Yong Seek Park

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.

MicroRNA and gene expression analysis of melatonin-exposed human breast cancer cell lines indicating involvement of the anticancer effect.

Abstract:  MicroRNAs (miRNAs) are small, noncoding RNAs that play a crucial role in regulation of gene expression. Recent studies have shown that miRNAs implicated in initiation and progression of various human cancers, including breast cancer and also analysis of miRNA expression profiles in cancer provide new insights into potential mechanisms of carcinogenesis. Melatonin, N‐acetyl‐5‐methoxytryptamine, is synthesized by the pineal gland in response to the dark/light cycle and has been known to act as a synchronizer of the biological clock. Melatonin has a variety of therapeutic effects, such as immunomodulatory actions, anti‐inflammatory effects, and antioxidant actions. Furthermore, melatonin is reported to have an anticancer function including suppression of the metabolism of tumor cells and induction of tumor suppressor genes in cancer cells, including breast cancer cells. In this study, we determined whether miRNAs play a role in regulation of various gene expression responses to melatonin in MCF‐7 human breast cancer cells. We examined whole‐genome miRNA and mRNA expression and found that 22 miRNAs were differentially expressed in melatonin‐treated MCF‐7 cells. We further identified a number of mRNAs whose expression level shows a high inverse correlation with miRNA expression. The Gene Ontology (GO) enrichment analysis and pathways analysis were performed for identification of the signaling pathways and biological processes affected by differential expression of miRNA and miRNA‐related genes. Our findings suggested that melatonin may modulate miRNA and gene expression as an anticancer mechanism in human breast cancer cells.

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.

Extracts from Citrus unshiu promote immune-mediated inhibition of tumor growth in a murine renal cell carcinoma model.

AIM OF THIS STUDY Citrus unshiu (Satsuma mandarin, SM) is a citrus fruit the peel of which has been used as a traditional Chinese medicine to treat common cold, relieve exhaustion, and cancer. In this study, we examined how effectively the content and peel extracts of SM can suppress cancer growth. The mechanism underlying cancer-suppressing properties of SM was investigated in tumor-bearing mice with renal carcinoma cell, Renca. MATERIALS AND METHODS Effectiveness of SM in tumor suppression was evaluated by measuring size of tumor mass in tumor-bearing mice treated with various doses of SM content and peel extracts. Proliferation of tumor cells and splenocytes was determined by MTT assay and [³H]TdR uptake, respectively. Relevant immunological mechanisms were chased by assaying cytokines including TGF-β, IL-6, IFN-γ, and TNF-α by ELISA. RESULTS The content and peel extracts of SM inhibited the growth of tumor cells in tumor-bearing mice. Especially, average tumor volume of two groups treated with 3 and 30 mg peel extracts per mouse weight (kg) were significantly decreased to 52.32% (p<0.05) and 68.72% (p<0.01), respectively. To identify tumor regression mechanism, anti-tumor cytokines measured in Con A-activated splenocytes from tumor-bearing mice. IFN-γ was increased in both of the peel extract-treated groups, while TNF-α, which had been decreased by tumor growth, was rescued to the normal level in SM content and peel extracts-treated groups. However, SM content and peel extracts did not inhibit proliferation and tumor-proliferative cytokines including TGF-β and IL-6 production of tumor cells. CONCLUSION These results indicate that SM content and peel extracts have anti-tumor properties in the tumor-bearing murine model. The mechanism underlying the anti-tumor effects of SM extracts is strongly suggested to be via boosting cytokines such as IFN-γ and TNF-α, enhancing immune-mediated anti-tumor properties.

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.

