Lutein Induces Reactive Oxygen Species-Mediated Apoptosis in Gastric Cancer AGS Cells via NADPH Oxidase Activation.

Disruption of apoptosis leads to cancer cell progression; thus, anticancer agents target apoptosis of cancer cells. Reactive oxygen species (ROS) induce apoptosis by activating caspases and caspase-dependent DNase, leading to DNA fragmentation. ROS increase the expression of apoptotic protein Bax, which is mediated by activation of nuclear factor-κB (NF--κB). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of endogenous ROS, and its activation is involved in apoptosis. Lutein, an oxygenated carotenoid and known antioxidant, is abundant in leafy dark green vegetables, such as spinach and kale, and in yellow-colored foods, such as corn and egg yolk. High amounts of lutein increase ROS levels and exhibit anticancer activity. However, its anticancer mechanism remains unclear. This study aimed to determine whether lutein activates NADPH oxidase to produce ROS and induce apoptosis in gastric cancer AGS cells. Lutein increased ROS levels and promoted the activation of NADPH oxidase by increasing the translocation of NADPH oxidase subunit p47 phox to the cell membrane. It increased NF-κB activation and apoptotic indices, such as Bax, caspase-3 cleavage, and DNA fragmentation, and decreased Bcl-2, cell viability, and colony formation in AGS cells. The specific NADPH oxidase inhibitor ML171, and the known antioxidant N-acetyl cysteine reversed lutein-induced cell death, DNA fragmentation, and NF-κB DNA-binding activity in AGS cells. These results suggest that lutein-induced ROS production is dependent on NADPH oxidase, which mediates NF-κB activation and apoptosis in gastric cancer AGS cells. Therefore, lutein supplementation may be beneficial for increasing ROS-mediated apoptosis in gastric cancer cells.

Astaxanthin Inhibits Interleukin-6 Expression in Cerulein/Resistin-Stimulated Pancreatic Acinar Cells.

Acute pancreatitis is a common clinical condition with increasing the proinflammatory mediators, including interleukin-6 (IL-6). Obesity is a negative prognostic factor in acute pancreatitis. Obese patients with acute pancreatitis have a higher systemic inflammatory response rate. Levels of serum resistin, an adipocytokine secreted by fat tissues, increase with obesity. Cerulein, a cholecystokinin analog, induces calcium (Ca2+) overload, oxidative stress, and IL-6 expression in pancreatic acinar cells, which are hallmarks of acute pancreatitis. A recent study showed that resistin aggravates the expression of inflammatory cytokines in cerulein-stimulated pancreatic acinar cells. We aimed to investigate whether resistin amplifies cerulein-induced IL-6 expression and whether astaxanthin (ASX), an antioxidant carotenoid with anti-inflammatory properties, inhibits ceruelin/resistin-induced IL-6 expression in pancreatic acinar AR42J cells. We found that resistin enhanced intracellular Ca2+ levels, NADPH oxidase activity, intracellular reactive oxygen species (ROS) production, NF-κB activity, and IL-6 expression in cerulein-stimulated AR42J cells, which were inhibited by ASX in a dose-dependent manner. The calcium chelator BAPTA-AM inhibited cerulein/resistin-induced NADPH oxidase activation and ROS production. Antioxidant N-acetyl cysteine (NAC) and ML171, a specific NADPH oxidase 1 inhibitor, suppressed cerulein/resistin-induced ROS production, NF-κB activation, and IL-6 expression. In conclusion, ASX inhibits IL-6 expression, by reducing Ca2+ overload, NADPH oxidase-mediated ROS production, and NF-κB activity in cerulein/resistin-stimulated pancreatic acinar cells. Consumption of ASX-rich foods could be beneficial for preventing or delaying the incidence of obesity-associated acute pancreatitis.

Lycopene Inhibits Oxidative Stress-Mediated Inflammatory Responses in Ethanol/Palmitoleic Acid-Stimulated Pancreatic Acinar AR42J Cells.

