- Inactivation of TPEF Gene by Aberrant Methylation in Hepatocellular Carcinoma.
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Woon Bok Chung, Soon Young Kim, So Young Chun, Ku Seong Kang, Hae Ahm Lee, Joung Ok Kim, Ji Young Park, Yoon Kyung Sohn, Jung Wan Kim
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Korean J Pathol. 2008;42(1):9-15.
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 Abstract
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- BACKGROUND
Abnormalities of genomic methylation patterns have been shown to play a role in the development of carcinoma, and the silencing of tumor suppressor genes is related to local de novo methylation.
METHODS
Using methylation specific arbitrarily primed-Polymerase Chain Reaction (Ms AP-PCR), we identified a 322 bp sequence that contained a 5' un-translated and exon1 regions of the TPEF gene. To evaluate the inactivation of the TPEF gene through hypermethylation in hepatocellular carcinoma (HCC), we investigated the correlation between methylation patterns and TPEF expression in tumor tissues of human HCC and cell lines via a Combined Bisulfite Restriction Assay (CoBRA) and RT-PCR.
RESULTS
A dense methylation pattern of the TPEF was detected in most cell lines, as well as in 10 of the 14 (71.4%) HCC tissues. In addition, loss of heterozygosity (LOH) from the TPEF gene was observed in 5 of the 14 (36%) HCC tissues. Furthermore, RT-PCR analysis revealed TPEF expression in 5 of 8 (62.5%) cell lines. Finally, treatment with a demethylating agent, 5-Aza- 2'-deoxycitidine (5-AzaC), increased the expression of TPEF mRNA.
CONCLUSION
These results indicate that inactivation of the TPEF gene through hypermethylation may be a mechanism by which tumorigenesis occurs in HCC.
- Genistein Induces G2/M Cell Cycle Arrest and Apoptosis in Rat Neuroblastoma B35 Cells; Involvement of p21(waf1/cip1), Bax and Bcl-2.
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Ismail A Ismail, Ku Seong Kang, Jung Wan Kim, Yoon Kyung Sohn
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Korean J Pathol. 2006;40(5):339-347.
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Abstract
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- BACKGROUND
The effect of genistein on different types of cells has been investigated. However, its effect on the nervous system is still unclear. The aim of the present work is to explore the effect of genistein on rat neuroblastoma B35 cells.
METHODS
The effect of genistein on the proliferation of B35 cells, its cytotoxicity, the cell-cycle distribution, the ultra-structural changes and the induction of apoptosis were determined using MTT assay, LDH assay, Flow-cytometric analysis, transmission electron microscopy and Hoechst staining, respectively. Furthermore, Real-time quantitative RT-PCR and Western blotting were used to examine the transcriptional and post-translational alterations of the G2/M cell-cycle arrest marker cyclin-dependent kinase inhibitor p21(waf1/cip1) and the apoptosis-related genes after genistein treatment.
RESULTS
Genistein significantly inhibits cell survival, slightly elevates the release of lactate dehydrogenase and induced apoptosis in B35 cells. Genistein increased the number of cells at S-phase and induced cells to accumulate at the G2/M phase. These G2/M arrested cells are associated with a marked up-regulation of p21(waf1/cip1) at both the mRNA and protein levels. We observed that genistein up-regulates pro-apoptotic Bax with concurrent down-regulation of the anti-apoptotic Bcl-2 protein.
CONCLUSION
These observations suggest that the anticancer effect of genistein on B35 neuroblastoma cells is mediated through multiple cellular pathways including G2/M cell-cycle arrest and the induction of apoptosis.
- Effects of Selective Cyclooxygenase-2 Inhibitor NS-398 Pretreatment on the Rat Spinal Cord after Contusion Injury.
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Hyeon Dae Cheong, Joo Kyung Sung, In Suk Ham, Ku Seong Kang, Joung Ok Kim, Jung Wan Kim, Tae In Park, Yoon Kyung Sohn
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Korean J Pathol. 2006;40(4):255-262.
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Abstract
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- BACKGROUND
Secondary spinal cord injury (SCI) that follows an initial mechanical insult can exacerbate the overall damage, limit the restorative processes and eventually lead to an in- creased neurological deficit. We hypothesized that selective inhibition of cyclooxygenase-2 (COX-2) may decrease the delayed cell death, and so this will contribute to decreased level of the secondary injury.
METHODS
The dorsal surface of the cord at the T9 level was subjected to weight drop impact using a 10 g rod. To block COX-2 activation, a selective COX-2 inhibitor (NS-398) was administered (5 mg/kg, i.p.) 15 min prior to SCI. The COX-1, COX-2, Caspase-3 and PGE2 expressions were measured by real time quantitative RT-PCR and fluorescence immunostaining.
