Ji Hoon Yu

Suppression of Cerulein-Induced Cytokine Expression by Antioxidants in Pancreatic Acinar Cells

Reactive oxygen species (ROS) has been considered to be an important regulator in the development and pathogenesis of pancreatitis and an activator of the transcription factor, nuclear factor-κB (NF-κB), regulating inflammatory cytokine gene expression. NF-κB activation was demonstrated in cerulein pancreatitis, which rapidly induces an acute, edematous form of pancreatitis. This study aimed to investigate whether cerulein induced ROS generation, lipid peroxide and hydrogen peroxide production, NF-κB activation, and expression of cytokines (IL-1β, IL-6) in pancreatic acinar cells. An additional aim was to establish whether these alterations were inhibited by antioxidants such as glutathione, superoxide dismutase, and catalase and an inhibitor of NF-κB activation, pyrrolidine dithiocarbamate (PDTC). To determine the possible interactions of the antioxidants and PDTC with cerulein-induced signaling, Ca2+ signal and amylase release were monitored in the pancreatic acinar cells treated with cerulein in the presence or absence of either the antioxidants or PDTC. The results showed that cerulein generated ROS and increased lipid peroxide and hydrogen peroxide production in the acinar cells, as determined by dichlorofluorescein diacetate dye. This resulted in NF-κB activation and the induction of cytokine gene expression in the cells. The cerulein-induced NF-κB activation was in parallel to IκBα degradation. Cerulein also induced Ca2+ signals and amylase release in acinar cells. Both antioxidants (glutathione, superoxide dismutase, catalase) and PDTC inhibited the cerulein-induced, oxidant-mediated alterations but did not affect the cerulein-evoked Ca2+ signals and amylase release in acinar cells. In conclusion, ROS, generated by cerulein, activates NF-κB, resulting in the up-regulation of inflammatory cytokine gene expression in acinar cells. NF-κB inhibition by scavenging ROS might alleviate the inflammatory response in pancreatic acinar cells by suppressing cytokine gene expression.

Oxidative stress and inflammatory signaling in cerulein pancreatitis.

Oxidative stress is considered to be an important regulator of the pathogenesis of acute pancreatitis. Reactive oxygen species (ROS) regulate the activation of inflammatory cascades, the recruitment of inflammatory cells and tissue damage in acute pancreatitis. A hallmark of the inflammatory response in pancreatitis is the induction of cytokine expression, which is regulated by a number of signaling molecules including oxidant-sensitive transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases (MAPKs). Cross-talk between ROS and pro-inflammatory cytokines is mediated by NF-κB, AP-1, STAT3, and MAPKs; this crosstalk amplifies the inflammatory cascade in acute pancreatitis. Therapeutic studies have shown that antioxidants and natural compounds can have beneficial effects for patients with pancreatitis and can also influence the expression of proinflammatory cytokines in cerulein-induced pancreatitis. Since oxidative stress may activate inflammatory signaling pathways and contribute to the development of pancreatitis, antioxidant therapy may alleviate the symptoms or prevent the development of pancreatitis. Since chronic administration of high doses of antioxidants may have deleterious effects, dosage levels and duration of antioxidant treatment should be carefully determined.

Mechanism of α-lipoic acid-induced apoptosis of lung cancer cells: Involvement of Ca2+

α‐Lipoic acid (LA) shows a protective effect on oxidative stress‐induced apoptosis while it induces apoptosis in various cancer cells. Intracellular Ca2+ plays a central role in triggering apoptotic pathways. In the present study, we aim to investigate whether LA induces apoptosis in lung cancer cells and whether Ca2+ is involved in LA‐induced apoptosis. We found that LA decreased cell viability and increased DNA fragmentation of the cells. LA activated the caspase‐independent pathway, determined by upregulation of poly(ADP‐ribose) polymerase (PARP) and increased the nuclear level of apoptosis‐inducing factor and caspase‐dependent apoptotic pathway, determined by increased levels of cytochrome c and PARP‐1 cleavage product. LA‐induced apoptotic alterations were inhibited in the cells treated with Ca2+ chelator BAPTA‐AM. In conclusion, LA induces apoptosis through caspase‐independent and caspase‐dependent pathways, which is mediated by intracellular Ca2+.