High alcohol intake results in the accumulation of non-oxidative ethanol metabolites such as fatty acid ethyl esters (FAEEs) in the pancreas. High FAEE concentrations mediate pancreatic acinar cell injury and are associated with alcoholic pancreatitis. Treatment with ethanol and the fatty acid palmitoleic acid (EtOH/POA) increased the levels of palmitoleic acid ethyl ester and induced zymogen activation and cytokine expression in pancreatic acinar cells. EtOH/POA induces nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production and pancreatic acinar cell injury. Lycopene, a bright-red carotenoid, is a potent antioxidant due to its high number of conjugated double bands. This study aimed to investigate whether lycopene inhibits the EtOH/POA-induced increase in ROS production, zymogen activation, and expression of the inflammatory cytokine IL-6 in EtOH/POA-stimulated pancreatic acinar AR42J cells. EtOH/POA increased the ROS levels, NADPH oxidase and NF-κB activities, zymogen activation, IL-6 expression, and mitochondrial dysfunction, which were inhibited by lycopene. The antioxidant N-acetylcysteine and NADPH oxidase 1 inhibitor ML171 suppressed the EtOH/POA-induced increases in ROS production, NF-κB activation, zymogen activation, and IL-6 expression. Therefore, lycopene inhibits EtOH/POA-induced mitochondrial dysfunction, zymogen activation, and IL-6 expression by suppressing NADPH oxidase-mediated ROS production in pancreatic acinar cells.

ß-Carotene Inhibits Expression of Matrix Metalloproteinase-10 and Invasion in Helicobacter pylori-Infected Gastric Epithelial Cells.

Matrix metalloproteinases (MMPs), key molecules of cancer invasion and metastasis, degrade the extracellular matrix and cell-cell adhesion molecules. MMP-10 plays a crucial role in Helicobacter pylori-induced cell-invasion. The mitogen-activated protein kinase (MAPK) signaling pathway, which activates activator protein-1 (AP-1), is known to mediate MMP expression. Infection with H. pylori, a Gram-negative bacterium, is associated with gastric cancer development. A toxic factor induced by H. pylori infection is reactive oxygen species (ROS), which activate MAPK signaling in gastric epithelial cells. Peroxisome proliferator-activated receptor γ (PPAR-γ) mediates the expression of antioxidant enzymes including catalase. ß-Carotene, a red-orange pigment, exerts antioxidant and anti-inflammatory properties. We aimed to investigate whether ß-carotene inhibits H. pylori-induced MMP expression and cell invasion in gastric epithelial AGS (gastric adenocarcinoma) cells. We found that H. pylori induced MMP-10 expression and increased cell invasion via the activation of MAPKs and AP-1 in gastric epithelial cells. Specific inhibitors of MAPKs suppressed H. pylori-induced MMP-10 expression, suggesting that H. pylori induces MMP-10 expression through MAPKs. ß-Carotene inhibited the H. pylori-induced activation of MAPKs and AP-1, expression of MMP-10, and cell invasion. Additionally, it promoted the expression of PPAR-γ and catalase, which reduced ROS levels in H. pylori-infected cells. In conclusion, ß-carotene exerts an inhibitory effect on MAPK-mediated MMP-10 expression and cell invasion by increasing PPAR-γ-mediated catalase expression and reducing ROS levels in H. pylori-infected gastric epithelial cells.

Lycopene Inhibits Toll-Like Receptor 4-Mediated Expression of Inflammatory Cytokines in House Dust Mite-Stimulated Respiratory Epithelial Cells.

House dust mites (HDM) are critical factors in airway inflammation. They activate respiratory epithelial cells to produce reactive oxygen species (ROS) and activate Toll-like receptor 4 (TLR4). ROS induce the expression of inflammatory cytokines in respiratory epithelial cells. Lycopene is a potent antioxidant nutrient with anti-inflammatory activity. The present study aimed to investigate whether HDM induce intracellular and mitochondrial ROS production, TLR4 activation, and pro-inflammatory cytokine expression (IL-6 and IL-8) in respiratory epithelial A549 cells. Additionally, we examined whether lycopene inhibits HDM-induced alterations in A549 cells. The treatment of A549 cells with HDM activated TLR4, induced the expression of IL-6 and IL-8, and increased intracellular and mitochondrial ROS levels. TAK242, a TLR4 inhibitor, suppressed both HDM-induced ROS production and cytokine expression. Furthermore, lycopene inhibited the HDM-induced TLR4 activation and cytokine expression, along with reducing the intracellular and mitochondrial ROS levels in HDM-treated cells. These results collectively indicated that the HDM induced TLR4 activation and increased intracellular and mitochondrial ROS levels, thus resulting in the induction of cytokine expression in respiratory epithelial cells. The antioxidant lycopene could inhibit HDM-induced cytokine expression, possibly by suppressing TLR4 activation and reducing the intracellular and mitochondrial ROS levels in respiratory epithelial cells.

