Journal of Pathology and Translational Medicine

Original Articles

Expression of Inducible Nitric Oxide Synthase and Nitric Oxide Mediated Apoptosis in Neuronal PC12 Cells after Lipopolysaccharide/Tumor Necrosis Factor-/Interferon- Treatment.: Jiyeon Kim, Jiyoung Kim, Kuseong Kang, Eunkyoung Kwak, Jiyoung Park, Taein Park, Yoonkyung Sohn; Korean J Pathol. 2002;36(4):249-256.

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Abstract PDF: BACKGROUND
Inducible nitric oxide synthase (iNOS) has been detected in a number of pathologic conditions in the central nervous system. This study was investigated the patterns of iNOS expression in the neuronal PC12 cell and the effects of nitric oxide on the apoptosis of PC12 cells.
METHODS
The stimulating agents for induction of iNOS expression in PC12 cells were bacterial lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-), and interferon-gamma (IFN-).
RESULTS
The expression iNOS mRNA and protein in PC12 cells stimulated with LPS/TNF-/IFN- were profoundly increased. The expression of iNOS mRNA arose at 6 hours, peaked at 12 hours, and declined to 48 hours after LPS/TNF-/ IFN- treatment. iNOS protein was increased up to 24 hours in LPS/TNF-/IFN- treated PC12 cells while the expression of nNOS was unaffected. Accumulation of NO derivatives in the culture media was markedly increased at least at up to 48 hours after LPS/TNF-/IFN- treatment. The induction of iNOS expression and NO production in differentiated PC12 cells was correlated with apoptotic cell death judged by transmission electron microscopy and DNA fragmentation from the results of the Terminal deoxynucleotidyl-transferase-mediated dUDP biotin nick end-labeling (TUNEL) method. After treatment with NOS inhibitor, N-monomethylarginine (NMMA), a profound decrease in NO production by LPS/TNF-/IFN- treated PC12 cells was noted. And the LPS/TNF-/IFN- induced apoptosis was prevented by the NMMA treatment.
CONCLUSIONS
From the above results it is concluded that the expression of iNOS in differentiated PC12 cells is induced by the combined application of LPS, TNF-, and IFN-. And the apoptosis of cultured PC12 cells is mediated by iNOS-derived NO.

Expression of Nitric Oxide Synthase Isotypes in Advanced Gastric Carcinoma.: Kyong Mee Kwon, Young Chae Chu, Tae Sook Hwang; Korean J Pathol. 2002;36(6):374-371.

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Abstract PDF: BACKGROUND
Increased expression of nitric oxide synthase (NOS) isotypes is present in human tumor cell lines and solid tumor tissues. Hypoxia upregulates NOS expression, and nitric oxide (NO) induces mitogenesis among endothelial cells. NO has been known to induce vascular endothelial growth factor (VEGF) expression in carcinoma cells and to induce neovascularization in tumors.
METHODS
The expression and cellular localization of 3 isotypes of NOS was detected by immunohistochemistry in 73 advanced gastric carcinoma tissues along with adjacent normal gastric mucosa; and the relationship to known clinicopathologic parameters, microvascular density, and VEGF expression was analysed.
RESULTS
Forty-four (60.3%), 56 (76.7%), and 52 (71.2%) of the 73 cases revealed eNOS, nNOS, and iNOS expression, respectively. Intestinal type adenocarcinomas tended to have higher activity of eNOS (p=0.000) and nNOS (p=0.001) activities than did the diffuse type adenocarcinomas. All isotypes of NOS (eNOS, p=0.001; nNOS, p=0.005; iNOS, p=0.044) tended to be highly expressed when the tumor was differentiated. There was no significant relationship between any of the 3 NOS isotypes and microvascular density, whereas VEGF was closely related with microvascular density (p=0.000). The expression of VEGF was not related to with any of the NOS isotype expressions.
CONCLUSIONS
From the above results, we speculated that NO may be implicated in the early stage of the gastric carcinogenesis rather than the growth and progression stages, and NO does not appear to affect angiogenesis or VEGF expression in the advanced gastric carcinoma.

Lipopolysaccharide/Interferon-gamma Induced Nitric Oxide Production in C6 Glioma Cells: Modulation by Dexamethasone.: Jong Heun Shin, Ku Seong Kang, Ji Yeoun Kim, Sun Zoo Kim, Ji Young Park, Eun Kyoung Kwak, Yoon Kyung Sohn; Korean J Pathol. 2002;36(6):406-411.

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Abstract PDF: 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.

Correlation Between Neuronal Apoptosis and Expression of Inducible Nitric Oxide Synthase after Transient Focal Cerebral Ischemia.: 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; Korean J Pathol. 2004;38(6):364-371.

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Abstract PDF: 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.: Hoon Kyu Oh, Ku Seong Kang, Ji Yeon Kim, Eun Kyoung Kwak, Jung Wan Kim, Ji Young Park, Yoon Kyung Sohn; Korean J Pathol. 2004;38(3):157-164.

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Abstract PDF: 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.

Nitric Oxide Synthase Expression in Early Stage of Aging Rat Kidney.: Kye Won Kwon, Hyeon Joo Jeong; Korean J Pathol. 2004;38(2):86-92.

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Abstract PDF: BACKGROUND
Nitric oxide synthase (NOS) has been suggested to have a role in renal injury of aging rats.
METHODS
Renal function and histology were compared between 12 month-and 7-9 week-old rats. Proliferating activity and cell death were evaluated by PCNA index and apoptosis. Three isoforms of NOS (eNOS, iNOS, and nNOS) were stained by immunohistochemistry.
RESULTS
Serum creatinine level was increased in old rats (1.0 mg/dL vs 0.5 mg/dL, p=0.000). 24 h proteinuria and urinary NO were comparable between the two groups. The percentage of global and segmental glomerulosclerosis increased in old rats. PCNA index decreased in the glomeruli (0.1 vs 0.6/glomerulus, p=0.005) and the tubulointerstitium (10.2 vs 19.2/mm2, p=0.019) of old rats compared to that of young rats. However, no difference was observed in the number of TUNEL positive cells. eNOS was not stained in young and old rat kidney, whereas iNOS was stained in the interstitial inflammatory cells of old rats (0.3 vs 0.0 of young rats/mm2, p=0.188). Macula densa nNOS staining significantly decreased in old rats compared to young rats (5.6 vs 9.5/mm2, p=0.009).
CONCLUSIONS
Proliferating activity is more affected than cell death with aging. Decreased nNOS expression without alteration of eNOS and iNOS expressions may implicate nNOS as a marker of renal injury in the early stage of aging.

The Effects of Ginsenoside Rb1 on the Apoptosis and the Production of Nitric Oxide in Rat C6 Glioma Cells.: Joo Hyeon Park, Yoon Hee Lee, Ku Seong Kang, Soo Kyoung Lee, Sun Zoo Kim, Ji Young Park, Eun Kyoung Kwak, Yoon Kyung Sohn; Korean J Pathol. 2004;38(1):1-7.

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Abstract PDF: 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

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