Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

BACKGROUND: Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. METHODS: Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. RESULTS: UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. CONCLUSIONS: UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer.

Anthocyanins From the Fruit of Vitis coignetiae Pulliat Potentiate the Cisplatin Activity by Inhibiting PI3K/Akt Signaling Pathways in Human Gastric Cancer Cells

BACKGROUND: Cisplatin (cis-diaminedichloroplatinum, CDDP) is a widely used chemotherapeutic agent for the treatment of many cancers. However, initial resistance to CDDP is a serious problem in treating these cancers. Vitis coignetiae Pulliat (Meoru in Korea) have shown anti-nuclear factor kappa B and anti-epidermal growth factor receptor activities in cancer cells. METHODS: In this study, in order to seeking an approach to increase the anti-cancer effects of CDDP with natural products. Here, we investigated anthocyanins isolated from Vitis coignetiae Pulliat (anthocyanidins isolated from meoru, AIMs) can enhance anti-cancer effects of cisplatin (CDDP) in stomach cancer cells. The cell viability of SNU-1 and SNU-16 cells after treated with AIMs and CDDP were analyzed by MTT assay. The expressions of Akt and X-linked inhibitor of apoptosis protein (XIAP) proteins were examined by western blot in AIMs- and CDDP-treated cells. RESULTS: We found that AIMs enhanced anticancer effects of CDDP, which activity was additive but not synergistic. AIMs suppressed Akt activity of the cancer cells activated by CDDP. AIMs also suppressed in XIAP an anti-apoptotic protein. CONCLUSIONS: This study suggests that the anthocyanins isolated from fruits of Vitis coignetiae Pulliat enhanced anti-cancer effects of CDDP by inhibiting Akt activity activated by CDDP.

Immunohistochemical study on cytokeratin expression on developing tongue in Korean native goats (Capra hircus) / 동물의과학연구지

Cytokeratin (CK) comprises the intermediate filament cytoskeleton of epithelial cells. Patterns of CK expression can be regarded as a specific marker for epithelial differentiation status. The aim of this study was to identify CK expression on tongues of Korean native goats ranging from 60-day-old fetuses to newborns during prenatal development using immunohistochemistry. The tongues of fetuses were removed from 2- to 4-year-old female Korean native goats by caesarean section performed under general anesthesia. Immunohistochemistry was performed to assess CK expression patterns on developing goat tongues using serial paraffin-embedded sections. Light zones signifying CK immunoreactivity in dorsal lingual epithelia were weakly positive in 60-day-old fetuses. In 90-day-old fetuses, deep areas in dorsal lingual epithelia were strongly positive for CK expression and superficial areas were moderately positive. In 120-day-old fetuses, light zones of lingual epithelia in the vallate papilla were strongly positive for CK expression, whereas ducts of von Ebner's glands were moderately positive. In neonates, taste buds were positive for CK expression, whereas non-taste epithelial cells and von Ebner's glands were negative. These findings indicate that goat tongues have different patterns of CK expression during development and provide a morphological basis for studies on the biological mechanism of epithelial differentiation.

Scanning electron microscopic observation of lingual papillae in a Bengal tiger (Panthera tigris tigris) / 동물의과학연구지

The morphology of the lingual papillae in a female Bengal tiger (Panthera tigris tigris) was examined by scanning electron microscopy (SEM). The tongue was 22.3 cm in length and 7.1 cm in width. Numerous filiform papillae were distributed over the entire dorsal surface of the tongue. SEM examination of the tongue revealed two types of mechanical papillae, i.e. filiform and conical papilla, and two types of gustatory papillae, i.e. fungiform and vallate papilla, on the dorsal surface of the tongue. Each filiform papilla consisted of one primary papilla and several secondary papillae. The filiform papillae on the anterior part of the tongue were divided into one primary and 6~14 secondary papillae. Unlike other mammalians, however, secondary papillae in the mid-part of the tongue showed pineal-like papillae. In the posterior part of the tongue, secondary papillae were rare or absent. Fungiform papillae were surrounded by filiform papillae and densely distributed on the lingual surface. There were two vallate papillae on the borderline between the lingual body and root of the tongue. A vallate papilla contained two secondary papillae inside the grooves. Conical papillae were located in the area of the vallate papillae and covered the posterior part of the tongue root. No foliate papillae were seen on both margins of the posterior part of the tongue. Our results indicate that the structure on the lingual papillae of the Bengal tiger is somewhat different from that of other mammals.