Role of NADPH oxidase and calcium in cerulein-induced apoptosis : Involvement of apoptosis-inducing factor

Abstract:  Apoptosis linked to oxidative stress has been implicated in pancreatitis. NADPH oxidase has been considered as a major source of reactive oxygen species (ROS) during inflammation and apoptosis in pancreatic acinar cells. Recently we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells and may contribute to apoptosis in pancreatic acinar AR42J cells stimulated with cerulein. The present study aims to investigate the apoptotic mechanism of pancreatic acinar cells stimulated with cerulein by determining whether cerulein induces apoptosis‐inducing factor (AIF) expression and whether cerulein‐induced expression of AIF is inhibited by transfection with antisense oligonucleotide (AS ODN) of p47phox or p67phox or treatment with a Ca2+ chelator BAPTA‐AM. As a result, cerulein induced the expression of apoptotic gene AIF. Transfection with AS ODN of p47phox or p67phox or treatment with BAPTA‐AM inhibited cerulein‐induced AIF expression in pancreatic acinar AR42J cells. These results demonstrate that NADPH oxidase and calcium have a role in cerulein‐induced apoptosis in pancreatic acinar AR42J cells by inducing the expression of AIF. In conclusion, the increase in intracellular Ca2+ and NADPH oxidase activity may be the upstream event of apoptotic gene (AIF) expression, which contributes to cerulein‐induced apoptosis in pancreatic acinar AR42 cells.

Oxidative stress and cytokines in the pathogenesis of pancreatic cancer.

Pancreatic cancer is one of the most aggressive, drug-resistant and lethal types of cancer with poor prognosis. Various factors including reactive oxygen species, cytokines, growth factors, and extracellular matrix proteins are reported to be involved in the development of pancreatic cancer. However, the pathogenesis of pancreatic cancer has not been completely elucidated. Oxidative stress has been shown to contribute to the development of pancreatic cancer. Evidences supporting the role of reactive oxygen species and cytokines as a risk for pancreatic cancer and the concept of antioxidant supplementation as a preventive approach for pancreatic cancer have been proposed. Here, we review the literature on oxidative stress, cytokine expression, inflammatory signaling, and natural antioxidant supplementation in relation to pancreatic cancer.

Role of Mitogen‐Activated Protein Kinases, NF‐κB, and AP‐1 on Cerulein‐Induced IL‐8 Expression in Pancreatic Acinar Cells

Abstract:  The cholecystokine (CCK) analogue cerulein causes pathophysiological, morphological, and biochemical events similar to various aspects of human pancreatitis. Doses of CCK or cerulein beyond those that cause the maximum pancreatic secretion of amylase and lipase result in pancreatitis, which is characterized by a dysregulation of the digestive enzyme production and cytoplasmic vacuolization and the death of acinar cells, edema formation, and an infiltration of inflammatory cells into the pancreas. This study aims to investigate whether cerulein induces IL‐8 expression in pancreatic acinar cells, and whether cerulein‐induced IL‐8 expression is inhibited in the cells transfected with mutant genes for c‐jun (TAM‐67), or IκBα (MAD‐3) or treated inhibitors of mitogen‐activated protein kinases (MAPKs). As a result, cerulein induced IL‐expression, which was inhibited in the cells transfected with TAM‐67 or MAD‐3 or treated inhibitors of MAPK. In conclusion, activation of MAPK, nuclear factor‐κB (NF‐κB), and activator protein‐1 (AP‐1) may be the upstream signaling for cerulein‐induced IL‐8 expression in pancreatic acinar cells.

Effects of omega-3 fatty acids on apoptosis of human gastric epithelial cells exposed to silica-immobilized glucose oxidase.

Oxidative stress plays a critical role in apoptosis of gastric epithelial cells. Omega‐3 fatty acids show anti‐inflammatory and/or anticancer effects and regulate apoptosis in various cells. In this study, we aimed to investigate whether omega‐3 fatty acids inhibit oxidative stress–induced apoptosis of gastric epithelial cells. The cells received oxidative stress caused by silica‐immobilized glucose oxidase acting on β‐d‐glucose and cultured in the absence or presence of α‐linolenic acid or docosahexanoic acid. Viable cell numbers, levels of H2O2 in the medium, DNA fragmentation, and protein levels of p53 and Bax were determined. As a result, silica‐immobilized glucose oxidase acting on β‐d‐glucose consistently and reproducibly produced H2O2, which decreased cell viability and increased DNA fragmentation of the cells. Omega‐3 fatty acids inhibited oxidative stress–induced cell death, DNA fragmentation, and induction of p53 and Bax of the cells. The silica‐immobilized glucose oxidase could be a useful tool for studies on oxidative stress–induced cellular events because it is reusable and forms a stable enzyme system acting on glucose. Omega‐3 fatty acids may be beneficial for preventing oxidative stress–induced apoptosis by inhibiting apoptotic gene expression and DNA fragmentation of gastric epithelial cells.