RESULTS
Many activated caspase-3 positive cells were observed at 6 h and they increased until 72 h after SCI. The expression of COX-2 peaked at 6 h after SCI, while the COX-1 expression was unaffected. The principal cells that showed a COX-2 expression were the neurons and microglia.
Pretreatment with NS-398 caused a significant decrease in the expression of prostaglandin E2 and activated caspase-3 positive cells after SCI.
CONCLUSION
These data suggest that COX-2 is one of the main factors related with the pathologic deficits from secondary SCI.
- Effects of Genistein and Daidzein on the Growth of Human Colon Cancer HCT-116 Cells.
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Jong Heon Shin, Ku Seong Kang, Joung Ok Kim, Ghil Suk Yoon, Tae Gyun Kwon, Jung Wan Kim, Yoon Kyung Sohn
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Korean J Pathol. 2006;40(1):46-51.
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Abstract
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- BACKGROUND
Genistein and daidzein are two major soybean isoflavones. They have received increasing attention because of their possible roles for cancer prevention. However, their mechanisms of action and molecular targets on the human colon cancer cells are not fully understood.
METHODS
Human colon cancer HCT-116 cells were treated with genistein and daidzein to investigate their effects on the cell growth and this was analyzed with MTT assay. TUNEL assay and Hoechst33342 stain were carried out to identify apotosis.
RESULTS
Daidzein was able to inhibit cell proliferation and induce apoptosis of the HCT-116 cells, but genistein didn't affect the cell growth. The ER antagonist ICI182780 didn't attenuate the antiproliferative and proapoptotic effects of daidzein: this means the effect of daidzein on the HCT-116 cells may not be dependent on the ER pathway. The other soybean isoflavone, genistein, attenuated the effects of daidzein on the HCT-116 cells and its mechanism should be elucidated.
CONCLUSIONS
These data suggest that daidzein may act as a preventive agent on human colon cancer, and its mechanism of action doesn't involve the ER-dependent pathway.
- Correlation Between Neuronal Apoptosis and Expression of Inducible Nitric Oxide Synthase after Transient Focal Cerebral Ischemia.
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Byoung Yuk Yi, Sung Kyoo Hwang, Ku Seong Kang, Hong Hua Quan, Young Mi Lee, Jung Wan Kim, Eun Kyoung Kwak, Ji Young Park, Yoon Kyung Sohn
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Korean J Pathol. 2004;38(6):364-371.
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Abstract
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- BACKGROUND
Neuronal death in acute-phase cerebral ischemic injury is caused by necrosis. However, neuronal injury after reperfusion can be associated with apoptosis.
METHODS
We used Sprague-Dawley rats whose brains were reperfused after middle cerebral artery occlusion for either 30 min or 2 h. We examined a relationship between apoptosis and the expression of inducible nitric oxide synthase (iNOS) in the brain tissue from 3 h to 14 days after reperfusion in both groups.
RESULTS
TUNEL and iNOS positivity were closely related in both groups. The 2-h ischemia group exhibited increases in the amount of TUNEL and iNOS-positive cells for up to 3 days after reperfusion, at which the TUNEL and iNOS-positive cells decreased. The 30-min ischemia group exhibited peak positivity 24 h after reperfusion, followed by a similar decrease. iNOS mRNA expression peaked 3 h after reperfusion in the 30-min ischemia group, at which time it decreased. In the 2-h ischemia group, iNOS mRNA increased 3 h after reperfusion, peaked 24 h after reperfusion, and then decreased.
CONCLUSION
These results indicated the occurrence of delayed apoptosis in transient cerebral ischemia. Increased expression of iNOS is closely associated with this apoptosis, and oxygen free radical-producing materials, such as nitric oxide, may play an important role in the induction of this apoptosis.
- Expression of Tumor Necrosis Factor-alpha, Interleukin-1beta and Inducible Nitric Oxide Synthase after Stereotaxic Injection of Lipopolysaccharide in Rat Hippocampus.
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Hoon Kyu Oh, Ku Seong Kang, Ji Yeon Kim, Eun Kyoung Kwak, Jung Wan Kim, Ji Young Park, Yoon Kyung Sohn
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Korean J Pathol. 2004;38(3):157-164.
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Abstract
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- BACKGROUND
Brain inducible nitric oxide synthase (iNOS) might be detectable in several pathologic conditions, and it is thought to play an important role in their pathophysiology. Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are believed to be essential factors of iNOS induction of the brain.
METHODS
After intrahippocampal stereotaxic injection of lipopoly-saccharide (LPS), the rat brains were removed at 6, 12 and 24 h. The rat brain tissues were examined to clarify the expression patterns of TNF-alpha, IL-1beta and iNOS.
