Seong Il Jeong

Fisetin induces Nrf2‐mediated HO‐1 expression through PKC‐δ and p38 in human umbilical vein endothelial cells

Fisetin is a natural flavonoid from fruits and vegetables that exhibits antioxidant, neurotrophic, anti‐inflammatory, and anti‐cancer effects in various disease models. Up‐regulation of heme oxygenase‐1 (HO‐1) expression protects against oxidative stress‐induced cell death, and therefore, plays a crucial role in cytoprotection in a variety of pathological models. In the present study, we investigated the effect of fisetin on the up‐regulation of HO‐1 in human umbilical vein endothelial cells (HUVECs). Small interfering RNA and pharmacological inhibitors of PKC‐δ and p38 MAPK attenuated HO‐1 induction in fisetin‐stimulated HUVECs. Fisetin treatment resulted in significantly increased NF‐E2‐related factor 2 (Nrf2) nuclear translocation, and antioxidant response element (ARE)‐luciferase activity, leading to up‐regulation of HO‐1 expression. In addition, fisetin pretreatment reduced hydrogen peroxide (H2O2)‐induced cell death, and this effect was reversed by ZnPP, an inhibitor of HO‐1. In summary, these findings suggest that induction of HO‐1 expression via Nrf2 activation may contribute to the cytoprotection exerted by fisetin against H2O2‐induced oxidative stress in HUVECs. J. Cell. Biochem. 112: 2352–2360, 2011.

Toxicologic evaluation of bacterial synthesized cellulose in endothelial cells and animals

Compared to plant cellulose, bacterial cellulose synthesized by Gluconacetobacter xylinus has features such as high crystallinity, tensile strength and water absorption capacity; biocompatibility; resistance to degradation and low solubility that may be advantageous for engineered tissue. However, little information is available concerning the potential toxicity of bacterial cellulose-based biomaterials. The present study investigated the toxicity of bacterial cellulose nanofibers in vitro in human umbilical vein endothelial cells (HUVECs) using viability and flow cytometric assays and in vivo using C57/Bl6 mice. The absence of toxicity in vitro and in vivo supports the view that bacterial cellulose may be amenable for use as a tissue engineering biomaterial.

Upregulation of heme oxygenase-1 as an adaptive mechanism for protection against crotonaldehyde in human umbilical vein endothelial cells.

Crotonaldehyde, a highly reactive α, β-unsaturated aldehyde, is a ubiquitous environmental pollutant and a product of endogenous lipid peroxidation. It is also a major component of cigarette smoke and is present in many foods and beverages, and has also been linked to development of various diseases. Activation of endothelial cells by stimuli such as cigarette smoke is an important risk factor for cardiovascular diseases, including atherosclerosis. Heme oxygenase-1 (HO-1) is a protective antioxidant enzyme with a critical role in resistance to oxidative stress and other cellular functions. In this study, we examined the effects of crotonaldehyde on HO-1 induction and determined the signaling pathways in human umbilical vein endothelial cells (HUVECs). Inhibition of the protein kinase C-δ (PKC-δ) and p38 pathways resulted in significant blockage of crotonaldehyde-mediated HO-1 induction. Crotonaldehyde treatment caused a dramatic increase in translocation of NF-E2 related factor (Nrf2), leading to induction of HO-1. In addition, small interfering RNA knockdown of Nrf2 and treatment with the specific HO-1 inhibitor ZnPP exhibited an obvious increase of apoptosis of crotonaldehyde-treated HUVECs. Taken together, our results demonstrated that crotonaldehyde-induced HO-1 expression is mediated by the PKC-δ-p38 MAPK-Nrf2-HO-1 pathway in HUVECs, which is an adaptive response to oxidative stress.

Up-regulation of Heme Oxygenase-1 by Korean Red Ginseng Water Extract as a Cytoprotective Effect in Human Endothelial Cells

Korean red ginseng (KRG) is used worldwide as a popular traditional herbal medicine. KRG has shown beneficial effects on cardiovascular diseases, such as atherosclerosis, diabetes, and hypertension. Up-regulation of a cytoprotective protein, heme oxygenase (HO)-1, is considered to augment the cellular defense against various agents that may induce cytotoxic injury. In the present study, we demonstrate that KRG water extract induces HO-1 expression in human umbilical vein endothelial cells (HUVECs) and possible involvement of the anti-oxidant transcription factor nuclear factor-eythroid 2-related factor 2 (Nrf2). KRG-induced HO-1 expression was examined by western blots, reverse transcriptase polymerase chain reaction and immunofluorescence staining. Specific silencing of Nrf2 genes with Nrf2-siRNA in HUVECs abolished HO-1 expression. In addition, the HO inhibitor zinc protoporphyrin blunted the preventive effect of KRG on H2O2-induced cell death, as demonstrated by terminal transferase dUTP nick end labeling assay. Taken together, these results suggest that KRG may exert a vasculoprotective effect through Nrf2- mediated HO-1 induction in human endothelial cell by inhibition of cell death.

