Acer mono Extract Inhibits Invasive Activities and G1/S Transition of HT1080 Fibrosarcoma Cells / 전남의대학술지

Acer mono is known to contain bioactive substances that exhibit beneficial effects in osteoporosis, gastric ulcers, hepatic damage, and pathologic angiogenesis. The current study aimed to investigate the effects of Acer mono extract on the invasive activities and cell-cycle progression of human fibrosarcoma cells. Cytotoxicity of Acer mono extract was assessed by MTT assay, in-vitro invasiveness of HT1080 fibrosarcoma cells was measured using matrigel assay, expression of invasion- and cell-cycle-related proteins was analyzed by western blot analysis, and that of E2F target genes was quantified using qRT-PCR. Acer mono extract did not show distinct cytotoxicity in the experimental concentrations used. Invasiveness of HT1080 fibrosarcoma cells and expression of cyclin D1 and CDK4 in them were significantly reduced in a dose-dependent manner after treatment with Acer mono extract. Acer mono extract showed inhibitory effects on the G1/S transition during cell-cycle progression; the active phosphorylated Rb protein level was decreased, and expression of E2F target genes was downregulated by the Acer mono extract. Our data collectively demonstrated that Acer mono extract exerts inhibitory effects on the invasiveness and cell-cycle progression of HT1080 human fibrosarcoma cells.

Acer mono Extract Inhibits Invasive Activities and G1/S Transition of HT1080 Fibrosarcoma Cells / 전남의대학술지

Acer mono is known to contain bioactive substances that exhibit beneficial effects in osteoporosis, gastric ulcers, hepatic damage, and pathologic angiogenesis. The current study aimed to investigate the effects of Acer mono extract on the invasive activities and cell-cycle progression of human fibrosarcoma cells. Cytotoxicity of Acer mono extract was assessed by MTT assay, in-vitro invasiveness of HT1080 fibrosarcoma cells was measured using matrigel assay, expression of invasion- and cell-cycle-related proteins was analyzed by western blot analysis, and that of E2F target genes was quantified using qRT-PCR. Acer mono extract did not show distinct cytotoxicity in the experimental concentrations used. Invasiveness of HT1080 fibrosarcoma cells and expression of cyclin D1 and CDK4 in them were significantly reduced in a dose-dependent manner after treatment with Acer mono extract. Acer mono extract showed inhibitory effects on the G1/S transition during cell-cycle progression; the active phosphorylated Rb protein level was decreased, and expression of E2F target genes was downregulated by the Acer mono extract. Our data collectively demonstrated that Acer mono extract exerts inhibitory effects on the invasiveness and cell-cycle progression of HT1080 human fibrosarcoma cells.

Decursin induces apoptosis in glioblastoma cells, but not in glial cells via a mitochondria-related caspase pathway

Decursin is a major biological active component of Angelica gigas Nakai and is known to induce apoptosis of metastatic prostatic cancer cells. Recently, other reports have been commissioned to examine the anticancer activities of this plant. In this study, we evaluated the inhibitory activity and related mechanism of action of decursin against glioblastoma cell line. Decursin demonstrated cytotoxic effects on U87 and C6 glioma cells in a dose-dependent manner but not in primary glial cells. Additionally, decursin increased apoptotic bodies and phosphorylated JNK and p38 in U87 cells. Decursin also down-regulated Bcl-2 as well as cell cycle dependent proteins, CDK-4 and cyclin D1. Furthermore, decursin-induced apoptosis was dependent on the caspase activation in U87 cells. Taken together, our data provide the evidence that decursin induces apoptosis in glioblastoma cells, making it a potential candidate as a chemotherapeutic drug against brain tumor.

