Publications by authors named "Reiko Akagi"

48 Publications

Heme oxygenase-1 induction by heat shock in rat hepatoma cell line is regulated by the coordinated function of HSF1, NRF2, AND BACH1.

J Biochem 2021 Jun 1. Epub 2021 Jun 1.

Department of Pharmacy, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University 1-1-1 Daigakudohri, Sanyo-onoda-shi 756-0884, Japan.

The mechanism of heme oxygenase-1 (HO-1) induction by heat shock (HS) loading remains unclear. Here, we investigated the contribution of transcription factors to HS-induced HO-1 expression, using a rat hepatoma cell line (H-4-II-E). Our results demonstrated that HS treatment resulted in a marked induction of HO-1. Immunohistochemical analysis showed a slight mismatch in the expression levels of HO-1 and HSP70 by HS among cells, suggesting a conflict between multiple induction mechanisms. We observed HS-induced nuclear localization of, not only phosphorylated HSF1, but also NRF2, which is a typical transcription factor activated by oxidative stress. HSF1 knockdown in H-4-II-E markedly reduced HO-1 induction by HS, while NRF2 knockdown resulted in a partial effect. The chromatin immunoprecipitation assay demonstrated that HS loading resulted in significant binding of HSF1 to the HSE in the promoter proximal region of HO-1 gene and another HSE located close to the MARE in the -4 kb upstream enhancer region 1, where NRF2 also bound, together with BACH1, a negative transcription factor of HO-1. These observations indicate that HO-1 induction by HS is mainly mediated by HSF1 binding to the proximal HSE. NRF2 binding to MARE by HS is predominantly suppressed by an increased binding of BACH1.
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http://dx.doi.org/10.1093/jb/mvab065DOI Listing
June 2021

Role of heme oxygenase-1 in human placenta on iron supply to fetus.

Placenta 2021 01 29;103:53-58. Epub 2020 Sep 29.

Department of Pharmacy, Faculty of Pharmacy, Yasuda Women's University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima-city, 731-0153, Japan. Electronic address:

Introduction: To date, details on how iron is supplied from the mother to the fetus through the placenta have remained unclear. Recently, increasing evidence has shown that heme oxygenase (HO)-1, which is an inducible isoform of the rate-limiting enzyme in the heme degradation pathway, may be involved in the effective reutilization of iron. In this study, we examined the distribution and gene expression of HO-1 in the villous tissue of human placenta at various periods of pregnancy.

Methods: Using the placenta of 38 samples for which consent was obtained, chronological changes in the localization of HO-1 protein were examined by histological examination. RT-PCR was also performed to examine the expression of HO-1, transferrin receptor-1, and ferroportin 1. Ferric iron in the tissues was analyzed by Prussian blue staining.

Results: Immunohistochemical studies showed that HO-1 protein was exclusively expressed in trophoblastic cells throughout gestation. In the miscarriage placenta in the first trimester, ho-1 mRNA levels were significantly higher than normal. Placenta with fetal death (miscarriage) in the first and second trimester indicate significantly higher ratio of ho-1 gene for iron production to the fpn-1 gene for iron excretion than normal. These suggest that the role of HO-1 with various physiological functions is changing throughout pregnancy.

Discussion: These findings suggest that HO-1 in placenta plays an important role in iron supplying system in the second trimester to support fetal development.
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http://dx.doi.org/10.1016/j.placenta.2020.09.065DOI Listing
January 2021

Visualization of the Redox Status of Cytosolic Glutathione Using the Organelle- and Cytoskeleton-Targeted Redox Sensors.

Antioxidants (Basel) 2020 Feb 3;9(2). Epub 2020 Feb 3.

Faculty of Pharmacy, Yasuda Women's University, Hiroshima 731-0153, Japan.