Genome-wide profiling in melatonin-exposed human breast cancer cell lines identifies differentially methylated genes involved in the anticancer effect of melatonin

Epigenetic alterations have emerged as an important mechanism involved in tumorigenesis. The epigenetic impact of DNA methylation in various types of human cancer is not completely understood. Previously, we observed melatonin‐induced differential expression of miRNA and miRNA‐related genes in human breast cancer cell lines that indicated an anticancer effect of melatonin. In this report, we further characterized epigenetic changes in melatonin‐exposed MCF‐7 cells through the analysis of DNA methylation profiles in breast cancer cells to provide new insights into the potential mechanisms of the anticancer effect of melatonin. Microarray‐based DNA methylation and gene expression profiling were carried out using human breast cancer cell lines. We further identified a number of mRNAs whose expression levels show an inverse correlation with DNA methylation levels. The mRNA expression levels and methylation status of candidate genes in melatonin‐exposed cells were confirmed by real‐time quantitative PCR and bisulfite PCR. This approach led to the detection of cancer‐related genes, which were oncogenic genes, including EGR3 and POU4F2/Brn‐3b were down‐regulated, while the tumor suppressor gene, GPC3, was up‐regulated by 1 nm melatonin‐treated MCF‐7 cells. Our results provide detailed insights into the DNA methylation patterns induced by melatonin and suggest a potential mechanism of the anticancer effect of aberrant DNA methylation in melatonin‐treated breast cancer cells.

Extract of Salvia miltiorrhiza (Danshen) induces Nrf2-mediated heme oxygenase-1 expression as a cytoprotective action in RAW 264.7 macrophages

ETHNOPHARMACOLOGICAL RELEVANCE Danshen (Salvia miltiorrhiza) is widely used in traditional herbal medicines for relief of a variety of symptoms related to complications arising from vascular diseases such as hypertension, diabetes, and atherosclerosis. Induction of heme oxygenase-1 (HO-1) expression protects against oxidative stress-induced cell damage, which plays an important role in cytoprotection in a variety of pathological models. MATERIALS AND METHODS In the present study, we investigated the effect of Danshen on the up-regulation of HO-1, an inducible and cytoprotective enzyme in RAW 264.7 macrophages. Molecular mechanisms underlying the effects, especially protective effects, was elucidated by analyzing the activation of transcription factors and their upstream signalling, and by evaluating the inhibitory effect of HO-1 on ROS production. RESULTS Danshen induced HO-1 mRNA expression and protein production, and nuclear translocation of NF-E2-related factor 2 in RAW 264.7 macrophages. Pharmacological inhibitors of PI3K/Akt and MEK1 attenuated HO-1 induction in Danshen-stimulated RAW 264.7 macrophages. Furthermore, Danshen pretreatment reduced intracellular production of reactive oxygen species after stimulation with hydrogen peroxide; this effect was reversed by the HO-1 inhibitor ZnPP. CONCLUSION Danshen induced HO-1 expression through PI3K/Akt-MEK1-Nrf2 pathway and reduced intracellular production of reactive oxygen species via induction of HO-1 expression. The results support a role of HO-1 in the cytoprotective effect of Danshen.