MicroRNA-29 Family Suppresses the Invasion of HT1080 Human Fibrosarcoma Cells by Regulating Matrix Metalloproteinase 2 Expression / 전남의대학술지

Matrix metalloproteinase 2 (MMP2) is a potent protumorigenic, proangiogenic, and prometastatic enzyme that is overexpressed in metastatic cancer. Although there have been various studies on the MMP2 gene, further studies of regulatory factors are required to achieve inhibition of MMP2 enzyme activities. MicroRNAs (miRNAs) play key roles in tumor metastasis. However, the specific functions of miRNAs in metastasis are unclear. In this study, we assessed the function of the microRNA-29 family (miR-29s) in HT1080 human fibrosarcoma cells and examined the regulatory mechanisms of these miRNAs on MMP2 activation. Using miRanda, TargetScan, and PicTar databases, miR-29s were identified as candidate miRNAs targeting MMP2. Gain-of-function studies showed that overexpression of miR-29s could inhibit the invasion of HT1080 cells, suggesting their tumor-suppressive roles in HT1080 cells. In addition, dual luciferase reporter assays indicated that miR-29s could inhibit the expression of the luciferase gene containing the 3'-untranslated region of MMP2 mRNA. Ectopic expression of miR-29s down-regulated the expression of MMP2. Moreover, ectopic expression of miR-29s reduced MMP2 enzyme activity. These results suggested that miR-29s could decrease the invasiveness of HT1080 cells by modulating MMP2 signaling. Taken together, our results demonstrated that miR-29s may serve as therapeutic targets to control tumor metastasis.

MicroRNA-29 Family Suppresses the Invasion of HT1080 Human Fibrosarcoma Cells by Regulating Matrix Metalloproteinase 2 Expression / 전남의대학술지

Matrix metalloproteinase 2 (MMP2) is a potent protumorigenic, proangiogenic, and prometastatic enzyme that is overexpressed in metastatic cancer. Although there have been various studies on the MMP2 gene, further studies of regulatory factors are required to achieve inhibition of MMP2 enzyme activities. MicroRNAs (miRNAs) play key roles in tumor metastasis. However, the specific functions of miRNAs in metastasis are unclear. In this study, we assessed the function of the microRNA-29 family (miR-29s) in HT1080 human fibrosarcoma cells and examined the regulatory mechanisms of these miRNAs on MMP2 activation. Using miRanda, TargetScan, and PicTar databases, miR-29s were identified as candidate miRNAs targeting MMP2. Gain-of-function studies showed that overexpression of miR-29s could inhibit the invasion of HT1080 cells, suggesting their tumor-suppressive roles in HT1080 cells. In addition, dual luciferase reporter assays indicated that miR-29s could inhibit the expression of the luciferase gene containing the 3'-untranslated region of MMP2 mRNA. Ectopic expression of miR-29s down-regulated the expression of MMP2. Moreover, ectopic expression of miR-29s reduced MMP2 enzyme activity. These results suggested that miR-29s could decrease the invasiveness of HT1080 cells by modulating MMP2 signaling. Taken together, our results demonstrated that miR-29s may serve as therapeutic targets to control tumor metastasis.

Phenolic Constituents from the Flowers of Hamamelis japonica Sieb. et Zucc

Hamamelis japonica (Hamamelidaceae), widely known as Japanese witch hazel, is a deciduous flowering shrub that produces compact clumps of yellow or orange-red flowers with long and thin petals. As a part of our ongoing search for phenolic constituents from this plant, eleven phenolic constituents including six flavonol glycosides, a chalcone glycoside, two coumaroyl flavonol glycosides and two galloylated compounds were isolated from the flowers. Their structures were elucidated as methyl gallate (1), myricitrin (2), hyperoside (3), isoquercitrin (4), quercitrin (5), spiraeoside (6), kaempferol 4'-O-beta-glucopyranoside (7), chalcononaringenin 2'-O-beta-glucopyranoside (8), trans-tiliroside (9), cis-tiliroside (10), and pentagalloyl-O-beta-D-glucose (11), respectively. These structures of the compounds were identified on the basis of spectroscopic studies including the on-line LCNMR- MS and conventional NMR techniques. Particularly, directly coupled LC-NMR-MS afforded sufficient structural information rapidly to identify three flavonol glycosides (2 - 4) with the same molecular weight in an extract of Hamamelis japonica flowers without laborious fractionation and purification step. Cytotoxic effects of all the isolated phenolic compounds were evaluated on HCT116 human colon cancer cells, and pentagalloyl-O-beta-D-glucose (11) was found to be significantly potent in inhibiting cancer cell growth.