Korean Red Ginseng Inhibits Amyloid-ß-Induced Apoptosis and Nucling Expression in Human Neuronal Cells.

INTRODUCTION: The accumulation of amyloid-ß (Aß) plaque in the brain is a characteristic feature of Alzheimer's disease (AD) and the cause of fatal oxidative damage to neuronal cells. Korean red ginseng (RG) is used extensively in traditional medicine and is known to have anti-oxidative and anti-inflammatory properties. OBJECTIVE: This study aims to investigate whether Korean RG extract inhibits Aß-induced neuronal apoptosis. METHODS: Human neuronal cells (SH-SY5Y cells) were stimulated with Aß (5 µmol/L) and treated with RG dissolved in phosphate-buffered saline (0.2, 2, 20 µg/mL). RESULTS: RG suppressed the reduction of cell viability and the increase in apoptotic factors (Bax/Bcl-2 ratio and caspase-3 activity) in Aß-treated cells. RG suppressed Aß-induced increases in intracellular and mitochondrial reactive oxygen species (ROS) levels and mitochondrial dysfunction (determined by low mitochondrial membrane potential and oxygen consumption rate) in a dose-dependent manner. RG inhibited nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) activation and expression of the pro-apoptotic gene Nucling in Aß-treated cells. CONCLUSION: RG confers protection against neuronal apoptosis by reducing ROS levels and suppressing mitochondrial dysfunction and NF-κB activation, which results in suppression of NF-κB-mediated activation of Nucling expression in Aß-treated cells. Supplementation with RG may be beneficial for preventing Aß-induced neuronal cell death associated with AD.

Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

OBJECTIVE: To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). MATERIALS: We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. TREATMENT: The cells were cultured with or without glutamine or GSH. METHODS: ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. RESULTS: While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and cultured without glutamine showed higher levels of ROS and IL-8 than those transfected with negative control siRNA; increased levels of ROS and IL-8 were suppressed by the treatment of glutamine. CONCLUSION: Glutamine deprivation induces ROS production, NF-κB activation, and IL-8 expression as well as a reduction in GSH in A-T fibroblasts, all of which are attenuated by glutamine supplementation.

α-Lipoic acid inhibits Helicobacter pylori-induced oncogene expression and hyperproliferation by suppressing the activation of NADPH oxidase in gastric epithelial cells.

Hyperproliferation and oncogene expression are observed in the mucosa of Helicobacter pylori- (H. pylori-) infected patients with gastritis or adenocarcinoma. Expression of oncogenes such as ß-catenin and c-myc is related to oxidative stress. α-Lipoic acid (α-LA), a naturally occurring thiol compound, acts as an antioxidant and has an anticancer effect. The aim of this study is to investigate the effect of α-LA on H. pylori-induced hyperproliferation and oncogene expression in gastric epithelial AGS cells by determining cell proliferation (viable cell numbers, thymidine incorporation), levels of reactive oxygen species (ROS), NADPH oxidase activation (enzyme activity, subcellular levels of NADPH oxidase subunits), activation of redox-sensitive transcription factors (NF-κB, AP-1), expression of oncogenes (ß-catenin, c-myc), and nuclear localization of ß-catenin. Furthermore, we examined whether NADPH oxidase mediates oncogene expression and hyperproliferation in H. pylori-infected AGS cells using treatment of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase. As a result, α-LA inhibited the activation of NADPH oxidase and, thus, reduced ROS production, resulting in inhibition on activation of NF-κB and AP-1, induction of oncogenes, nuclear translocation of ß-catenin, and hyperproliferation in H. pylori-infected AGS cells. DPI inhibited H. pylori-induced activation of NF-κB and AP-1, oncogene expression and hyperproliferation by reducing ROS levels in AGS cells. In conclusion, we propose that inhibiting NADPH oxidase by α-LA could prevent oncogene expression and hyperproliferation occurring in H. pylori-infected gastric epithelial cells.

Anti-inflammatory mechanism of polyunsaturated fatty acids in Helicobacter pylori-infected gastric epithelial cells.

