Ki Cheon Kim

Endoplasmic reticulum stress signaling is involved in silver nanoparticles-induced apoptosis.

Although silver nanoparticles (AgNPs) have been reported to exert strong acute toxic effects on various cultured cells by inducing oxidative stress, the molecular mechanisms by which AgNPs-damaged cells are unknown. Because the endoplasmic reticulum (ER) may play an important role in the response to oxidative stress-induced damage and is quite sensitive to oxidative damage, we hypothesized that AgNPs may exert cytotoxic effects on cells by modulating ER stress. In our study, AgNPs resulted in cytotoxicity and apoptotic cell death when analyzing cell viability, DNA fragmentation and the apoptotic sub-G(1) population. Flow cytometry and confocal microscopy indicated that the cells were sensitive to AgNPs with respect to the induction of mitochondrial Ca(2+) overloading and enhancement of ER stress. AgNPs induced a number of signature ER stress markers, including phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and its downstream eukaryotic initiation factor 2α, phosphorylation of inositol-requiring protein 1 (IRE1), splicing of ER stress-specific X-box transcription factor-1, cleavage of activating transcription factor 6 (ATF6) and up-regulation of glucose-regulated protein-78 and CCAAT/enhancer-binding protein-homologous protein (CHOP/GADD153). Down-regulation of PERK, IRE1 and ATF6 expression using siRNA significantly decreased AgNPs-induced the enhancement of ER stress. In addition, down-regulation of CHOP expression with siRNA CHOP attenuated AgNPs-induced apoptosis. Taken together, the present study supports an important role for the ER stress response in mediating AgNPs-induced apoptosis.

Up-regulation of Nrf2-mediated heme oxygenase-1 expression by eckol, a phlorotannin compound, through activation of Erk and PI3K/Akt.

The aim of the present study was to examine the cytoprotective effect of eckol, a phlorotannin found in Ecklonia cava and to elucidate underlying mechanisms. Heme oxygenase-1 (HO-1) is an important antioxidant enzyme that plays a role in cytoprotection against oxidative stress. Eckol-induced HO-1 expression both at the level of mRNA and protein in Chinese hamster lung fibroblast (V79-4) cells, resulting in increased HO-1 activity. The transcription factor NF-E2-related factor 2 (Nrf2) is a critical regulator of HO-1, achieved by binding to the antioxidant response element (ARE). Eckol treatment resulted in the enhanced level of phosphorylated form, nuclear translocation, ARE-binding, and transcriptional activity of Nrf2. Extracellular regulated kinase (Erk) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, Akt) contributed to ARE-driven HO-1 expression. Eckol activated both Erk and Akt, and treatments with U0126 (an Erk kinase inhibitor), LY294002 (a PI3K inhibitor), specific Erk1 siRNA, and Akt siRNA suppressed the eckol-induced activation of Nrf2, resulting in a decrease in HO-1 expression. ZnPP (a HO-1 inhibitor), HO-1 siRNA, and Nrf2 siRNA markedly abolished the cytoprotective effect of eckol against hydrogen peroxide-induced cell damage. Likewise, U0126 and LY294002 inhibited the eckol-induced cytoprotective effect against oxidative cell damage. These studies demonstrate that eckol attenuates oxidative stress by activating Nrf2-mediated HO-1 induction via Erk and PI3K/Akt signaling.

Silver nanoparticles down-regulate Nrf2-mediated 8-oxoguanine DNA glycosylase 1 through inactivation of extracellular regulated kinase and protein kinase B in human Chang liver cells

Recently, we reported that silver nanoparticles (AgNPs) induced reactive oxygen species (ROS) generation and the resultant oxidative stress contributes to the cell damage associated with AgNPs. 8-Oxoguanine (8-oxoG) is sensitive marker of ROS-induced DNA damage. 8-Oxoguanine DNA glycosylase 1 (OGG1) is an important DNA repair enzyme that recognizes and excises 8-oxoG. The aim of the present study was to examine the effect of AgNPs-induced oxidative stress on OGG1 and to elucidate mechanisms underlying AgNPs toxicity. AgNPs decreased OGG1 mRNA and protein expression, resulting in decreased OGG1 activity. Decreased OGG1 activity in AgNPs-treated cells led to increased 8-oxoG levels. The transcription factor NF-E2-related factor 2 (Nrf2) is an important factor in the inducible regulation of OGG1. AgNPs treatment decreased nuclear Nrf2 expression, translocation into nucleus, and transcriptional activity of Nrf2. Extracellular regulated kinase (ERK) and protein kinase B (PKB, AKT), which are upstream of Nrf2, contribute to OGG1 expression. AgNPs attenuated both active forms of ERK and AKT protein expression, resulting in suppression of Nrf2 and decrease of OGG1 expression. These studies demonstrate that down-regulation of Nrf2-mediated OGG1 in exposure to AgNPs occurs through ERK and AKT inactivation.