Adhesion of Weissella cibaria to the Epithelial Cells and Factors Affecting its Adhesion

We evaluated the ability of lactic acid bacteria, Weissella cibaria, isolated from the oral cavity to adhere to epithelial cells. W. cibaria efficiently adhered to KB cells and HeLa cells. In addition, W. cibaria efficiently adhered to Fusobacterium nucleatum. But the adhesiveness of W. cibaria disappeared upon exposure to LiCl or pronase, suggesting that the S-layer proteins of W. cibaria mediated the adhesiveness. The molecular mass of the S-layer proteins extracted from W. cibaria was approximately 50 kDa. When W. cibaria strains were washed with 0.45% saline, the bacteria were efficiently adhered to the epithelial cells. In conclusion, W. cibaria has the ability to adhere to epithelial cells through the S-layer proteins.

The effect of YB-1 antisense oligonucleotides on tumor cell growth / 대한내과학회지

BACKGROUND: Human YB-1 is a transcription factor that binds to the inverted CCAAT box in the promoter region of a variety of genes such as PCNA, DNA polymerase and MDR. In this study we evaluated the effect of YB-1 antisense oligonucleotides on tumor cell growth. METHODS: Chang liver, HepG2 and CT-26 cells were cultured as immortalized cell lines. The MTT (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide) assay, Northern blot and flow cytometric analyses were used to determine cell growth, gene expression and cell cycle changes. In an animal model, CT-26 cells were injected into Balb/c mice to induce tumor; YB-1 antisense oligonucleotides were injected into the tail vein or tumor tissue of the mice; change of tumor size was then measured. RESULTS: Phosphorothioated YB-1 antisense oligonucleotides suppressed the proliferation of the immortalized liver cells (Chang liver cells) and a variety of cancer cells (HepG2 and CT-26 cells); however, it did not inhibit normal cell growth. The DOTAP/antisense oligonucleotide mixture showed stronger effects on cell proliferation than did the antisense oligonucleotide alone. The YB-1 antisense oligonucleotide decreased specific expression of the YB-1 mRNA in the immortalized cancer cell lines. Flow cytometric analysis revealed that the inhibition of cell proliferation might have been due to a decrease in the S phase of the cell cycle. We found that in an animal tumor model, the administration of the YB-1 antisense oligonucleotide, in the vein or tumor tissues, decreased the tumor size significantly. CONCLUSIONS: These results suggest that the YB-1 antisense oligonucleotide may inhibit growth of a variety of cancer cells.

Antisense Deoxyoligonucleotides Inhibit Activities of Matrix Metalloproteinase-2 in Human Fibrosarcoma HT1080 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: MMP-2, 72 kDa-type IV collagenase, plays a major role in the migration and growth of tumor cells, a process that requires the disintegration of basement membrane. Activation of MMP-2 is correlated with the invasiveness of various tumors. The aim of this study was to determine the sequence-specific phosphorothioated oligodeoxynucleotides (ODNs) inhibiting the translation of MMP-2 mRNA and the subsequent invasiveness of tumor cells. MATERIALS AND METHODS: Eight types of antisense ODNs were designed and each (8micro gram/ml) were transfected into HT1080 cells. The effects of these antisense ODNs on MMP expression were examined by gelatin zymography, Western blot, Northern blot and matrigel assay. RESULTS: Antisense-5 (+904~923), antisense-6 (+1274~+1293) and antisense-7 (+1646~+1665) reduced the MMP-2 activity of the culture supernatant in HT1080 fibrosarcoma cells. Treatment with antisense-6 showed inhibition of MMP-2 mRNA and protein, and in vitro invasion in a dose-dependent manner. CONCLUSION: Antisense-6 might be one of the therapeutic candidates for tumor invasion and metastasis.