Helicobacter pylori is an important risk factor for gastric inflammation, which is mediated by multiple signaling pathways. The aim of this study was to investigate the effects of polyunsaturated fatty acids (PUFAs), such as linoleic acid (LA), alpha-linolenic acid (ALA), and docosahexaenoic acid (DHA), on the expression of the proinflammatory chemokine interleukin-8 (IL-8) in H. pylori-infected gastric epithelial AGS cells. To investigate whether PUFAs modulate H. pylori-induced inflammatory signaling, we determined the activation of epidermal growth factor receptor (EGFR), protein kinase C-δ (PKC δ), mitogen-activated protein kinases (MAPKs), nuclear factor-kappa B (NF- κB), and activator protein-1 (AP-1) as well as IL-8 expression in H. pylori-infected gastric epithelial cells that had been treated with or without PUFAs. We found that PUFAs inhibited IL-8 mRNA and protein expression in H. pylori-infected cells. ω-3 fatty acids (ALA, and DHA) suppressed the activation of EGFR, PKC δ, MAPK, NF- κB, and AP-1 in these infected cells. LA did not prevent EGFR transactivation and exhibited a less potent inhibitory effect on IL-8 expression than did ALA and DHA. In conclusion, PUFAs may be beneficial for prevention of H. pylori-associated gastric inflammation by inhibiting proinflammatory IL-8 expression.

Astaxanthin inhibits integrin α5 expression by suppressing activation of JAK1/STAT3 in Helicobacter pylori­stimulated gastric epithelial cells.

Integrins act as cell­matrix adhesion molecules involved in cell attachment to the extracellular matrix and generate signals that respond to cancer metastasis. Integrin α5ß1 is a heterodimer with α5 and ß1 subunits and mediates cell adhesion and migration of cancer cells. Integrins are transcriptionally regulated by the Janus kinase (JAK)/STAT signaling pathways. Our previous study revealed that Helicobacter pylori increased the levels of reactive oxygen species (ROS), which activate JAK1/STAT3 in gastric cancer AGS cells in vitro. Astaxanthin (ASX) has been reported to be an effective antioxidant and anticancer nutrient. The present study investigated whether ASX suppresses H. pylori­induced integrin α5 expression, cell adhesion and migration and whether ASX reduces ROS levels and suppresses phosphorylation of JAK1/STAT3 in gastric cancer AGS cells stimulated with H. pylori. The effect of ASX was determined using a dichlorofluorescein fluorescence assay, western blot analysis, adhesion assay and wound­healing assay in AGS cells stimulated with H. pylori. The results demonstrated that H. pylori increased the expression levels of integrin α5, without affecting integrin ß1, and increased cell adhesion and migration of AGS cells. ASX reduced ROS levels and suppressed JAK1/STAT3 activation, integrin α5 expression, cell adhesion and migration of H. pylori­stimulated AGS cells. In addition, both a JAK/STAT inhibitor, AG490, and an integrin α5ß1 antagonist, K34C, suppressed cell adhesion and migration in H. pylori­stimulated AGS cells. AG490 inhibited integrin α5 expression in AGS cells stimulated with H. pylori. In conclusion, ASX inhibited H. pylori­induced integrin α5­mediated cell adhesion and migration by decreasing the levels of ROS and suppressing JAK1/STAT3 activation in gastric epithelial cells.

Reactive oxygen species mediate Jak2/Stat3 activation and IL-8 expression in pulmonary epithelial cells stimulated with lipid-associated membrane proteins from Mycoplasma pneumoniae.

OBJECTIVE: To investigate the involvement of reactive oxygen species (ROS) in the activation of Janus kinase2 (Jak2)/signal transducers and activators of transcription3 (Stat3), and IL-8 expression in pulmonary epithelial cells stimulated with lipid-associated membrane proteins (LAMP) from Mycoplasma pneumoniae using a known antioxidant, N-acetylcysteine (NAC). METHODS: Pulmonary epithelial A549 cells were treated with or without NAC in the presence or absence of LAMP. Intracellular ROS levels were detected by fluorescent analysis for fluorescent dichlorofluorescein. mRNA expression of IL-8 was analyzed by reverse transcription-polymerase chain reaction. IL-8 protein in the medium was determined by enzyme-linked immunosorbent assay. Activation of Jak2/Stat3 was determined by the increases in phospho-specific forms of Jak2/Stat3 compared to total forms of Jak2/Stat3 by western blotting. Stat3-DNA binding activity was assessed by electrophoretic mobility shift assay. RESULTS: LAMP increased the level of ROS, phosphorylation of Jak2/Stat3, Stat3-DNA binding activity, and IL-8 expression in A549 cells, which were inhibited by NAC dose-dependently. CONCLUSION: LAMP of M. pneumoniae induces the production of ROS, Jak2/Stat3 activation, and IL-8 induction in A549 cells. Antioxidants such as NAC may be beneficial for preventing pulmonary inflammation caused by M. pneumoniae.