RESULTS
The inflammatory cells which were stained with anti-TNF-alpha antibody, appeared in 6 h and increased for 24 h after LPS injection. The iNOS positive cells appeared after 12 h of LPS injection. A semiquantitative analysis of reverse transcription-polymerase chain reaction (RT-PCR) revealed that the TNF-alpha and IL-1beta mRNA arose at 1 h, peaked at 6 h and then declined until 48 h after LPS injection. The iNOS mRNA arose after 6 h, peaked at 12 h, and declined until 48 h after LPS injection.
CONCLUSIONS
We conclude that the induction of inflammatory events by intrahippocampal injection of LPS activates TNF-alpha and IL-1beta secretion, and this is followed by an induction of iNOS expression. TNF-alpha and IL-1beta seem to be related with iNOS expression in brain inflammation.
- The Effects of Ginsenoside Rb1 on the Apoptosis and the Production of Nitric Oxide in Rat C6 Glioma Cells.
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Joo Hyeon Park, Yoon Hee Lee, Ku Seong Kang, Soo Kyoung Lee, Sun Zoo Kim, Ji Young Park, Eun Kyoung Kwak, Yoon Kyung Sohn
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Korean J Pathol. 2004;38(1):1-7.
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Abstract
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- BACKGROUND
Ginsenosides, the extract of Panax ginseng, exert various pharmacological effects such as anticancer activity by the mechanism that is not yet defined. In this study, we proposed that the anticancer effect of ginsenoside Rb1 is related to tumor cell apoptosis and ginsenoside Rb1 induces the tumor cell apoptosis via the nitric oxide (NO) production.
METHODS
Rat C6 glioma cells were activated by treating with lipopolysaccharide (LPS), interferon (IFN)-gamma , and tumor necrosis factor (TNF)-alpha on the culture medium to investigate the effects of ginsenoside Rb1.
RESULTS
Compared with C6 glioma cells treated with LPS/IFN-gamma/TNF-alpha, C6 glioma cells treated with LPS/IFN-gamma/TNF-alpha/ginsenoside Rb1 showed marked increase in the NO production and apoptosis. Ginsenoside Rb1 induces the NO production in C6 glioma cells in dose-dependent manner. When C6 glioma cells treated with LPS/IFN-gamma/TNF-alpha/ginsenoside Rb1 were incubated with the specific inhibitor of iNOS, S-Methyl-2-thiopseudoureasulfate (SMT), both NO production and apoptosis in C6 glioma cells was significantly decreased. Ginsenoside Rb1 induced the expression of iNOS mRNA and iNOS protein in C6 glioma cells.
CONCLUSIONS
These results suggest that the induction of iNOS expression and subsequent
- Lipopolysaccharide/Interferon-gamma Induced Nitric Oxide Production in C6 Glioma Cells: Modulation by Dexamethasone.
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Jong Heun Shin, Ku Seong Kang, Ji Yeoun Kim, Sun Zoo Kim, Ji Young Park, Eun Kyoung Kwak, Yoon Kyung Sohn
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Korean J Pathol. 2002;36(6):406-411.
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Abstract
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- BACKGROUND
Glial cell-derived nitric oxide (NO), and its regulation has significant implications for central nervous system pathophysiology. The aim of the present study was to see the production of NO in lipopolysaccharide (LPS)/interferon-gamma (IFN-)-treated C6 glioma cells and the effect of dexamethasone on NO production and apoptosis of LPS/IFN--treated C6 glioma cells.
METHODS
The apoptosis of LPS/IFN- treated C6 glioma cell was examined with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method and the production of NO in culture medium was measured. The expression of iNOS mRNA was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The effect of the N-monomethyl L-arginine (NMMA) and dexamethasone on the apoptosis and NO production was also examined.
RESULTS
Inducible nitric oxide synthase (iNOS) mRNA and NO production were markedly increased in LPS/IFN--treated C6 glioma cells. The expression of iNOS mRNA arose at 3 hours, peaked at 12 hours, and declined 24 hours after LPS/IFN--treatment. Accumulation of NO derivatives in the culture media was increased at least upto 48 hours after LPS/IFN-. The induction of iNOS expression and NO production in LPS/IFN--treated C6 cells was correlated with apoptotic cell death judged by TUNEL staining. After treatment of NMMA, one of the NOS inhibitors, NO production and apoptosis were markedly decreased. Dexamehasone treatment suppressed the NO production by concentration depenedent manner.
CONCLUSIONS
From the above results it is concluded that the LPS/IFN- induced apoptosis of C6 cells is mediated by iNOS-derived NO and NO production and apoptosis was suppressed by dexamethasone.
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