Glutathione is a small thiol-containing peptide that plays a central role in maintaining cellular redox homeostasis. Glutathione serves as a physiologic redox buffer by providing thiol electrons for catabolizing harmful oxidants and reversing oxidative effects on biomolecules. Recent evidence suggests that the balance of reduced and oxidized glutathione (GSH/GSSG) defines the redox states of Cys residues in proteins and fine-tunes their stabilities and functions. To elucidate the redox balance of cellular glutathione at subcellular resolution, a number of redox-sensitive green fluorescent protein (roGFP) variants have been developed. In this study, we constructed and functionally validated organelle- and cytoskeleton-targeted roGFP and elucidated the redox status of the cytosolic glutathione at a subcellular resolution. These new redox sensors firmly established a highly reduced redox equilibrium of cytosolic glutathione, wherein significant deviation was observed among cells. By targeting the sensor to the cytosolic and lumen sides of the Golgi membrane, we identified a prominent redox gradient across the biological membrane at the Golgi body. The results demonstrated that organelle- and cytoskeleton-targeted sensors enable the assessment of glutathione oxidation near the cytosolic surfaces of different organelle membranes.
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http://dx.doi.org/10.3390/antiox9020129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070464PMC
February 2020

Antitumor effect of palmitic acid-conjugated DsiRNA for colon cancer in a mouse subcutaneous tumor model.

Chem Biol Drug Des 2019 04 11;93(4):570-581. Epub 2019 Jan 11.

Department of Life Science, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan.

In this study, we synthesized Dicer-substrate siRNA conjugated with palmitic acid at the 5'-end of the sense strand (C16-DsiRNA), and examined its RNAi effect on β-catenin as a target gene in a colon cancer cell line, HT29Luc, both in vitro and in vivo. We examined the in vitro RNAi effect in HT29Luc cells and found that C16-DsiRNA strongly inhibited expression of the β-catenin gene in comparison with non-modified DsiRNA. Also, high membrane permeability of C16-DsiRNA was exhibited, and it was confirmed that most of the C16-DsiRNA was localized in cytoplasm of HT29Luc cells. In regard to the in vivo RNAi effect, C16-DsiRNA complexed with Invivofectamine targeting the β-catenin gene was locally administered to a subcutaneous tumor formed by implantation of HT29Luc cells into the subcutis of nude mice; we evaluated the effect by measuring the bioluminescence increase, which reflects tumor growth, using an in vivo imaging system. As a result, C16-DsiRNA strongly inhibited the growth of tumors formed in subcutis of nude mice compared with non-modified DsiRNA, and this in vivo RNAi effect lasted up to 15 days. Our results suggest that C16-DsiRNA should be vigorously pursued as a novel nucleic acid medicine for clinical treatment of cancer.
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http://dx.doi.org/10.1111/cbdd.13454DOI Listing
April 2019

NRF2 and HSF1 coordinately regulate heme oxygenase-1 expression.

Biochem Biophys Res Commun 2018 11 11;506(1):7-11. Epub 2018 Oct 11.

Department of Pharmacy, Yasuda Women's University, Japan.

Heme oxygenase-1 (HO-1) is an inducible enzyme responding to various stresses and has cytoprotective activities. Although HO-1 has been referred to as heat shock protein (HSP) 32, the heat-mediated induction of HO-1 varies among different species and cell lines. We examined the effects of heat shock on HO-1 expression in mouse embryonic fibroblast (MEF) cells deficient in heat shock factor 1 (HSF1) or nuclear factor-erythroid-2-related factor 2 (NRF2). Heme-induced expression of HO-1 was 2-fold higher in Hsf1 cells than in the wild-type cells at both mRNA and protein levels. In Nrf2 cells, heme-induced expression of HO-1 was not detected. In contrast, HO-1 expression was markedly induced by heat shock at 40-42 °C in Nrf2 cells while the wild-type cells were not responsive. The heat-induced expression of HO-1 in Nrf2 cells were almost completely diminished by transfection of siRNA against Hsf1 gene. These results suggest that HSF1 and NRF2 suppress heme-induced and heat-induced HO-1 expression, respectively.
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http://dx.doi.org/10.1016/j.bbrc.2018.10.030DOI Listing
November 2018

Local redox environment beneath biological membranes probed by palmitoylated-roGFP.

Redox Biol 2018 04 21;14:679-685. Epub 2017 Nov 21.

Department of Pharmacy, Yasuda Women's University, Hiroshima, Japan.