Lycopene Inhibits IL-6 Expression by Upregulating NQO1 and HO-1 via Activation of Nrf2 in Ethanol/Lipopolysaccharide-Stimulated Pancreatic Acinar Cells.

In alcoholic pancreatitis, alcohol increases gut permeability, which increases the penetration of endotoxins, such as lipopolysaccharides (LPS). LPS act as clinically significant triggers to increase pancreatic damage in alcoholic pancreatitis. Ethanol or LPS treatment increases reactive oxygen species (ROS) production in pancreatic acinar cells. ROS induce inflammatory cytokine production in pancreatic acinar cells, leading to pancreatic inflammation. The nuclear erythroid-2-related factor 2 (Nrf2) pathway is activated as a cytoprotective response to oxidative stress, and induces the expression of NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). Lycopene exerts anti-inflammatory and antioxidant effects in various cells. We previously showed that lycopene inhibits NADPH oxidase to reduce ROS and IL-6 levels, and zymogene activation in ethanol or palmitoleic acid-treated pancreatic acinar cells. In this study, we examined whether lycopene inhibits IL-6 expression by activating the Nrf2/NQO1-HO-1 pathway, and reducing intracellular and mitochondrial ROS levels, in ethanol and LPS-treated pancreatic AR42J cells. Lycopene increased the phosphorylated and nuclear-translocated Nrf2 levels by decreasing the amount of Nrf2 sequestered in the cytoplasm via a complex formation with Kelch-like ECH1-associated protein 1 (Keap1). Using exogenous inhibitors targeting Nrf2 and HO-1, we showed that the upregulation of activated Nrf2 and HO-1 results in lycopene-induced suppression of IL-6 expression and ROS production. The consumption of lycopene-rich foods may prevent the development of ethanol and LPS-associated pancreatic inflammation by activating Nrf2-mediated expression of NQO1 and HO-1, thereby decreasing ROS-mediated IL-6 expression in pancreatic acinar cells.

Lutein inhibits IL­6 expression by inducing PPAR­Î³ activation and SOCS3 expression in cerulein­stimulated pancreatic acinar cells.

Acute pancreatitis is a severe inflammatory disease of the pancreas. In experimental acute pancreatitis, cerulein induces the expression of interleukin­6 (IL­6) by activating Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 in pancreatic acinar cells. Ligands of peroxisome proliferator activated receptor­Î³ (PPAR­Î³) and suppressor of cytokine signaling (SOCS) 3 inhibit IL­6 expression by suppressing JAK2/STAT3 in cerulein­stimulated pancreatic acinar AR42J cells. Lutein, an oxygenated carotenoid, upregulates and activates PPAR­Î³ to regulate inflammation in a renal injury model. The present study aimed to determine whether lutein activated PPAR­Î³ and induced SOCS3 expression in unstimulated AR42J cells, and whether lutein inhibited activation of JAK2/STAT3 and IL­6 expression via activation of PPAR­Î³ and SOCS3 expression in cerulein­stimulated AR42J cells. The anti­inflammatory mechanism of lutein was determined using reverse transcription­quantitative PCR, western blot analysis and enzyme­linked immunosorbent assay in AR42J cells stimulated with or without cerulein. In another experiment, cells were treated with lutein and the PPAR­Î³ antagonist GW9662 or the PPAR­Î³ agonist troglitazone prior to cerulein stimulation to determine the involvement of PPAR­Î³ activation. The results indicated that lutein increased PPAR­Î³ and SOCS3 levels in unstimulated cells. Cerulein increased phospho­specific forms of JAK2 and STAT3, and mRNA and protein expression of IL­6, but decreased SOCS3 levels in AR42J cells. Cerulein­induced alterations were suppressed by lutein or troglitazone. GW9662 alleviated the inhibitory effect of lutein on JAK2/STAT3 activation and IL­6 expression in cerulein­stimulated cells. In conclusion, lutein inhibited the activation of JAK2/STAT3 and reduced IL­6 levels via PPAR­Î³­mediated SOCS3 expression in pancreatic acinar cells stimulated with cerulein.

