Satoko Adachi

Astaxanthin Protects Mesangial Cells From Hyperglycemia-Induced Oxidative Signaling

Astaxanthin (ASX) is a carotenoid that has potent protective effects on diabetic nephropathy in mice model of type 2 diabetes. In this study, we investigated the protective mechanism of ASX on the progression of diabetic nephropathy using an in vitro model of hyperglycemia, focusing on mesangial cells. Normal human mesangial cells (NHMCs) were cultured in the medium containing normal (5 mM) or high (25 mM) concentrations of D‐glucose. Reactive oxygen species (ROS) production, the activation of nuclear transcription factors such as nuclear factor kappa B (NFκB) and activator protein‐1 (AP‐1), and the expression/production of transforming growth factor‐beta 1 (TGFβ1) and monocyte chemoattractant protein‐1 (MCP‐1) were evaluated in the presence or absence of ASX. High glucose (HG) exposure induced significant ROS production in mitochondria of NHMCs, which resulted in the activation of transcription factors, and subsequent expression/production of cytokines that plays an important role in the mesangial expansion, an important event in the pathogenesis of diabetic nephropathy. ASX significantly suppressed HG‐induced ROS production, the activation of transcription factors, and cytokine expression/production by NHMCs. In addition, ASX accumulated in the mitochondria of NHMCs and reduced the production of ROS‐modified proteins in mitochondria. ASX may prevent the progression of diabetic nephropathy mainly through ROS scavenging effect in mitochondria of mesangial cells and thus is expected to be very useful for the prevention of diabetic nephropathy. J. Cell. Biochem. 103: 1925–1937, 2007.

Lansoprazole, a Proton Pump Inhibitor, Mediates Anti-Inflammatory Effect in Gastric Mucosal Cells through the Induction of Heme Oxygenase-1 via Activation of NF-E2-Related Factor 2 and Oxidation of Kelch-Like ECH-Associating Protein 1

Induction of heme oxygenase-1 (HO-1) expression has been associated with cytoprotective and anti-inflammatory actions of lansoprazole, a proton pump inhibitor, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigate the role of transcriptional NF-E2-related factor 2 (Nrf2), its phosphorylation/activation, and oxidation of Kelch-like ECH-associating protein 1 (Keap1) in lansoprazole-induced HO-1 up-regulation using cultured gastric epithelial cells (rat gastric mucosal cell line, RGM-1). HO-1 expression of RGM-1 cells was markedly enhanced in a time- and dose-dependent manner by the treatment with lansoprazole, and this up-regulation of HO-1 contributed to the inhibition of chemokine production from stimulated RGM-1 cells. Transfection of Nrf2-siRNA suppressed the lansoprazole-induced HO-1. An electrophoretic mobility shift assay showed increases in the nuclear translocation and stress-response elements (StRE) binding activity of Nrf2 proteins in RGM-1 cells treated with lansoprazole. Furthermore, in RGM-1 cells transfected with HO-1 enhancer luciferase reporter plasmid containing mutant StRE, lansoprazole-induced HO-1 reporter gene activity was diminished. Lansoprazole promoted the phosphorylation of extracellular signal-regulated kinase (ERK), and lansoprazole-induced HO-1 up-regulation was suppressed by U0126, an ERK-specific inhibitor. Phosphorylated Nrf2 protein was detected in the phosphoprotein fraction purified by a Pro-Q Diamond Phosphoprotein Enrichment kit. Finally, an oxidative form of the Keap1 protein was detected in lansoprazole-treated RGM-1 cells by analyzing S-oxidized proteins using biotinylated cysteine as a molecular probe. These results indicate that lansoprazole up-regulates HO-1 expression in rat gastric epithelial cells, and the up-regulated HO-1 contributes to the anti-inflammatory effects of the drug. Phosphorylation of ERK and Nrf2, activation and nuclear translocation of Nrf2, and oxidation of Keap1 are all involved in the lansoprazole-induced HO-1 up-regulation.

Whole body hyperthermia improves obesity-induced insulin resistance in diabetic mice

Aim: In this study, we examined the efficacy of whole body hyperthermia (WBH) on obesity-induced insulin resistance in diabetic mice. Methods: Male db/db mice were treated with WBH 3 times per week for 12 weeks. The rectal temperature of mice reached 38.0°C 5 min after heating, and was kept at 38.0°C for 30 min. At the end of each week, tail snip glucose levels were determined under fasting conditions. The GLUT-4 gene expression of muscle tissue was analyzed by real-time PCR. Results: (1) WBH-treated db/db mice showed a significant decrease in fasting blood glucose level as compared with untreated db/db mice (p < 0.01). (2) Plasma insulin levels in untreated db/db mice at the age of 10 weeks were significantly increased compared with those of db/+ mice (p < 0.0001). On the other hand, the reduction (31%) in insulin levels in WBH-treated mice indicated improved insulin sensitivity. (3) The ability of WBH to increase insulin sensitivity was further established in glucose tolerance tests and insulin tolerance tests. (4) Urine albumin of db/db mice significantly increased compared with those of db/+ mice at 18 weeks of age (p < 0.001). This increase in urinary albumin was significantly inhibited by WBH (p < 0.01). (5) WBH up-regulated the expression of GLUT4 mRNA in skeletal muscle. Conclusion: Although the mechanisms have not yet been completely investigated, WBH may provide a new therapeutic or preventive modality against obesity-related diseases such as T2DM and metabolic or insulin resistance syndrome.