No Association of Factor XIII Val34Leu Polymorphism with Primary Intracerebral Hemorrhage and Healthy Controls in Korean Population

The polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recognized as a risk factor for primary intracerebral hemorrhage (PICH). In addition, FXIII Val34Leu has a significant ethnic heterogeneity. FXIII Val34Leu was detected in 41.7-54.8% of the Westerners, but in 2.5% of the Asians. We aimed to evaluate the prevalence of FXIII Val34Leu in patients with PICH and in healthy controls among Koreans. We recruited 58 in-patients with PICH, defined by brain computed tomography or magnetic resonance imaging, and 48 controls matched for age, sex, and risk factors for cerebrovascular diseases. Genomic DNA was extracted from blood. A 183-bp fragment of exon 2/intron B of the factor XIII Asubunit gene was amplified by polymerase chain reaction (PCR). The factor XIII genotype was determined through a single-stranded conformational polymorphism. Fifty-eight patients and 48 controls showed the same band patterns on SSCP. In addition, we directly sequenced six random-selected DNA segments using DNA auto-sequencer. In conclusion, the results of this study suggest that FXIII Val34Leu be absent or rare both in patients with PICH and in healthy controls among Koreans.

SB203580, a P38 MAPK Inhibitor, Blocks in vitro Invasion by Human Gastric SNU-638 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The role of P38 mitogen-activated protein kinase (MAPK) in gastric cancer invasion has not yet been determined. In this study, we examined the effects of SB203580, a specific P38 MAPK inhibitor, on the in vitro invasion of gastric cancer and upon the molecules involved in this process. MATERIALS AND METHODS: Human gastric cancer SNU-638 cells were maintained in RPMI 1640 supplemented with 10% FBS. BIOCOAT matrigel invasion chambers were used to examine in vitro invasiveness, zymography for gelatinase activity, CAT assay for uPA promoter activity and Western and Northern blotting to determine protein and mRNA levels, respectively. RESULTS: Treatment of SNU-638 cells with SB203580, a specific P38 MAPK inhibitor, reduced in vitro invasiveness, dose-dependently. SB203580 treatment was found to decrease both mRNA expression and uPA promoter activity in gastric SNU-638 cells. In vitro invasion of SNU-638 cells was partially abrogated by uPA-neutralizing antibodies. The activities of MMPs were not significantly altered by SB203580. CONCLUSION: Our results suggest that P38 MAPK is a potential therapeutic target for inhibiting uPA-dependent gastric tumor invasiveness and metastasis.

Downregulation of TGF-beta1 Expression in Penile Corpus Cavernosum by Antisense TGF-beta1 Oligonucleotides in Diabetic Rat / 대한남성과학회지

PURPOSE: We investigated the effect of antisense TGF-beta1 oligonucleotides on the expression of TGF-beta1 in the penile corpus cavernosum of streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Forty Sprague-Dawley male rats (200-210 g) were divided into two groups: control (n=10) and experimental group (n=30). The experimental group was received intravenous injection of streptozotocin (50 mg/kg). After 2 weeks, development of diabetes was verified by measuring body weight and blood sugar levels. The experimental group was divided further into 3 groups: vehicle (liposome only), sense oligomer group, and antisense oligomer (5'-CCGAGGGCGGCATGGGGGA-3') group. Penile tissues were used to perform Western analysis and immunohistochemistry for the TGF-beta1 expression. RESULTS: The mean glucose concentrations were 86 9 mg/dl in the control group and 485 119 mg/dl in the experimental group. Downregulation of TGF-beta1 protein expression was noted in the antisense oligomer group compared to sense oligomer group. In the immunohistochemistry, control group showed weak immunoreactivity whereas vehicle or sense oligomer group showed strong immunoreactivity both after 1 and 5 days of treatment. Antisense oligomer treated diabetic group showed weak immunoreactivity similar to control group after 1 day of treatment, even though they showed similar immunoreactivity as vehicle or sense oligomer group after 5 days of treatment. CONCLUSIONS: Antisense TGF-beta1 oligonucleotides suppressed the expression of TGF-beta1 in the corpus cavernosum of diabetic rats. It implies that penile corpus cavernosal fibrosis may be prevented by the application of antisense TGF-beta1 oligonucleotides.