Oxidative stress causes epigenetic alteration of CDX1 expression in colorectal cancer cells.

The intestine-specific transcription factor, caudal type homeobox-1 (CDX1), is a candidate tumor suppressor gene that plays key roles in regulating intestinal epithelial differentiation and proliferation. It is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines by promoter hypermethylation. Since the effects of oxidative stress on the transcription of tumor suppressor genes are largely unknown, this study explored the epigenetic alterations that occur during reactive oxygen species (ROS)-induced silencing of CDX1 in colorectal cancer cells. Oxidative stress by hydrogen peroxide (H2O2) down-regulated CDX1 mRNA levels and protein expression in the human colorectal cancer cell line, T-84. This down-regulation was abolished by pretreatment with the ROS scavenger, N-acetylcysteine. In addition, the DNA methylation inhibitor, 5-aza-2-deoxycytidine (5-Aza-dC) markedly attenuated the decrease in mRNA and protein expression levels induced by H2O2. Moreover, methylation-specific PCR data revealed that H2O2 treatment increased CDX1 promoter methylation, and treatment with 5-Aza-dC reversed this effect, suggesting that an epigenetic regulatory mechanism triggered by ROS-induced methylation may be involved in CDX1 expression. Furthermore, H2O2 treatment resulted in up-regulation of DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) expression and activity, and enhanced the association between DNMT1 and HDAC1. Taken together, these results suggest that ROS-induced oxidative stress silences the tumor suppressor CDX1 through epigenetic regulation, and may therefore be associated with the progression of colorectal cancer.

Myricetin Protects Cells against Oxidative Stress-Induced Apoptosis via Regulation of PI3K/Akt and MAPK Signaling Pathways

Recently, we demonstrated that myricetin exhibits cytoprotective effects against H2O2-induced cell damage via its antioxidant properties. In the present study, myricetin was found to inhibit H2O2-induced apoptosis in Chinese hamster lung fibroblast (V79-4) cells, as shown by decreased apoptotic bodies, nuclear fragmentation, sub-G1 cell population, and disruption of mitochondrial membrane potential (Δψm), which are increased in H2O2-treated cells. Western blot data showed that in H2O2-treated cells, myricetin increased the level of Bcl-2, which is an anti-apoptotic factor, and decreased the levels of Bax, active caspase-9 and -3, which are pro-apoptotic factors. And myricetin inhibited release of cytochrome c from mitochondria to cytosol in H2O2-treated cells. Myricetin-induced survival correlated with Akt activity, and the rescue of cells by myricetin treatment against H2O2-induced apoptosis was inhibited by the specific PI3K (phosphoinositol-3-kinase) inhibitor. Myricetin-mediated survival also inhibited the activation of p38 mitogen activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), which are members of MAPK. Our studies suggest that myricetin prevents oxidative stress-induced apoptosis via regulation of PI3K/Akt and MAPK signaling pathways.

Cytoprotective effect of the fruits of Lycium chinense Miller against oxidative stress-induced hepatotoxicity.

AIM OF THE STUDY Fruits of Lycium chinense Miller (Solanaceae), distributed in northeast Asia, have gained attraction for their hepatoprotective role in traditional oriental medicine. The excessive production of reactive oxygen species (ROS) is hazardous for living organisms and damage major cellular constituents such as DNA, lipid, and protein. The cytoprotective effect of Lycium chinense fruits (Lycium extract) was assessed against H(2)O(2)-induced Chang liver cell damage. MATERIALS AND METHODS The effect of Lycium extract against H(2)O(2)-induced cell death was determined by the MTT assay. Radical scavenging activity was determined through the assessments of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, intracellular ROS, hydroxyl radicals, and superoxide. The inductions of antioxidant enzymes were determined via their protein expressions and activities. DNA damage was measured using comet assay and expression of phospho-histone H2A.X. Lipid peroxidation was measured using 8-isoprostane level and fluorescent probe. Protein modification was measured using protein carbonyl moiety. RESULTS AND CONCLUSION Lycium extract scavenged the DPPH free radicals, intracellular ROS, hydroxyl radicals, and superoxide. Lycium extract recovered activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) decreased by H(2)O(2). Lycium extract decreased DNA damage, lipid peroxidation and protein carbonyl values increased by H(2)O(2) exposure. In addition, Lycium extract increased the cell viability of Chang liver cells exposed to H(2)O(2) via inhibition of apoptosis. These results show that Lycium extract protected Chang liver cells against oxidative stressed cell damage by H(2)O(2) via scavenging ROS and enhancing antioxidant enzyme activity.

Compound K, a metabolite of ginseng saponin, induces mitochondria-dependent and caspase-dependent apoptosis via the generation of reactive oxygen species in human colon cancer cells.