Production of reactive oxygen species (ROS) and consequent glutathione oxidation are associated with various physiological processes and diseases, including cell differentiation, senescence, and inflammation. GFP-based redox sensors provide a straight-forward approach to monitor ROS levels and glutathione oxidation within a living cell at the subcellular resolution. We utilized palmitoylated versions of cytosolic glutathione and hydrogen peroxide sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively) and demonstrated a unique redox environment near biological membranes. In HeLa cells, cytosolic glutathione was practically completely reduced (E = - 333mV) and hydrogen peroxide level was under the detectable range. In contrast, the cytoplasmic milieu near membranes of intracellular vesicles exhibited significant glutathione oxidation (E > - 256mV) and relatively high HO production, which was not observed for the plasma membrane. These vesicles colocalized with internalized EGFR, suggesting that HO production and glutathione oxidation are characteristics of cytoplasmic surfaces of the endocytosed vesicles. The results visually illustrate local redox heterogeneity within the cytosol for the first time.
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http://dx.doi.org/10.1016/j.redox.2017.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704182PMC
April 2018

Thiol-based copper handling by the copper chaperone Atox1.

IUBMB Life 2017 04 15;69(4):246-254. Epub 2017 Mar 15.

Department of Pharmacy, Yasuda Women's University, Yasuhigashi, Asaminami-ku, Hiroshima, Japan.

Human antioxidant protein 1 (Atox1) plays a crucial role in cellular copper homeostasis. Atox1 captures cytosolic copper for subsequent transfer to copper pumps in trans Golgi network, thereby facilitating copper supply to various copper-dependent oxidereductases matured within the secretory vesicles. Atox1 and other copper chaperones handle cytosolic copper using Cys thiols which are ideal ligands for coordinating Cu(I). Recent studies demonstrated reversible oxidation of these Cys residues in copper chaperones, linking cellular redox state to copper homeostasis. Highlighted in this review are unique redox properties of Atox1 and other copper chaperones. Also, summarized are the redox nodes in the cytosol which potentially play dominant roles in the redox regulation of copper chaperones. © 2016 IUBMB Life, 69(4):246-254, 2017.
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http://dx.doi.org/10.1002/iub.1620DOI Listing
April 2017

Prevention of Barrier Disruption by Heme Oxygenase-1 in Intestinal Bleeding Model.

Biol Pharm Bull 2016 ;39(6):1007-12

Department of Pharmacy, Faculty of Pharmacy, Yasuda Women's University.

In this study we investigated the effect of free heme, the local level of which was increased by bleeding, on the intestinal barrier function, using human epithelial colorectal adenocarcinoma cells (Caco-2). Our results show that the addition of hemin to the culture medium markedly disrupted the barrier function, which was significantly improved by glutamine supplementation. Although hemin treatment caused the increased expression of heme oxygenase (HO)-1, the inhibition of HO activity resulted in the aggravation of hemin-induced barrier dysfunction. Up-regulation of HO-1 by pretreatment with a low concentration of hemin almost completely prevented hemin-induced barrier dysfunction. Taken together, these observations indicate that an abnormally high level of intracellular free heme causes barrier dysfunction, probably through the modulation of proteins forming tight junctions.
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http://dx.doi.org/10.1248/bpb.b15-01028DOI Listing
January 2017

Nonpeptide neurotrophic agents useful in the treatment of neurodegenerative diseases such as Alzheimer's disease.

J Pharmacol Sci 2015 Feb 13;127(2):155-63. Epub 2015 Jan 13.

Faculty of Pharmacy, Yasuda Women's University, Yasuhigashi, Asaminami-ku, Hiroshima 731-0153, Japan.

Developed regions, including Japan, have become "aged societies," and the number of adults with senile dementias, such as Alzheimer's disease (AD), Parkinson's disease, and Huntington's disease, has also increased in such regions. Neurotrophins (NTs) may play a role in the treatment of AD because endogenous neurotrophic factors (NFs) prevent neuronal death. However, peptidyl compounds have been unable to cross the blood-brain barrier in clinical studies. Thus, small molecules, which can mimic the functions of NFs, might be promising alternatives for the treatment of neurodegenerative diseases. Natural products, such as or nutraceuticals or those used in traditional medicine, can potentially be used to develop new therapeutic agents against neurodegenerative diseases. In this review, we introduced the neurotrophic activities of polyphenols honokiol and magnolol, which are the main constituents of Magnolia obovata Thunb, and methanol extracts from Zingiber purpureum (BANGLE), which may have potential therapeutic applications in various neurodegenerative disorders.
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http://dx.doi.org/10.1016/j.jphs.2014.12.015DOI Listing
February 2015

Glutamine protects intestinal barrier function of colon epithelial cells from ethanol by modulating Hsp70 expression.

Pharmacology 2013 17;91(1-2):104-11. Epub 2013 Jan 17.