The cytotoxic effect of Vibrio vulnificus hemolysin on the mouse peritoneal macrophages

V. vulnificus is an estuarine bacterium which causes septicemia and shock in susceptible patients. The organism produces a hemolytic cytolysin (VvH), which has a membrane damaging effect on erythrocytes. To clarify the mechanisms by which VvH might contribute to virulence, we examined its effect on macrophages. When mouse peritoneal macrophages were harvested and co-cultured with hemolysin-positive V. vulnificus strains (100 bacteria/ cell), about 60% of the macrophages were killed; macrophages were not killed when co-cultured V. vulnificus strain CVD 707, a VvH-negative deletion mutant. Exposure of macrophages to filtered culture supernatants (2.5 HU/ml) and purified VvH (3 HU/ml) resulted in an increase in dead cells (80 and 90%, respectively), as determined by the trypan blue dye exclusion method and LDH release from macrophages was also increased (70 and 65.5%, respectively). The cytotoxic effect of VvH on macrophages was both the dose- and time-dependent. The VvH caused damage to the macrophage membrane and was blocked significantly by preincubation with cholesterol (p<0.01). Fetal bovine serum showed remarkable inhibition of VvH synthesis by V. vulnificus and inhibited VvH activity in culture supernatant. Cell viability was increased by 35% (p<0.01) and LDH release decreased by 28% (P<0.01) when macrophages were incubated with V. vulnificus (100 bacteria/ cell) in DMEM-10% FBS for 2 hr. Bacterial clearance activity of mice against V. vulnificus CVD 707 was decreased by pretreatment with 10 HU of VvH. This result suggests that the VvH can impair the membrane of macrophages and may play a role in the pathogenesis of V. vulnificus septicemia.

The Role of Vibrio vulnificus Capsular Polysaccharide as a Virulence Factor

To evaluate the role of capsular polysaccharide (CPS) as a virulence factor, the interaction of V. vulnificus with mouse peritoneal macrophages and serum, which are involved in the clearance of bacteria from blood and other tissues, were examined. In this study, MO6-24/0 (wild strain; hemolysin- and capsule-positive), MO6-24/I' (acapsular spontaneous mutant), CVD 752 (acapsular transposon mutant), and CVD 707 (hemolysin-negative and capsule-positive mutant) were used. The strain with CPS (MO6-24/0 and CVD 707) were more resistant to phagocytosis by mouse peritoneal macrophages compared with acapsular strains (MO6-24/T and CVD 752), and the resistance to phagocytosis was not changed by serum opsonin in the capsular strains. Acapsular strains were more susceptible to serum bactericidal activity than the capsular strains through the classical complement pathway. MO6-24/0 strain were detected in blood, spleen, liver and lung at 4 hours after intraperitoneally infection, whereas CVD 752 were not detected. All tested strains could induced the transcription of inflammatory cytokine gene such as IL-1, IL-6, IL-10 and TNF-u, and their inductions were not decreased by cytochalasin B treatment. This results demonstrate that CPS of V. vulnificus plays an important role in V. vulnificus infection through interfering nonspecific host defense system such as blood clearance and phagocytosis.

Expression of Rous Sarcoma Virus Enhancer Factor Gene in Human Hepatocellular Carcinoma / 대한암학회지

PURPOSE: We have previously cloned three enhancer factor genes encoding proteins that bind to long terminal repeats (LTRs) of Rous sarcoma virus. Among these genes, RSV- EF-I gene is expressed in rat hepatoma tissues and several proliferating cell lines but not in normal rat liver tissues. We have isolated the human homologue of RSV-EF-I gene and examined its expression in human hepatocellular carcinoma tissues. MATERIALS AND METHODS: We have screened the human genomic library and cDNA library of Hep G2 cell line derived from human hepatocellular carcinoma to isolate the human homologue of RSV-EF-I gene. RESULTS: We have isolated one cDNA clone containing about 1.5 kb insert and sequenced. Sequence analysis reveals that this human homologue of RSV-EF-I gene has a high similarities to human YB-1 mRNA, human DNA-binding protein B (dbpB) gene and other Y-box protein genes. It is expressed in human hepatocellular carcinoma but very slightly in normal human liver tissues in Northern blot analysis. CONCLUSION: Our data suggest that the human homologue of RSV-EF-I gene presumably belongs to Y-box protein family genes and plays a role in the transformation of the human hepatoma cells.