Effect of hyperthermia combined with gemcitabine on apoptotic cell death in cultured human pancreatic cancer cell lines

Background and aim: It is reported that NF-κB is activated by chemotherapy in some cancer cell lines and NF-κB activation is one of the mechanisms by which tumors are induced to become resistant to chemotherapy. We reported that heat-treatment-induced heat shock protein 70 (Hsp70) could inhibit I-kappa-B kinase, resulting in the inhibition of NF-κB activation. Therefore, we speculated that activated NF-κB in a pancreatic cell line might be inhibited by heat treatment, resulting in the enhancement of gemcitabine-induced cytotoxicity. Methods: We used the human pancreatic carcinoma cell lines AsPC-1 and MIAPaCa-2. Both cell lines were treated with various concentrations (0, 5, 10, 20, and 30 μM) of gemcitabine for 24 h. Heat treatment (43°C, 1 h) was performed at various times relative to gemcitabine treatment. The effect of gemcitabine and heat treatment on cell survival was determined by WST-8 assay. The status of NF-κB in carcinoma cells exposed to gemcitabine was investigated by electrophoretic mobility shift assay and immunocytochemistry. We analyzed apoptosis and necrosis in AsPC-1 and MIAPaCa-2 cells by flow cytometry. Furthermore, the levels of Hsp70, cyclin D1, caspase-3, and vascular endothelial growth factor in each treatment group were detected by western blotting. Results: (1) Significant cytotoxicity was observed with gemcitabine. (2) Gemcitabine activated NF-κB binding activity in both cell lines. (3) Heat treatment inhibited the gemcitabine-induced activation of NF-κB. (4) Heat treatment enhanced the cytotoxicity of gemcitabine, especially when heat treatment was performed 24 h before gemcitabine was given. (5) The levels of Hsp70 were increased by heat treatment. Gemcitabine did not affect the protein level of Hsp70. The levels of pro-caspase-3 were decreased by heat treatment combined with gemcitabine. Conclusions: Heat treatment inhibited gemcitabine-induced activation of NF-κB, resulting in the enhancement of the cytotoxicity of gemcitabine.

Involvement of reactive oxygen species in indomethacin-induced apoptosis of small intestinal epithelial cells

BackgroundThe precise pathogenic mechanism of nonsteroidal antiinflammatory drug-induced small intestinal injury is still unknown. In the present study, we investigated the mechanism by which indomethacin induced mucosal injury by using an in vitro model of small intestine.MethodsThe colon cancer cell line Caco-2, exhibiting a small intestinal phenotype starting as a crypt cell and differentiating to a villous phenotype, and RIE, a rat intestinal epithelial cell line, were employed. Indomethacin was added to differentiated the Caco-2 and RIE monolayer, and cell death was quantified by MTT assay and LDH release in the cell culture supernatant. Indomethacin-induced cell death was also qualified by fluorescent probes under the fluorescent microscope. As a functional study, the permeability of the Caco-2 monolayer was assessed by measuring transepithelial electrical resistance (TEER) and the flux of FITC-conjugated dextran across the monolayer. Indomethacin-induced reactive oxygen species production in Caco-2 and RIE was evaluated by redoxsensitive fluorogenic probes using a fluorometer. In some experiments, antioxidants were used to clarify the role of reactive oxygen species on indomethacin-induced Caco-2 cell death.ResultsIndomethacin caused cell death (mainly apoptosis) of Caco-2 and RIE in a dose-and time-dependent manner that was correlated with increased permeability of the Caco-2 monolayer. Exposure of Caco-2 and RIE with indomethacin also resulted in a significant reactive oxygen species production that was inhibited by the pretreatment of these cells with antioxidants.ConclusionsTaken together, reactive oxygen species production is one of the mechanisms by which indomethacin induced small intestinal injury.

Acetyl salicylic acid induces damage to intestinal epithelial cells by oxidation-related modifications of ZO-1

Acetyl salicylic acid (ASA) is one of the most frequently prescribed medications for the secondary prevention of cardiovascular and cerebrovascular events. It has recently been reported to cause small intestinal mucosal injury at a considerably higher rate than previously believed. The aim of this study is to investigate the mechanism by which this occurs using an in vitro small intestine model focusing on the role of oxidative stress and cell permeability. Differentiated Caco-2 exhibits a phenotype similar to human small intestinal epithelium. We measured whether ASA induced the increase of differentiated Caco-2 permeability, the decrease of tight junction protein expression, the production of reactive oxygen species (ROS), and the expression of ROS-modified zonula occludens-1 (ZO-1) protein. In some experiments, Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, a superoxide dismutase mimetic) was used. The nontoxic concentration of ASA decreased transepithelial electrical resistance and increased the flux of fluorescein isothiocyanate-conjugated dextran across Caco-2 in a time-dependent manner. The same concentration of ASA significantly decreased ZO-1 expression among TJ proteins as assessed by Western blot and immunocytochemistry and increased ROS production and the expression of oxidative stress-modified ZO-1 protein. However, MnTMPyP suppressed the ASA-induced increased intercellular permeability and the ASA-induced ROS-modified ZO-1 expression. Our findings indicate that ASA-induced ROS production can specifically modify the expression of ZO-1 protein and induce increased cell permeability, which may ultimately cause small intestinal mucosal injury.