Department of Pharmacy, Faculty of Pharmacy, Yasuda Women's University, Hiroshima, Japan.

In this study, we investigated the protective effect of glutamine on barrier dysfunction induced by ethanol, by using human epithelial colorectal adenocarcinoma cells (Caco-2). Our results show that addition of glutamine to culture medium significantly improved the disruption of integrity caused by ethanol, which was associated with increased expression of heat shock protein 70 (Hsp70). Ethanol exposure moderately activates heat shock factor 1 (HSF1), which was characterized by increased DNA-binding activity and phosphorylation status of HSF1. Remarkably, glutamine treatment enhanced ethanol-mediated expression of Hsp70 and activation of HSF1. Up-regulation of Hsp70 by pretreatment with heat stress also promoted recovery from the ethanol-induced barrier dysfunction. Taken together, these observations indicate that glutamine protects the intestinal barrier function in Caco-2 cells, in part by modulating HSF1-mediated Hsp70 expression.
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http://dx.doi.org/10.1159/000345930DOI Listing
August 2013

Heme and non-heme iron transporters in non-polarized and polarized cells.

BMC Cell Biol 2010 Jun 4;11:39. Epub 2010 Jun 4.

Department of Molecular Genetics, Kawasaki Medical School, Okayama 701-0192, Japan.

Background: Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs), and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs.

Results: In this study, we analyzed the subcellular localizations of 2 isoenzymes of HOs, 4 isoforms of divalent metal transporter 1 (DMT1), and 2 candidate heme transporters--heme carrier protein 1 (HCP1) and heme responsive gene-1 (HRG-1)--in non-polarized and polarized cells. In non-polarized cells, HCP1, HRG-1, and DMT1A-I are located in the plasma membrane. In polarized cells, they show distinct localizations: HCP1 and DMT1A-I are located in the apical membrane, whereas HRG-1 is located in the basolateral membrane and lysosome. 16Leu at DMT1A-I N-terminal cytosolic domain was found to be crucial for plasma membrane localization. HOs are located in smooth endoplasmic reticulum and colocalize with NADPH-cytochrome P450 reductase.

Conclusions: HCP1 and DMT1A-I are localized to the apical membrane, and HRG-1 to the basolateral membrane and lysosome. These findings suggest that HCP1 and DMT1A-I have functions in the uptake of dietary heme and non-heme iron. HRG-1 can transport endocytosed heme from the lysosome into the cytosol. These localization studies support a model in which cytosolic heme can be degraded by HOs, and the resulting iron is exported into tissue fluids via the iron transporter ferroportin 1, which is expressed in the basolateral membrane in enterocytes or in the plasma membrane in macrophages. The liberated iron is transported by transferrin and reutilized for hemoglobin synthesis in the erythroid system.
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http://dx.doi.org/10.1186/1471-2121-11-39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3224662PMC
June 2010

Site-specific induction of intestinal hypoxia-inducible factor-1α after hemorrhagic shock.

Mol Med Rep 2009 Mar-Apr;2(2):149-52

Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama 700-8558, Japan.

The intestine is a major target organ in hemorrhagic shock (HS)-induced tissue injury. Hypoxia-inducible factor (HIF)-1α is the primary transcription factor responsible for regulating cellular response to changes in oxygen tension. Since HS is an acute hypoxic insult, the present study examined changes in the gene expression of HIF-1α in various regions of the intestine, as well as the distribution of HIF-1α protein in the intestinal cells of a rat model of HS. Levels of HIF-1α mRNA were marginally detectable in the intestine of sham-operated control animals, but obviously induced following HS. Duodenal, jejunal and colonic levels of HIF-1α mRNA robustly increased and reached a maximum during the ischemic phase of HS, followed by a rapid decrease almost to control levels during the early phase of resuscitation. The induction of HIF-1α mRNA was maximal in the duodenum. In contrast to the duodenum, jejunum and colon, in the ileum the HIF-1α mRNA level did not increase after HS. Consistent with enhanced HIF-1α gene expression, HIF-1α protein was expressed in the mucosal cells of the duodenum, jejunum and colon, but not in the ileum following HS. These findings indicate that intestinal HIF-1α expression was up-regulated at both the transcriptional and protein level in a site-specific manner in this rat model of HS. Differential regulation of HIF-1α expression along the longitudinal axes of the intestine might be a determinant of the adaptive response to HS-induced intestinal damage.
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http://dx.doi.org/10.3892/mmr_00000075DOI Listing
October 2012

Heme Oxygenase-1 is an Essential Cytoprotective Component in Oxidative Tissue Injury Induced by Hemorrhagic Shock.