Effect of BCG Immunotherapy on the Cytokine Production and Antitumor Activity against MBT - 2 Mouse Bladder Tumor / 대한면역학회지

Antitumor effects of Bacillus Calmette-Guerin (BCG) against superficial urinary bladder cancer is known to be strong when BCG is directly infused into the bladder, but its immunological mechanisms are poorly understood. These experiments were performed to elucidate the effects of intralesional or systemic administration of BCG on the antitumor activity in murine transitional cell carcinoma (MBT-2) model and on the production of cytokines by the activated splenocytes or macrophages. ...continue...

A Basic Study for the Development of Effective Preventive Measure against Vibrio vulnificus Septicemia: Bactericidal Mechanism of Osmotic Shock

The establishment of effective preventive measure against V. vulnificus septicemia is urgently required. It was reported that V. vulnificus osmotically shocked by distilled water lost viability rapidly but regain viability after appropriate resuscitation (RS) procedure. But V. vulnificus was reported to be completely killed when osmotically shocked in the presence of ethylenediaminetetraacetic acid (EDTA). This study was carried out to uncover the bactericidal mechanism of osmotic shock and the mechanism of potentiation of osmotic shock by EDTA. When about 2.0 x 10(7) CFU/ml of V. vulnificus were inoculated in distilled water, the number of viable cells abruptly decreased to 2.5 x 10(3) CFU/ml in 1 min. and slowly thereafter to 1.0 x 10(1) CFU/ml in 5 min. After RS, there was a increase in the number of surviving bacteria by 10(3) to 10(4) fold. When the bacteria were inoculated in 1 mM EDTA solution, osmotic concentration of which is about 30 mEq./1, no colony could be observed even in 1 minute. The turbidity decreased abruptly as soon as the bacteria were inoculated in distilled water or in the 1 mM EDTA solution, but rather slowly thereafter. When V. vulnificus whose cellular constituents were labeled with 3H-L-amino acid mixture was inoculated in distilled water or in the 1 mM EDTA solution, about 35% of the whole cell radioactivity was released in the 1 mM EDTA solution in 30 sec while about 6% of the whole cell radioactivity was released to the supernatant in distilled water in 5 minutes. The cell surface hydrophilicity decreased significantly by osmotic shock. The decrease was more significant when the bacteria were inoculated in 1 mM EDTA solution than in distilled water. Bacterial cell volume analysis with a flow cytometer revealed that the osmotic shock balloons V. vulnificus. The increase in the cell volume was more prominent in 1 mM EDTA solution. When the cytoplasmic RNA content in the osmotically shocked bacteria was measured by a flow cytometer, the frequency of the cells with decreased RNA content increased after osmotic shock, and the degree of increase was more prominent in 1 mM EDTA solution. Number of non-staining cells also increased after osmotic shock, and the degree of increase was more prominent in the 1 mM EDTA solution. To see whether the susceptibility to osmotic shock is unique to V. vulnificus, bactericidal kinetic curves of other Vibrio species were observed after inoculating in distilled water. V. cholerae and V. mimicus were more resistant to the osmotic shock than V. vulnificus. V. parahaemolyticus, V. furnissii, V. fluvialis, V. damsela, and V. harveyi showed similar susceptibility to osmotic shock as V. vulnificus. V. alginolyticus and V. hollisae were more susceptible than V. vulnificus. The concentration of NaCl in culture media influenced the susceptibility of V. vulnificus to osmotic shock. V. vulnificus grown in 0.5% NaCl was more resistant to the osmotic shock than that grown in 2.5% NaCl. Taken together, it was concluded that osmotic shock causes leakage of the cytoplasmic contents(ribosomes etc.). And EDTA was supposed to quantitatively potentiate the bactericidal effect of the osmotic shock. Susceptibility to osmotic shock was influenced by the osmolarity of culture media and appeared to be a phenotypic property of V. vulnificus.

Effect of 1,25 dihydroxy-vitamin D3 and physiological inducers on the expression of interleukin-1?gene in the human myeloid leukemic cells

No abstract available.

In vitro effect of ciprofloxacin on vibrio vulnificus

No abstract available.

Expression of cell surface antigens and oncogene of leukemic U-937 cells by differentiation inducers / 대한암학회지

No abstract available.