Omega-3 fatty acid-derived mediator, Resolvin E1, ameliorates 2,4-dinitrofluorobenzene-induced atopic dermatitis in NC/Nga mice

Atopic dermatitis (AD) is a common inflammatory skin disease for which few effective treatments are available. Resolvin E1 (RvE1; 5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an endogenous lipid mediator derived from omega-3 fatty eicosapentaenoic acid, which is a potent inhibitor of inflammation. AD-like skin lesion was induced by repetitive skin contact with DNFB in NC/Nga mice and the effects of RvE1 were evaluated on the basis of histopathological findings of skin, ear swelling and cytokine production of CD4(+) T cells. Intraperitoneal injection of RvE1 for one week after DNFB challenge significantly lowered ear swelling and improved back skin lesions. In addition, RvE1 significantly suppressed production of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) by activated CD4(+) T cells and serum IgE level. Furthermore, RvE1 reduced DNFB-induced infiltration of eosinophils, mast cells, CD4(+) T cells, and CD8(+) T cells in skin lesions. Therefore, RvE1 may suppress the development of AD-like skin lesions in DNFB-treated NC/Nga mice by reducing IL-4 and IFN-γ of activated CD4(+) T cells and serum IgE levels and infiltration of immune cells to skin lesion.

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.

Effect of Perilla frutescens var. acuta Kudo and rosmarinic acid on allergic inflammatory reactions

Allergy is characterized by an overreaction of the immune system. Perilla frutescens leaf extract has been reported to exhibit antiallergic inflammatory activity. To investigate precisely the effect and mechanism of 30% ethanol extract powder of P. frutescens var. acuta Kudo (EPPF) and rosmarinic acid (RA), a component of EPPF in allergic rhinitis and rhinoconjunctivitis, the antiallergic effects of EPPF and RA were analyzed using in vivo and in vitro models. Cytokine production was analyzed by means of an enzyme-linked immunosorbent assay. Cytokine expression was analyzed via reverse transcription-polymerase chain reaction and Western blotting. Transcription factor and caspase-1 activity were analyzed by a luciferase assay and caspase-1 assay, respectively. The number of nasal, ear and eye rubs after an ovalbumin (OVA) challenge in OVA-sensitized mice was significantly higher than that in OVA-unsensitized mice. Increased number of rubs was inhibited by administration of EPPF or RA. Increased levels of IgE in the serum, spleen and nasal mucosa of OVA-sensitized mice were reduced by EPPF or RA administration. The histamine level was also reduced by EPPF or RA administration in the serum of OVA-sensitized mice. Protein levels and mRNA expressions of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α were inhibited by EPPF or RA administration in the nasal mucosa tissue or spleen of OVA-sensitized mice. In EPPF or RA-administered mice, the mast cell and eosinophil infiltration increase as caused by OVA-sensitization was decreased. In addition, EPPF or RA inhibited both cyclooxygenase-2 protein expression and caspase-1 activity in the same nasal mucosa tissue. In activated human mast cells, nuclear factor-kappa B (NF-κB)/Rel A and caspase-1 activation increased, whereas NF-κB/Rel A and caspase-1 activation was inhibited after a treatment of EPPF or RA. These results indicate that EPPF and RA ameliorate allergic inflammatory reactions such as allergic rhinitis and allergic rhinoconjunctivitis.

Production and applications of rosmarinic acid and structurally related compounds

Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyllactic acid; RA) is a naturally occurring hydroxylated compound commonly found in species of the subfamily Nepetoideae of the Lamiaceae and Boraginaceae, such as Rosmarinus officinalis, Salvia officinalis, and Perilla frutescens. RA is biosynthesized from the amino acids l-phenylalanine and l-tyrosine by eight enzymes that include phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase. RA can also be chemically produced by the esterification of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA and its numerous derivatives containing one or two RA with other aromatic moieties are well known and include lithospermic acid, yunnaneic acid, salvianolic acid, and melitric acid. Recently, RA and its derivatives have attracted interest for their biological activities, which include anti-inflammatory, anti-oxidant, anti-angiogenic, anti-tumor, and anti-microbial functions. Clinically, RA attenuates T cell receptor-mediated signaling, attenuates allergic diseases like allergic rhinitis and asthma, and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like symptoms, protects from neurotoxicity, and slows the development of Alzheimer’s disease. These attributes have increased the demand for the biotechnological production and application of RA and its derivatives. The present review discusses the function and application of RA and its derivatives including the molecular mechanisms underlying clinical efficacy.

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.