Induction of Heme Oxygenase-1 Inhibits Cell Death in Crotonaldehyde-Stimulated HepG2 Cells via the PKC-δ -p38 -Nrf2 Pathway

Background Crotonaldehyde, an alpha, beta-unsaturated aldehyde present in cigarette smoke, is an environmental pollutant and a product of lipid peroxidation. It also produces adverse effects to humans and is considered as a risk factor for various diseases. Heme oxygenase-1 (HO-1) plays important roles in protecting cells against oxidative stress as a prime cellular defense mechanism. However, HO-1 may be associated with cell proliferation and resistance to apoptosis in cancer cells. The aim of this study was to examine the effects of HO-1 induction on cell survival in crotonaldehyde-stimulated human hepatocellular carcinoma (HepG2) cells. Methods To investigate the signaling pathway involved in crotonaldehyde-induced HO-1 expression, we compared levels of inhibition efficiency of specific inhibitors and specific small interfering RNAs (siRNAs) of several kinases. The cell-cycle and cell death was measured by FACS and terminal dUTP nick-end labeling (TUNEL) staining. Results Treatment with crotonaldehyde caused a significant increase in nuclear translocation of NF-E2 related factor (Nrf2). Treatment with inhibitors of the protein kinase C-δ (PKC-δ) and p38 pathways resulted in obvious blockage of crotonaldehyde-induced HO-1 expression. Furthermore, treatment with HO-1 siRNA and the specific HO-1 inhibitor zinc-protoporphyrin produced an increase in the G0/G1 phase of the cell cycle in crotonaldehyde-stimulated HepG2 cells. Conclusions Taken together, the results support an anti-apoptotic role for HO-1 in crotonaldehyde-stimulated human hepatocellular carcinoma cells and provide a mechanism by which induction of HO-1 expression via PKC-δ–p38 MAPK–Nrf2 pathway may promote tumor resistance to oxidative stress.

Effect of α,β-unsaturated aldehydes on endothelial cell growth in bacterial cellulose for vascular tissue engineering

Cardiovascular disease is the leading global cause of death. Cigarette smoking is a major risk factor for cardiovascular disease. And α,β-unsaturated aldehydes such as acrolein and crotonaldehyde are major component of cigarette smoke. Use of replacement grafts materials is one of the cardiovascular disease treatments. However, currently available synthetic materials generally produce poor outcomes including hyperplasia and thrombogenicity. Recently, bacterial synthesized cellulose has received interest as a new functional vascular graft biomaterial owing to its biocompatibility. However, the association of a bacterial cellulose-based scaffold and cigarette smoke is not known. The present study investigated the alteration of function of human umbilical vein endothelial cells (HUVECs) treated with α,β-unsaturated aldehyde on bacterial cellulose-based material. The data suggest that α,β-unsaturated aldehydes in cigarette smoke induce altered endothelial cell functions including morphology, adhesion, proliferation, viability and growth on bacterial cellulose. These results may provide the view that cigarette smoking of cardiovascular disease patients applied to bacterial cellulose-based vascular grafts is risk.

Genome-wide analysis of gene expression by crotonaldehyde in human umbilical vein endothelial cells

Crotonaldehyde (CRA) is an α,β-unsaturated aldehyde that is genotoxic, carcinogenic, and mutagenic, and forms 1,N(2)-propanodeoxyguanosine. Humans are exposed to CRA in work places, and from tobacco smoke, environmental pollution, food, and beverages. In addition, CRA is a product of endogenous lipid peroxidation, arising as a consequence of oxidative stress and electrophilic stress, which may be related to development of cardiovascular diseases. In this study, we used 24 k whole human DNA chips for identification of gene expression profiles by treatment of CRA in human umbilical vein endothelial cells (HUVECs). Among the 24,000 genes of cDNA microarrays, we identified up- and down-regulated genes that changed by more than 1.8 and 2 fold by CRA. Our data showed that CRA changed gene expression patterns at the genome-wide transcriptional level in HUVECs. And several genes showed association with cardiovascular diseases.

MicroRNA microarray analysis of human umbilical vein endothelial cells exposed to benzo(a)pyrene

MicroRNAs (miRNAs) are small non-coding RNA molecules that function as potent negative regulators of gene expression. They play a critical role in the regulation of genes involved in the cellular and molecular responses to toxicants. Benzo(a)pyrene (BaP) is an environmental contaminant found in cigarette smoke and is one of the polycyclic aromatic hydrocarbons (PAHs). BaP is considered a major contributor of vascular disease. In this study, we investigated whether miRNAs play a role in regulation of various gene expression responses to BaP in human umbilical vein endothelial cells (HUVECs). We performed a pair-wise correlation coefficient analysis on expression levels of 105 miRNAs and 2335 mRNA, and observed anti-correlations between miRNA and mRNA. The Gene Ontology enrichment analysis on the differential expression of miRNA-related genes revealed significant enrichment in the biological processes and signaling pathways. Our results first revealed the expression profiling of miRNAs in endothelial cells and contribute to the improvement of vascular disease.