Anti-carcinogenic actions of glycoprotein conjugated with isoflavones from submerged-liquid culture of Agaricus blazei mycelia through reciprocal expression of Bcl-2 and Bax proteins / 동물의과학연구지

Glycoproteins isolated from fruit bodies and mycelial cultures of mushrooms exhibit anti-carcinogenic actions in human cancer cells and animal tumor cells by induction of apoptosis. Here, we report that isoflavone-conjugated glycoproteins (designate Gluvone), exhibit strong anti-carcinogenic effects on human breast cancer MCF-7 cells by induction of apoptosis. Gluvone with 9.4 kDa of molecular weight was isolated from submerged-liquid culture of Agaricus blazei mycelia (ABM) in soy flake-containing liquid medium. MCF-7 cells were incubated with various amounts of Gluvone (0~250 microM) for a period of 6 days. Gluvone exhibited anti-proliferative actions in a dose-dependent manner and 62% growth inhibition at 200 microM for 4 days relative to control. Hoechst 33258 staining analysis revealed that Gluvone induced formation of apoptotic bodies. Gluvone was associated with down-regulation of anti-apoptotic Bcl-2 protein expression as well as up-regulation of pro-apoptotic Bax protein expression. Gluvone treatment induced proteolytic activation of caspase-9 and caspase-3 through cytochrome c release from mitochondria to cytosol as well as concomitant degradation of poly (ADP-ribose) polymerase (PARP). In addition, Gluvone induced activation of caspase-8. Taken all together, these results indicate that the anti-proliferative effect of Gluvone is associated with induction of apoptotic cell death through the mitochondrial dysfunction pathway mediated by enhancement of Bax protein expression and suppression of Bcl-2 protein expression.

Differentially expressed proteins in the liver of Gulo-/- mice following treatments with Helicobacter pylori and diethylnitrosamine / 동물의과학연구지

Vitamin C (ascorbic acid) is an essential nutrient of most living tissues. We established a strain of Gulo-/- mice with known deficiency, in which vitamin C intake can be controlled by diet, like humans, and investigated the differentially expressed proteins following treatments with Helicobacter pylori and diethylnitrosamine (DENA) in the liver of Gulo-/- mice using a proteomic approach. Expression of p53, 14-3-3epsilon and 14-3-3delta in Gulo-/- mice liver tissue was analyzed by immunohistochemistry. 2-DE maps constructed from Gulo-/- mice liver and differentially expressed proteins in liver tissue were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/MS). In Gulo-/- mice after H. Pylori infection, followed by treatment with DENA, no differences in p53, 14-3-3epsilon and 14-3-3delta were observed by immunohistochemistry. Proteome analyses using MALDI-TOF/MS resulted in successful identification of 12 proteins (nine proteins were up-regulated and three were down-regulated). Specifically, peroxiredoxin-6 and Alpha-1-antitrypsin 1-4 were up-regulated in liver after H. Pylori infection followed by treatment with DENA. These results indicated that oral supplementation with vitamin C led to rescue of Gulo-/- mice from vitamin deficiency, and protected the liver from H.pylori infection and/or DENA effect, and vitamin C also protected the liver against oxidative stress.