Calcium-dependent apoptotic gene expression in cerulein-treated AR42J cells.

Abstract: Elevated Ca2+ concentrations within the pancreatic acinar cells represent a risk factor for the development of acute pancreatitis. Apoptosis is an important characteristic of pancreatitis, with induction of apoptotic genes and intraceullar increase of calcium, endonucleases, and protease. The present study, which aims to investigate whether (1) cerulein induces apoptotic gene expression (bax, bid, p53) in pancreatic acinar AR42J cells and (2) cerulein‐induced gene expression is mediated by intracellular Ca2+, monitored the gene expression profile in the cells treated with the Ca2+ chelator BAPTA‐AM. Results showed that cerulein (10−7 M) evoked an initial peak Ca2+ signal; a further Ca2+ signal was induced with second treatment of cerulein. Cerulein‐induced Ca2+ signal could not be detected in the cells treated with the Ca2+ chelator BAPTA‐AM. Cerulein dose‐dependently induced apoptosis, determined by DNA fragmentation and pro‐apoptotic bid expression in AR42J cells. Cerulein induced bid, bax, and p53 mRNA expression, which was inhibited in the cells treated with cerulein and cultured in the presence of BAPTA‐AM. The present results suggest that increase in the free cytosolic Ca2+ may be the upstream event of apoptotic gene (bax, bid, p53) expression, which contribute to cerulein‐induced apoptosis in pancreatic acinar cells.

Ataxia telangiectasia mutated inhibits oxidative stress-induced apoptosis by regulating heme oxygenase-1 expression.

Ataxia telangiectasia (AT) is caused by mutational inactivation of the ataxia telangiectasia mutated (Atm) gene, which is involved in DNA repair. Increased oxidative stress has been shown in human AT cells and neuronal tissues of Atm-deficient mice. Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme and protects cells against oxidative stress. The purpose of this study is to determine whether ATM induces antioxidant enzyme HO-1 and protects cells from oxidative stress-mediated apoptosis by driving the activation of PKC-δ and NF-κB, by increasing cell viability, and by downregulating DNA fragmentation and apoptotic indicators (apoptosis-inducing factor and cleaved caspase-3). AT fibroblasts stably transfected with human full-length ATM cDNA (YZ5 cells) or the empty vector (MOCK cells) were treated with H2O2 as a source of reactive oxygen species (ROS). As a result, transfection with ATM inhibited ROS-induced cell death and DNA fragmentation in MOCK cells. Transfection with ATM induced expression of HO-1 which was mediated by PKC-δ and NF-κB in H2O2-treated MOCK cells. ZnPP, an HO-1 inhibitor, and transfection with HO-1 siRNA increased ROS levels and apoptosis, whereas hemin, an HO-1 activator, reduced ROS levels and apoptosis in H2O2-treated YZ5 cells. Rottlerin, a PKC-δ inhibitor, inhibited NF-κB activation and HO-1 expression in H2O2-treated YZ5 cells. MOCK cells showed increased cell death, DNA fragmentation, and apoptotic indicators compared to YZ5 cells exposed to H2O2. In addition, transfection with p65 siRNA increased ROS levels and DNA fragmentation, but decreased HO-1 protein levels in H2O2-treated YZ5 cells. In conclusion, ATM induces HO-1 expression via activation of PKC-δ and NF-κB and inhibits oxidative stress-induced apoptosis. A loss of HO-1 induction may explain why AT patients are vulnerable to oxidative stress.

Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts

ObjectiveTo 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).MaterialsWe 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.TreatmentThe cells were cultured with or without glutamine or GSH.MethodsROS 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.ResultsWhile 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.ConclusionGlutamine 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.