Korean Red Ginseng Extract Inhibits IL-8 Expression via Nrf2 Activation in Helicobacter pylori-Infected Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) causes gastric diseases by increasing reactive oxygen species (ROS) and interleukin (IL)-8 expression in gastric epithelial cells. ROS and inflammatory responses are regulated by the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) and the expression of Nrf2 target genes, superoxide dismutase (SOD) and heme oxygenase-1 (HO-1). We previously demonstrated that Korean red ginseng extract (RGE) decreases H. pylori-induced increases in ROS and monocyte chemoattractant protein 1 in gastric epithelial cells. We determined whether RGE suppresses the expression of IL-8 via Nrf2 activation and the expression of SOD and HO-1 in H. pylori-infected gastric epithelial AGS cells. H. pylori-infected cells were treated with RGE with or without ML385, an Nrf2 inhibitor, or zinc protoporphyrin (ZnPP), a HO-1 inhibitor. Levels of ROS and IL-8 expression; abundance of Keap1, HO-1, and SOD; levels of total, nuclear, and phosphorylated Nrf2; indices of mitochondrial dysfunction (reduction in mitochondrial membrane potential and ATP level); and SOD activity were determined. As a result, RGE disturbed Nrf2-Keap1 interactions and increased nuclear Nrf2 levels in uninfected cells. H. pylori infection decreased the protein levels of SOD-1 and HO-1, as well as SOD activity, which was reversed by RGE treatment. RGE reduced H. pylori-induced increases in ROS and IL-8 levels as well as mitochondrial dysfunction. ML385 or ZnPP reversed the inhibitory effect of RGE on the alterations caused by H. pylori. In conclusion, RGE suppressed IL-8 expression and mitochondrial dysfunction via Nrf2 activation, induction of SOD-1 and HO-1, and reduction of ROS in H. pylori-infected cells.

Astaxanthin Inhibits Oxidative Stress-Induced Ku Protein Degradation and Apoptosis in Gastric Epithelial Cells.

Oxidative stress induces DNA damage which can be repaired by DNA repair proteins, such as Ku70/80. Excess reactive oxygen species (ROS) stimulate the activation of caspase-3, which degrades Ku 70/80. Cells with decreased Ku protein levels undergo apoptosis. Astaxanthin exerts antioxidant activity by inducing the expression of catalase, an antioxidant enzyme, in gastric epithelial cells. Therefore, astaxanthin may inhibit oxidative stress-induced DNA damage by preventing Ku protein degradation and thereby suppressing apoptosis. Ku proteins can be degraded via ubiquitination and neddylation which adds ubiquitin-like protein to substrate proteins. We aimed to determine whether oxidative stress decreases Ku70/80 expression through the ubiquitin-proteasome pathway to induce apoptosis and whether astaxanthin inhibits oxidative stress-induced changes in gastric epithelial AGS cells. We induced oxidative stress caused by the treatment of ß-D-glucose (G) and glucose oxidase (GO) in the cells. As a result, the G/GO treatment increased ROS levels, decreased nuclear Ku protein levels and Ku-DNA-binding activity, and induced the ubiquitination of Ku80. G/GO increased the DNA damage marker levels (γ-H2AX; DNA fragmentation) and apoptosis marker annexin V-positive cells and cell death. Astaxanthin inhibited G/GO-induced alterations, including Ku degradation in AGS cells. MLN4924, a neddylation inhibitor, and MG132, a proteasome inhibitor, suppressed G/GO-mediated DNA fragmentation and decreased cell viability. These results indicated that G/GO-induced oxidative stress causes Ku protein loss through the ubiquitin-proteasome pathway, resulting in DNA fragmentation and apoptotic cell death. Astaxanthin inhibited oxidative stress-mediated apoptosis via the reduction of ROS levels and inhibition of Ku protein degradation. In conclusion, dietary astaxanthin supplementation or astaxanthin-rich food consumption may be effective for preventing or delaying oxidative stress-mediated cell damage by suppressing Ku protein loss and apoptosis in gastric epithelial cells.