The objective of this study was to elucidate the cytotoxic mechanism of Compound K, with respect to the involvement of reactive oxygen species (ROS) and the mitochondrial involved apoptosis, in HT-29 human colon cancer cells. Compound K exhibited a concentration of 50% growth inhibition (IC50) at 20 μg/mL and cytotoxicity in a time dependent manner. Compound K produced intracellular ROS in a time dependent fashion; however, N-acetylcysteine (NAC) pretreatment resulted in the inhibition of this effect and the recovery of cell viability. Compound K induced a mitochondria-dependent apoptotic pathway via the modulation of Bax and Bcl-2 expressions, resulting in the disruption of the mitochondrial membrane potential (Δψm). Loss of the Δψm was followed by cytochrome c release from the mitochondria, resulting in the activation of caspase-9, -3, and concomitant poly ADP-ribosyl polymerase (PARP) cleavage, which are the indicators of caspase-dependent apoptosis. The apoptotic effect of Compound K, exerted via the activation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), was abrogated by specific MAPK inhibitors. This study demonstrated that Compound K-mediated generation of ROS led to apoptosis through the modulation of a mitochondria-dependent apoptotic pathway and MAPK pathway.

7,8-Dihydroxyflavone protects human keratinocytes against oxidative stress-induced cell damage via the ERK and PI3K/Akt-mediated Nrf2/HO-1 signaling pathways.

This study investigated the effect of 7,8-dihydroxyflavone (DHF) on the expression and activity of heme oxygenase-1 (HO-1), an enzyme with potent antioxidant properties, as well as the molecular mechanisms involved. DHF markedly upregulated HO-1 mRNA and protein expression in human keratinocytes (HaCaT cells), resulting in increased HO-1 activity. DHF also increased the protein level of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates HO-1 expression by binding to the antioxidant response element (ARE) within the HO-1 gene promoter, in a time-dependent manner. Moreover, DHF decreased the expression of Kelch-like ECH-associated protein 1, a repressor of Nrf2 activity, and induced the translocation of Nrf2 from the cytosol into the nucleus, thereby allowing its association with the ARE site. DHF activated extracellular-regulated kinase (ERK) and protein kinase B (PKB, Akt) in keratinocytes, while the ERK and Akt inhibitors attenuated DHF-enhanced Nrf2 and HO-1 expression. DHF also protected the keratinocytes against hydrogen peroxide- and ultraviolet B-induced oxidative damage, while HO-1, ERK and Akt inhibitors markedly suppressed DHF-mediated cytoprotection. Taken together, the results suggested that DHF activates ERK- and Akt-Nrf2 signaling cascades in HaCaT cells, leading to the upregulation of HO-1 and cytoprotection against oxidative stress.

The Polyphenol Chlorogenic Acid Attenuates UVB-mediated Oxidative Stress in Human HaCaT Keratinocytes.

We investigated the protective effects of chlorogenic acid (CGA), a polyphenol compound, on oxidative damage induced by UVB exposure on human HaCaT cells. In a cell-free system, CGA scavenged 1,1-diphenyl-2-picrylhydrazyl radicals, superoxide anions, hydroxyl radicals, and intracellular reactive oxygen species (ROS) generated by hydrogen peroxide and ultraviolet B (UVB). Furthermore, CGA absorbed electromagnetic radiation in the UVB range (280–320 nm). UVB exposure resulted in damage to cellular DNA, as demonstrated in a comet assay; pre-treatment of cells with CGA prior to UVB irradiation prevented DNA damage and increased cell viability. Furthermore, CGA pre-treatment prevented or ameliorated apoptosis-related changes in UVB-exposed cells, including the formation of apoptotic bodies, disruption of mitochondrial membrane potential, and alterations in the levels of the apoptosis-related proteins Bcl-2, Bax, and caspase-3. Our findings suggest that CGA protects cells from oxidative stress induced by UVB radiation.

Inhibition of matrix metalloproteinase-1 induced by oxidative stress in human keratinocytes by mangiferin isolated from Anemarrhena asphodeloides.

Oxidative stress is related to the synthesis of matrix metalloproteinases (MMPs), which cause skin aging. The protective effects of mangiferin derived from Anemarrhena asphodeloides were investigated against hydrogen peroxide (H2O2)-induced damage using human skin keratinocyte (HaCaT) cells. Mangiferin was found to scavenge intracellular reactive oxygen species (ROS), superoxide radicals, and hydroxyl radicals. ROS regulate MMPs gene expression and activation of proenzymes. Mangiferin inhibited H2O2-induced MMP-1 gene expression and protein levels as well as its activity. Moreover, it abrogated mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway and stress-activated protein kinase/extracellular signal-regulated kinase (SEK)-c-JUN N-terminal kinase (JNK) pathway, which are induced by H2O2 treatment. And, it inhibited DNA binding activity of activator protein-1 (AP-1), a transcription factor of MMP-1 and downstream of ERK and JNK. Finally, it protected the human skin keratinocytes from H2O2-induced cell death. Taken together, these results indicate that mangiferin attenuated H2O2-induced MMP-1 activation via inhibition of ERK and JNK pathway and AP-1.