Heat-Shock Protein 70-Overexpressing Gastric Epithelial Cells Are Resistant to Indomethacin-Induced Apoptosis

Background/Aims: Protecting intestinal mucosa from nonsteroidal anti-inflammatory drugs is still an unsolved problem. It has been revealed that apoptosis in epithelial cells as a result of mitochondrial injury is an important pathogenesis in indomethacin-induced gastric mucosal injury. In this study, we revealed the effect of overexpressed heat-shock protein 70 (HSP70) in indomethacin-induced apoptosis and oxidative stress. Methods: HSP70-overexpressing rat gastric mucosal cells (7018-RGM-1 cells) and control cells (pBK-CMV-12 cells) were used and treated with 0–500 μM of indomethacin for 24 h. Cell viability and cytotoxity were measured by a WST-8 assay and a lactate dehydrogenase release assay, respectively. Apoptosis was observed by fluorescence microscopy staining with Hoechst 33342 and propidium iodide. The expression of Bcl-2 family proteins, activation of caspase-3, and 4-hydroxy-2-nonenal (4-HNE)-modified proteins were assessed by Western blot analysis. Results: Indomethacin caused apoptosis of gastric epithelial cells. The 7018-RGM-1 cells survived significantly after indomethacin treatment compared to the control cells. The increase in pro-apoptotic Bad proteins, the decrease in anti-apoptotic Bcl-2 proteins, and caspase activation were all suppressed in the 7018-RGM-1 cells. A lower level of indomethacin-induced 4-HNE-modification was detected in the 7018-RGM-1 cells than in the control cells. Conclusion: Overexpressed HSP70 may potentiate resistance to apoptosis and oxidative stress in indomethacin-induced gastric epithelial cell injury.

Relationship between circulating cytokine levels and physical or psychological functioning in patients with advanced cancer.

OBJECTIVE To investigate the relation between functional impairments of cancer patients and circulating cytokines using a multiplex technique. DESIGN AND METHODS 50 patients with cancer were assessed using the quality of life (QOL) questionnaire. 27 plasma cytokine levels were determined by using the Bio-Plex array system. The relation to QOL scores was assessed using Chi-square test for categorical variables and univariate linear regression analysis for cytokine levels. RESULTS Multivariate analysis showed that interleukin-6 (IL-6) level is a significant independent determinant of physical (β=-0.238, P=0.0126) and cognitive functioning (β=-0.462, P=0.0006) and that vascular endothelial growth factor (VEGF) level is a significant independent determinant of emotional functioning (β=-0.414, P=0.039). CONCLUSION This study, in which 27 cytokines are simultaneously tested with cutting edge technology, demonstrates that plasma IL-6 and VEGF are significant independent determinants of functional impairments in patients with cancer.

Tumor inoculation site affects the development of cancer cachexia and muscle wasting

The phenotype and severity of cancer cachexia differ among tumor types and metastatic site in individual patients. In this study, we evaluated if differences in tumor microenvironment would affect the development of cancer cachexia in a murine model, and demonstrated that body weight, adipose tissue and gastrocnemius muscle decreased in tumor‐bearing mice. Interestingly, a reduction in heart weight was observed in the intraperitoneal tumor group but not in the subcutaneous group. We evaluated 23 circulating cytokines and members of the TGF‐β family, and found that levels of IL‐6, TNF‐α and activin A increased in both groups of tumor‐bearing mice. Eotaxin and G‐CSF levels in the intraperitoneal tumor group were higher than in the subcutaneous group. Atrogin 1 and MuRF1 mRNA expressions in the gastrocnemius muscle increased significantly in both groups of tumor‐bearing mice, however, in the myocardium, expression of these mRNAs increased in the intraperitoneal group but not in subcutaneous group. Based on these results, we believe that differences in microenvironment where tumor cells develop can affect the progression and phenotype of cancer cachexia through alterations in various circulating factors derived from the tumor microenvironment.

Ecabet sodium promotes the healing of trinitrobenzene-sulfonic-acid-induced ulceration by enhanced restitution of intestinal epithelial cells.

Background and Aims:  Ecabet sodium (ES) is a gastric mucosal protective and ulcer‐healing agent. Recently enema therapy with ES was found to be effective for the treatment of human ulcerative colitis as well as experimental colitis in an animal model. Whereas ES possesses potential as a novel treatment for ulcerative colitis, its precise mechanism of action remains to be elucidated. In this study, we investigated the therapeutic efficacy of ES in an experimental rat model of colitis, and evaluated the restitution of intestinal epithelial cells treated with ES in vitro.