A light microscopic study on tongue development in Korean native goats (Capra hircus) / 동물의과학연구지

The purpose of this study was to identify the composition and organization of lingual tissues underlying the histo-structural change of developing tongue in Korean native goats by light microscopy (LM). Tongues of the fetuses on days 60, 90, 120 and neonate were examined for the morphological development. In the 60-day-old fetuses, the tongue tissues were differentiated into epithelium, lamina propria and muscle layer. Primordia of filiform, conical, lentiform, fungiform and vallate papillae appeared and rudiments of taste bud were observed in the epithelia of the primordia of the gustatory papillae. The dorsal surface of the lingual epithelia showed a weak PAS positive reaction. Collagenous fibers and small blood vessels were shown in the connective tissues. In the 90-day-old fetuses, Von Ebner's glands were moderately PAS positive while the muscle fibers and connective tissue were strongly positive for PAS. The collagenous fibers increased and came to have a more complex arrangement in the tongue. The muscle fibers were spread out at various directions and developed in striated muscle bundles. In the 120-day-old fetuses, taste buds were observed in the epithelia of the gustatory papillae, and several well-developed tissues visible such as blood vessels, collagenous fibers, muscle fiber bundles and Von Ebner's glands. In the neonates, many taste buds were found in a transverse section of the vallate papilla. The muscle layers, Von Ebner's glands, collagenous fibers and blood vessels were more developed than those of the 120-day old fetuses. These findings indicate that goat tongues have a variety of different shapes during prenatal development.

Anti-proliferative efficacy comparison of conjugated linoleic acid on human cancer cell lines / 동물의과학연구지

The anti-proliferative efficacy of t,t-conjugated linoleic acid (t,t-CLA), c9,t11-CLA, and t10,c12-CLA was compared in several human cancer cell lines. Gastric NCI-N87, liver Hep3B, pancreas Capan-2, and lung NCI-H522 cancer cells were incubated with 50 microM CLA isomers over a period of 6 days. The t,t-CLA inhibited the growth of all cancer cell lines to different extents, but c9,t11-CLA and t10,c12-CLA inhibited or stimulated the growth of the cancer cell lines. NCI-N87 cells were the most sensitive to growth inhibition and apoptosis from all CLA isomers tested. In NCI-N87 cells, CLA isomers reduced the release of arachidonic acid (AA) via the inhibition of cytosolic phospholipase A2 (cPLA2) activity, consequently reducing the production of PGE2 through the inhibition of cyclooxygenase-2 (COX-2). The efficacies of CLA isomers were in the following order (from most to least effective): t,t-CLA, t10,c12-CLA and c9,t11-CLA. Overall, these results imply that the anti-proliferative efficacy of t,t-CLA on cancer cells, especially NCI-N87 cells, was greater than other CLA isomers due to its induction of apoptosis through the inhibition of cPLA2 and COX-2 activities.

Two-dimensional gel electrophoresis and immunoblot analysis of Neospora caninum tachyzoites

Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.

Reference Map of Soluble Proteins from Salmonella enterica Serovar Enteritidis by Two-Dimensional Electrophoresis

Protein identification by peptide mass fingerprinting using matrix-assisted laser desorption ionization time of fight (MALDI-TOF) mass spectrometry (MS) can analyze unambiguously identity of the spots from a 2-dimensional electrophoresis (2-DE) gel. This study developed a technique for 2-DE of Salmonella enterica serovar Enteritidis (S. enteritidis) by improving the dissolution conditions by 2-DE using a pH 4 - 7 immobilized pH gradient (IPG) strip. This report examines the protein components from the patterns of the S. enteritidis protein. The most abundant protein displayed a great number of clusters within the pH 4.5 - 7 range with a molecular mass ranging from 35-80 kDa. Some of these spots were identified as metabolic related enzymes. The protein fraction was also analyzed using an immobilized pH gradient strip. Different proteins were identified on the spot according to the elongation factors. In addition, this study showed that the 2-DE analysis of S. enteritidis provides useful information regarding the S. enteritidis proteome, and this approach might provide a strategy for identifying bacterial proteins using a proteome technology.