Astaxanthin Inhibits Matrix Metalloproteinase Expression by Suppressing PI3K/AKT/mTOR Activation in Helicobacter pylori-Infected Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) increases production of reactive oxygen species (ROS) and activates signaling pathways associated with gastric cell invasion, which are mediated by matrix metalloproteinases (MMPs). We previously demonstrated that H. pylori activated mitogen-activated protein kinase (MAPK) and increased expression of MMP-10 in gastric epithelial cells. MMPs degrade the extracellular matrix, enhancing tumor invasion and cancer progression. The signaling pathway of phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase B (AKT)/mammalian target of rapamycin (mTOR) is associated with MMP expression. ROS activates PIK3/AKT/mTOR signaling in cancer. Astaxanthin, a xanthophyll carotenoid, shows antioxidant activity by reducing ROS levels in gastric epithelial cells infected with H. pylori. This study aimed to determine whether astaxanthin inhibits MMP expression, cell invasion, and migration by reducing the PI3K/AKT/mTOR signaling in H. pylori-infected gastric epithelial AGS cells. H. pylori induced PIK3/AKT/mTOR and NF-κB activation, decreased IκBα, and induced MMP (MMP-7 and -10) expression, the invasive phenotype, and migration in AGS cells. Astaxanthin suppressed these H. pylori-induced alterations in AGS cells. Specific inhibitors of PI3K, AKT, and mTOR reversed the H. pylori-stimulated NF-κB activation and decreased IκBα levels in the cells. In conclusion, astaxanthin suppressed MMP expression, cell invasion, and migration via inhibition of PI3K/AKT/mTOR/NF-κB signaling in H. pylori-stimulated gastric epithelial AGS cells.

α­lipoic acid inhibits cerulein/resistin­induced expression of interleukin­6 by activating peroxisome proliferator­activated receptor­Î³ in pancreatic acinar cells.

Cerulein­induced pancreatitis resembles human acute pancreatitis in terms of pathological events, such as enzymatic activation and inflammatory cell infiltration in the pancreas. Cerulein is a cholecystokinin analog that increases levels of reactive oxygen species (ROS) and interleukin­6 (IL­6) expression level in pancreatic acinar cells. Serum levels of resistin, which is secreted from adipocytes, are reportedly higher in patients with acute pancreatitis than in healthy individuals. Previously, it was shown that the adipokine resistin can aggravate the cerulein­induced increase in ROS levels and IL­6 expression level in pancreatic acinar cells. Peroxisome proliferator­activated receptor­gamma (PPAR­Î³) is a key regulator of the transcription and expression of antioxidant enzymes, including heme oxygenase 1 (HO­1) and catalase. α­lipoic acid, a naturally occurring dithiol antioxidant, can prevent cerulein­induced pancreatic damage in rats. In the present study, it was aimed to investigate whether α­lipoic acid can attenuate the cerulein/resistin­induced increase in IL­6 expression and ROS levels via PPAR­Î³ activation in pancreatic acinar AR42J cells. The anti­inflammatory mechanism of α­lipoic acid was determined using reverse transcription­quantitative PCR, western blot analysis, enzyme­linked immunosorbent assay, immunofluorescence staining and fluorometry. Treatment with cerulein and resistin increased ROS levels and IL­6 expression level, which were inhibited by α­lipoic acid in pancreatic acinar cells. α­lipoic acid increased the nuclear translocation and expression level of PPAR­Î³ and the expression levels of its target genes: HO­1 and catalase. The PPAR­Î³ antagonist GW9662 and HO­1 inhibitor zinc protoporphyrin reversed the inhibitory effect of α­lipoic acid on cerulein/resistin­induced increase in ROS and IL­6 levels. In conclusion, α­lipoic acid inhibits the cerulein/resistin­induced increase in ROS production and IL­6 expression levels by activating PPAR­Î³ and inducing the expression of HO­1 and catalase in pancreatic acinar cells.

