Sang Pil Yoon

Human 8-oxoguanine DNA glycosylase suppresses the oxidative stress induced apoptosis through a p53-mediated signaling pathway in human fibroblasts.

Human 8-oxoguanine DNA glycosylase (hOGG1) is the main defense enzyme against mutagenic effects of cellular 7,8-dihydro-8-oxoguanine. In this study, we investigated the biological role of hOGG1 in DNA damage–related apoptosis induced by hydrogen peroxide (H2O2)–derived oxidative stress. The down-regulated expression of hOGG1 by its small interfering RNA prominently triggers the H2O2-induced apoptosis in human fibroblasts GM00637 and human lung carcinoma H1299 cells via the p53-mediated apoptotic pathway. However, the apoptotic responses were specifically inhibited by hOGG1 overexpression. The p53–small interfering RNA transfection into the hOGG1-deficient GM00637 markedly inhibited the H2O2-induced activation of p53-downstream target proteins such as p21, Noxa, and caspase-3/7, which eventually resulted in the increased cell viability. Although the cell viability of hOGG1-knockdown H1299 p53 null cells was similar to that of the hOGG1 wild-type H1299, after the overexpression of p53 the hOGG1-knockdown H1299 showed the significantly decreased cell viability compared with that of the hOGG1 wild-type H1299 at the same experimental condition. Moreover, the array comparative genome hybridization analyses revealed that the hOGG1-deficient GM00637 showed more significant changes in the copy number of large regions of their chromosomes in response to H2O2 treatment. Therefore, we suggest that although p53 is a major modulator of apoptosis, hOGG1 also plays a pivotal role in protecting cells against the H2O2-induced apoptosis at the upstream of the p53-dependent pathway to confer a survival advantage to human fibroblasts and human lung carcinomas through maintaining their genomic stability. (Mol Cancer Res 2007;5(10):1083–98)

Silibinin Inhibits LPS-Induced Macrophage Activation by Blocking p38 MAPK in RAW 264.7 Cells.

We demonstrate herein that silibinin, a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), inhibits LPS-induced activation of macrophages and production of nitric oxide (NO) in RAW 264.7 cells. Western blot analysis showed silibinin inhibits iNOS gene expression. RT-PCR showed that silibinin inhibits iNOS, TNF-α, and IL1β. We also showed that silibinin strongly inhibits p38 MAPK phosphorylation, whereas the ERK1/2 and JNK pathways are not inhibited. The p38 MAPK inhibitor abrogated the LPS-induced nitrite production, whereas the MEK-1 inhibitor did not affect the nitrite production. A molecular modeling study proposed a binding pose for silibinin targeting the ATP binding site of p38 MAPK (1OUK). Collectively, this series of experiments indicates that silibinin inhibits macrophage activation by blocking p38 MAPK signaling.

Long-term effects of chitosan oligosaccharide in streptozotocin-induced diabetic rats.

Streptozotocin has been used to induce an experimental model for diabetes to study the activity of anti-diabetic agents. The cholesterol-lowering effect of chitosan makes a continued issue in the field of diabetes, but the hypoglyecemic effect is inconclusive to date. Unlike chitosan, the water soluble chitosan oligosaccharide may possess various biological properties for diabetes. The present study was designed to investigate the long-term effects of chitosan oligosaccharide in normal and streptozotocin-induced diabetic rats using glycated hemoglobin and C-peptide. Chitosan oligosaccharide feeding did not cause any harmful effect on plasma glucose as well as plasma lipid metabolism in normal rats, although slightly elevated triglyceride was observed. As compared with the diabetic control rats, effects of chitosan oligosaccharide for 12 weeks in the diabetic rats were summarized as follows; 1) the blood glucose concentrations fell significantly and it was confirmed by decreased glycated hemoglobin, 2) the plasma C-peptide was increased and provided elevated degree of insulin secretion, and 3) relatively well reconstructed pancreatic islet with β cells and additional insulin-immunolabeled cells in the pancreatic acinus and in the intercalated duct were observed. These results suggested that chitosan oligosaccharide could improve the altered blood glucose metabolism in the diabetic rats by various mechanisms such as accelerated proliferation or neogenesis of β cells and increased secretory capacity of insulin.

Colon cancer progression is driven by APEX1-mediated upregulation of Jagged

Aberrant expression of apurinic-apyrimidinic endonuclease-1 (APEX1) has been reported in numerous human solid tumors and is positively correlated with cancer progression; however, the role of APEX1 in tumor progression is poorly defined. Here, we show that APEX1 contributes to aggressive colon cancer behavior and functions as an upstream activator in the Jagged1/Notch signaling pathway. APEX1 overexpression or knockdown in human colon cancer cell lines induced profound changes in malignant properties such as cell proliferation, anchorage-independent growth, migration, invasion, and angiogenesis in vitro and in tumor formation and metastasis in mouse xenograft models. These oncogenic effects of APEX1 were mediated by the upregulation of Jagged1, a major Notch ligand. Furthermore, APEX1 expression was associated with Jagged1 in various colon cancer cell lines and in tissues from colon cancer patients. This finding identifies APEX1 as a positive regulator of Jagged1/Notch activity and suggests that it is a potential therapeutic target in colon cancers that exhibit high levels of Jagged1/Notch signaling.

Immunohistochemical detection of StarD6 in the rat nervous system.

Steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain 6 (StarD6) is known to be exclusively expressed in germ cells of testis. As little is known about StarD6 expression in the nervous system, we investigated the distribution of StarD6 in rat neural tissues. Immunoreactivity of StarD6 was detected in the brain, spinal cord and dorsal root ganglia; particularly cerebral cortex (layer V and VI), hippocampus, substantia gelatinosa of the spinal cord. We provided compelling evidence that multiple neuronal and glial populations were immunolabelled with anti-StarD6 antibody throughout the nervous system. We postulate that StarD6 might play an important role in lipid sensing of the nervous system based on its immunolocalization in this study.

Protective effects of epigallocatechin gallate (EGCG) on streptozotocin-induced diabetic nephropathy in mice.

There is increasing evidence suggesting that antioxidants in green tea extracts may protect kidneys on the progression of end-stage renal disease. We investigated the protective impacts of (-)-epigallocatechin 3-O-gallate (EGCG) against streptozotocin (STZ)-induced diabetic nephropathy in mice. The mice were divided into 5 groups (n=10 per group): control (saline, i.p.), STZ (200mg/kg, i.p.), EGCG50 (50mg/kg, S.Q.), EGCG100 (100mg/kg, S.Q.), and EGCG200 (200mg/kg, S.Q.). Animals were sacrificed at scheduled times after EGCG administration and then quantitative and qualitative analysis were performed. Compared with the control group, the STZ group showed an increase in levels of blood glucose, blood urea nitrogen, creatinine and urine protein amounts with a decrease in body weight. All the above parameters were significantly reversed with EGCG treatment, especially in the EGCG100 group. After STZ injection, there was a mesangial proliferation with increased renal osteopontin accumulation and its protein expression in the glomeruli and the proximal tubules. Mice kidneys after EGCG-treatment showed a reduced expression of above parameters and relatively improved histopathological findings. These results indicated that EGCG 100mg/kg might provide an effective protection against STZ-induced diabetic nephropathy in mice by osteopontin suppression.

The changed immunoreactivity of StarD6 after pilocarpine-induced epilepsy.

This study was designed whether StarD6 is also affected by neurodegenerative stimuli, as other START proteins may be one of the delivery proteins in cholesterol metabolism of neurosteroid synthesis. Increased immunoreactivity of StarD6 was observed in all zones of the hippocampus by the administration of pilocarpine (350 mg/kg, intraperitoneally). More intense StarD6 immunolocalization was seen in the strata radiatum and lacunosum-moleculare, particularly in CA1-2 areas until 12 h after pilocarpine-induced epilepsy. StarD6 protein level was transiently increased at 3 h after pilocarpine-treated rat hippocampus comparing with normal rat. This raises the possibility that StarD6 might have nuclear roles and contribute to subsequent formation of neuroprotective steroids after excitotoxic brain injury.

Senescence-dependent MutS alpha dysfunction attenuates mismatch repair.

DNA damage and mutations in the genome increase with age. To determine the potential mechanisms of senescence-dependent increases in genomic instability, we analyzed DNA mismatch repair (MMR) efficiency in young and senescent human colonic fibroblast and human embryonic lung fibroblast. It was found that MMR activity is significantly reduced in senescent cells. Western blot and immunohistochemistry analysis revealed that hMSH2 and MSH6 protein (MutSα complex), which is a known key component in the MMR pathway, is markedly down-regulated in senescent cells. Moreover, the addition of purified MutSα to extracts from senescent cells led to the restoration of MMR activity. Semiquantitative reverse transcription-PCR analysis exhibited that MSH2 mRNA level is reduced in senescent cells. In addition, a decrease in E2F transcriptional activity in senescent cells was found to be crucial for MSH2 suppression. E2F1 small interfering RNA expression reduced hMSH2 expression and MMR activity in young human primary fibroblast cells. Importantly, expression of E2F1 in quiescent cells restored the MSH2 expression as well as MMR activity, whereas E2F1-infected senescent cells exhibited no restoration of MSH2 expression and MMR activity. These results indicate that the suppression of E2F1 transcriptional activity in senescent cells lead to stable repression of MSH2, followed by a induction of MutSα dysfunction, which results in a reduced cellular MMR capacity in senescent cells. (Mol Cancer Res 2008;6(6):978–89)

NEDDylation promotes stress granule assembly

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

Acute responses of DNA repair proteins and StarD6 in rat hippocampus after domoic acid-induced excitotoxicity.

StarD6, which might be considered to be neuroprotective, and DNA repair proteins can play a role against oxidative damages by excitotoxin in the nervous system. In order to investigate the relationship between StarD6 and DNA repair proteins, excitotoxicity was induced by domoic acid in male Sprague-Dawley rats. Western blot analysis revealed transitorily elevated levels in StarD6, apurinic/apyrimidinic endonuclease (APE) and 8-oxoguanine DNA-glycosylase (Ogg1) in accord with the DNA damage marker phosphorylated H2AX. Immunohistochemistry revealed that increased intensity was transiently seen not only in the Stratum (Str.) radiatum and Str. lacunosum-moleculare with StarD6 and APE, but also in the Str. pyramidale with Ogg1. Intensities decreased 24h after domoic acid injection in CA3 with APE and Ogg1 as well as in the Str. radiatum and Str. lacunosum-moleculare with StarD6 and APE. These results suggested that StarD6 may not be closely related with DNA repair proteins in the hippocampus after domoic acid-induced excitotoxicity, although the activities of these proteins might be positively affected by excitotoxic stimuli.