Spontaneous Bone Regeneration in Surgically Induced Bone Defects in Young Rabbits / 대한정형외과학회잡지

PURPOSE: To evaluate and compare the spontaneous regeneration repair process in femoral bone defects in 6-week-old rabbits and to compare the healing time periods between different rabbit groups. MATERIALS AND METHODS: Bone defects were created at the femur mid-shaft and an external fixator was applied in 50 rabbits. The periosteum was resected in 10 rabbits (defect size: 15%, 25%) and left untouched in the remaining rabbits. Forty rabbits were divided into four groups according to the percentage of bone defects (15%, 20%, 25%, 30%). Radiographs were taken weekly to evaluate the bone regeneration and union. The bone union time was measured between the osteotomy and the cortico-medullary differentiation by examining radiographs. The healing index was defined as the union time (week) per amount (cm) of bone defect. Eight rabbits, 2 from each groups with the bone defects, were investigated by histopathologic examination. RESULTS: The mean union time was approximately 7.0 to 7.3 weeks. The healing index in groups that had a large percentage of bone defects was less than in groups that had a small percentage of bone defects. The periosteum-resected group did not show bone regeneration. Histopathologic examinations showed intramembranous and atypical endochondral ossifications along the periosteum and typical endochondral ossification at the center of the bone defects. CONCLUSION: Spontaneous bone regeneration may be used in children to fill the bone defect instead of performing an internal bone transport. Spontaneous bone regeneration is useful in cases of mid-shaft bone defects or when the remaining bone fragments are large enough for an external fixation application.

The thrombolytic effect of lumbrokinase is not as potent as urokinase in a rabbit cerebral embolism model

The purpose of the present study is to determine whether lumbrokinase has an in vivo thrombolytic effect in a rabbit cerebral embolism model. In our previous studies, we found that lumbrokinase, an extract from Korean earth worms, has a strong in vitro fibrinolytic effect without the presence of plasminogen and significant in vivo thrombolytic effects of lumbrokinase in a rat human-clot-induced cerebral embolism model. We established the cerebral embolism model in rabbits by injecting a piece of human clot into the internal carotid artery via the external carotid artery and confirmed the occlusion with angiography. Twenty one rabbits were divided into three groups and 5cc of saline, urokinase of 50,000 u/ml, and equipotent LK were injected intraarterially for 30 minutes into each group of 7 animals. Ten minutes after the end of infusion, an angiogram was performed to confirm the recanalization. Clot lysis occurred in one, six, and one animals in the saline, urokinase and lumbrokinase treated groups respectively. With regard to its in vitro effect, lumbrokinase is not as potent in vivo. Further investigation should be performed to determine the cause of its weakened in vivo effect and to develop a method to potentiate it.

Primary culture of osteoblast / 대한정형외과학회잡지

No abstract available.

Effects of 2-Deoxy-D-Glucose on Metabolic Status, Proliferative Capacity and Growth Rate of FSall Tumor: Observations made by In Vivo 31P-Nuclear Magnetic Resonance Spectroscopy and Flow Cytometry / 대한치료방사선과학회지

The effect of 2-deoxy-d-glucose (2-DDG) on C3H mouse fibrosarcoma (FSall) was studied. Metabolic status, especially for energy metabolism, was studied using in vivo 31P-MRS, proliferative capacity was observed on flow cytometry (FC) and growth rate was measured after transplantation of 106 viable tumor cells in the dorsum of foot of C3Hf/Sed mice. One gram of 2-DDG per kg of body weight was injected intraperitoneally on 12th day of implantation. Average tumor size on 12th day of implantation was 250mm3. Growth rate of FSall tumor was measured by tumor doubling time between tumor age 5-12 days was 0.84 days with slope 0.828 and tumor doubling time between tumor age 13-28 days was 3.2 days with slope 0.218 in control group. After 2-DDG injection, tumor doubling time was elongated to 5.1 days with slope 0.136. The effect of 2-DDG studied in vivo 31P-MRS suggested that the increase of phosphomonoester (PME) and inorganic phosphate (Pi) by increasing size of tumor, slowed down after 2-DDG injection. Flow cytometry showed significantly increased S-phase and G2+M phase fraction suggesting increased proliferative capacity of tumor cells in the presence of 2-DDG. Authors observed an interesting effect 2-DDG on FSall tumor and attempt to utilize as an adjunct for radiotherapy.