α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa and triggers various stomach diseases. H. pylori induces reactive oxygen species (ROS) production and DNA damage. The heterodimeric Ku70/Ku80 protein plays an essential role in the repair of DNA double-strand breaks (DSB). Oxidative stress stimulate apoptosis and DNA damage that can be repaired by Ku70/80. However, excessive reactive oxygen species (ROS) can cause Ku protein degradation, resulting in DNA fragmentation and apoptosis. α-lipoic acid (α-LA), which is found in organ meats such as liver and heart, spinach, broccoli, and potatoes, quenches free radicals, chelates metal ions, and reduces intracellular DNA damage induced by oxidative stress. Here, we investigated whether H. pylori decreases Ku70/80 and induces apoptosis, and whether α-LA inhibits changes induced by H. pylori. We analyzed ROS, DNA damage markers (γ-H2AX, DNA fragmentation), levels of Ku70/80, Ku-DNA binding activity, Ku80 ubiquitination, apoptosis indices (Bcl-2, Bax, apoptosis-inducing factor (AIF), and caspase-3), and viability in a human gastric epithelial adenocarcinoma cell line (AGS). H. pylori increased ROS, DNA damage markers, Ku80 ubiquitination, and consequently induced apoptosis. It also decreased nuclear Ku70/80 levels and Ku-DNA-binding activity; increased Bax expression, caspase-3 cleavage, and truncated AIF; but decreased Bcl-2 expression. These H. pylori-induced alterations were inhibited by α-LA. The antioxidant N-acetylcysteine and proteasome inhibitor MG-132 suppressed H. pylori-induced cell death and decreased nuclear Ku70/80 levels. The results show that oxidative stress induced Ku70/80 degradation via the ubiquitin-proteasome system, leading to its nuclear loss and apoptosis in H. pylori-infected cells. In conclusion, α-LA inhibited apoptosis induced by H. pylori by reducing ROS levels and suppressing the loss of Ku70/80 proteins in AGS cells.

Potential role of NADPH oxidase-mediated activation of Jak2/Stat3 and mitogen-activated protein kinases and expression of TGF-ß1 in the pathophysiology of acute pancreatitis.

OBJECTIVE: NADPH oxidase is potentially associated with acute pancreatitis by producing reactive oxygen species (ROS). We investigated whether NADPH oxidase mediates the activation of Janus kinase (Jak)2/signal transducers and activators of transcription (Stat)3 and mitogen-activated protein kinases (MAPKs) to induce the expression of transforming growth factor-ß1 (TGF-ß1) in cerulein-stimulated pancreatic acinar cells. TREATMENT: AR42J cells were treated with an NADPH oxidase inhibitor diphenyleneiodonium (DPI) or a Jak2 inhibitor AG490. Other cells were transfected with antisense or sense oligonucleotides (AS or S ODNs) for NADPH oxidase subunit p22(phox) or p47(phox). METHODS: TGF-ß1 was determined by enzyme-linked immonosorbent assay. STAT3-DNA binding activity was measured by electrophoretic mobility shift assay. Levels of MAPKs as well as total and phospho-specific forms of Jak1/Stat3 were assessed by Western blot analysis. RESULTS: Cerulein induced increases in TGF-ß1, Stat3-DNA binding activity and the activation of MAPKs in AR42J cells. AG490 suppressed these cerulein-induced changes, similar to inhibition by DPI. Cerulein-induced activation of Jak2/Stat3 and increases in MAPKs and TGF-ß1 levels were inhibited in the cells transfected with AS ODN for p22(phox) and p47(phox) compared to S ODN controls. CONCLUSION: Inhibition of NADPH oxidase may be beneficial for prevention and treatment of pancreatitis by suppressing Jak2/Stat3 and MAPKs and expression of TGF-ß1 in pancreatic acinar cells.

Diphenyleneiodonium inhibits the activation of mitogen-activated protein kinases and the expression of monocyte chemoattractant protein-1 in Helicobacter pylori-infected gastric epithelial AGS cells.

OBJECTIVE: To investigate whether NADPH oxidase induces MCP-1 expression and the activation of mitogen-activated protein kinases (MAPKs) in H. pylori-infected gastric epithelial cells. MATERIAL: H. pylori in Korean isolates, human gastric epithelial AGS cells TREATMENT: AGS cells pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) are cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. METHODS: Reactive oxygen species (ROS) and MCP-1 were determined by confocal microscopy and enzyme-linked immonosorbent assay. NADPH oxidase activity was measured by lucigenin assay. mRNA expression of MCP-1 was analyzed by reverse transcription-polymerase chain reaction. Levels of MAPKs were assessed by Western blot analysis. RESULTS: H. pylori induced increase in ROS, NADPH oxidase activity, MCP-1 expression, and the activation of MAPKs including extracellular signal-regulated kinases, p38, and jun N-terminal kinases in AGS cells, which was inhibited by DPI. CONCLUSION: Inhibiting NADPH oxidase by DPI suppresses H. pylori-induced activation of MAPKs and MCP-1 expression in AGS cells.