AP‐1) target genes in human colorectal cancer tissues. The tumor cell invasion was decreased by knockdown of KITENIN in three tested cell lines. The mRNA expression of cyclin D1 and COX‐2 was decreased in KITENIN knockdown Caco2 and the mRNA expression of MMP‐3 and COX‐2 was decreased in KITENIN knockdown DLD1 and SW480. The extracellular‐signal protein kinase 1/2 (ERK1/2) phosphorylation was decreased in KITENIN knockdown in three tested cell lines. Expression of KITENIN and AP‐1 target genes was significantly increased in human colorectal cancer tissues. The ERK1/2, c‐Jun N‐terminal kinase (JNK) and p38 phosphorylations were increased in human colorectal cancer tissues. Expression of KITENIN was significantly associated with lymphovascular invasion, depth of invasion, lymph node metastasis, tumor stage and poor survival. These results indicate that KITENIN is associated with human colorectal cancer progression including invasion and metastasis.

Small interfering RNA-directed targeting of RON alters invasive and oncogenic phenotypes of human hepatocellular carcinoma cells

The recepteur dorigine nantais (RON) receptor tyrosine kinase is highly expressed in various cancers including human hepatocellular carcinoma (HCC) and involved in tumor progression. The aims of the current study were to evaluate whether RON affects tumor cell behavior and oncogenic signaling cascades in HCC cells. We investigated the biologic role of RON on tumor cell behavior and oncogenic signaling cascades including Akt, c-Raf and extracellular signal-regulated kinase (ERK) by using the small interfering RNA (siRNA) in HCC cell lines, chang, HepG2 and Huh7. Knockdown of RON suppressed tumor cell migration and invasion in all tested HCC cell lines. The proportion of apoptotic cells induced by knockdown of RON was greater than that induced by transfection of the scramble siRNA in all tested HCC cell lines. Knockdown of RON resulted in cell cycle arrest in the G2/M phase of chang and Huh7 cells, and sub G1 phase of HepG2 cells. Knockdown of RON activated cleaved caspase-3 and PARP, and down-regulated the expression of Bcl-2, Bcl-xL and survivin, leading to induction of apoptosis in all tested cell lines. Knockdown of RON negatively regulates the progression of the cell cycle by decreasing cyclin D1 and D3, and increasing p21 and p27 in all tested cell lines. The phosphorylation of Akt, c-Raf and ERK1/2 signal proteins was significantly blocked by knockdown of RON in all tested cell lines. These results suggest that RON is associated with invasive and oncogenic phenotypes such as tumor cell migration, invasion, resistance to apoptosis and cell cycle arrest through the modulation of Akt, c-Raf and ERK signaling cascades in HCC cells.

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.

Alumina coating on 5 V lithium cobalt fluorophosphate cathode material for lithium secondary batteries – synthesis and electrochemical properties

The high voltage cathode material, Li2CoPO4F was successfully synthesized and coated with various amounts of Al2O3 for enhanced electrochemical performance. X-ray diffraction data revealed that the unit cell had an orthorhombic structure with the Pnma space group. An initial discharge capacity of ∼127 mA h g−1 was obtained between 2 and 5.1 V. The capacity retention, ratio of discharge capacity at the 15th cycle to that at the 1st cycle, at a current rate of C/2 was increased from 53% for the pristine sample to 73% for 1 wt% Al2O3 coated Li2CoPO4F. Cyclic voltammetry and charge–discharge studies showed an increased operating voltage for Li2CoPO4F after Al2O3 coating. Electrochemical impedance spectroscopy results suggested a reduction in charge transfer resistance in the as-prepared samples. Moreover, the reduction in irreversible capacity loss, defined as the difference between charge capacity and discharge capacity of the same cycle, in the initial cycle suggested a decrease in electrolyte oxidation after the coating process. A uniform amorphous layer of Al2O3 coating of ∼3 nm thickness protected the surface of Li2CoPO4F during high voltage operation.