J Clin Biochem Nutr 2009 Jan 27;44(1):28-40. Epub 2008 Dec 27.

Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama 700-8558, Japan.

Hemorrhagic shock causes oxidative stress that leads to tissue injuries in various organs including the lung, liver, kidney and intestine. Excess amounts of free heme released from destabilized hemoproteins under oxidative conditions might constitute a major threat because it can catalyze the formation of reactive oxygen species. Cells counteract this by rapidly inducing the rate-limiting enzyme in heme breakdown, heme oxygenase-1 (HO-1), which is a low-molecular-weight stress protein. The enzymatic HO-1 reaction removes heme. As such, endogenous HO-1 induction by hemorrhagic shock protects tissues from further degeneration by oxidant stimuli. In addition, prior pharmacological induction of HO-1 ameliorates oxidative tissue injuries induced by hemorrhagic shock. In contrast, the deletion of HO-1 expression, or the chemical inhibition of increased HO activity ablated the beneficial effect of HO-1 induction, and exacerbates tissue damage. Thus, HO-1 constitutes an essential cytoprotective component in hemorrhagic shock-induced oxidative tissue injures. This article reviews recent advances in understanding of the essential role of HO-1 in experimental models of hemorrhagic shock-induced oxidative tissue injuries with emphasis on the role of its induction in tissue defense.
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http://dx.doi.org/10.3164/jcbn.08-210-HODOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613496PMC
January 2009

Co-synthesis of Human delta-Aminolevulinate Dehydratase (ALAD) Mutants with the Wild-type Enzyme in Cell-free System-Critical Importance of Conformation on Enzyme Activity-.

J Clin Biochem Nutr 2008 Nov 31;43(3):143-53. Epub 2008 Oct 31.

Department of Nutritional Science, Okayama Prefectural University, 111 Kuboki, Soja-city 719-1197, Japan.

Properties of mutant delta-aminolevulinate dehydratase (ALAD) found in patients with ALAD porphyria were studied by enzymological and immunological analyses after the synthesis of enzyme complexes using a cell-free system. Enzyme activities of homozygous G133R, K59N/G133R, V153M, and E89K mutants were 11%, 22%, 67%, and 75% of the wild-type ALAD, respectively, whereas that of K59N, a normal variant, was 112%. Enzyme activities of L273R, C132R and F12L were undetectable. Co-synthesis of F12L, L273R, G133R, K59N/G133R, or C132R mutants with the wild-type at various ratios showed that ALAD activity was proportionally decreased in the amount of the wild-type in the complex. In contrast, co-synthesis of V153M, K59N, and E89K with the wild-type did not influence enzyme activity of the wild-type. Surface charge changes in K59N, E89K, C132R and G133R predicted by mutations were also confirmed by native polyacrylamide gel electrophoresis. A compound E89K and C132R complex showed ALAD activity similar to that was found in erythrocytes of the patient. These findings indicate that cell-free synthesis of ALAD proteins reflects enzymatic activities found in patients, and suggest that, in addition to the direct effect of mutations on the catalytic activity, conformational effects play an important role in determining enzyme activity.
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http://dx.doi.org/10.3164/jcbn.2008035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581755PMC
November 2008

Prevention of hemorrhagic shock-induced intestinal tissue injury by glutamine via heme oxygenase-1 induction.

Shock 2009 Jan;31(1):40-9

Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan.

Hemorrhagic shock (HS) is an oxidative stress that causes intestinal tissue injury. Heme oxygenase 1 (HO-1) is induced by oxidative stress and is thought to play an important role in the protection of tissues from oxidative injury. We previously reported the ileum to be the most susceptible to HS-induced tissue injury site in the intestine because HO-1 induction is the lowest at this site. We also previously demonstrated that glutamine (GLN) significantly induced HO-1 in the lower intestinal tract. In the present study, we investigated whether GLN pretreatment improves HS-induced intestinal tissue injury in the ileum by HO-1 induction. Treatment of rats with GLN (0.75 g/kg, i.v.) markedly induced functional HO-1 protein in mucosal epithelial cells in the ileum. Glutamine treatment before HS (MAP of 30 mmHg for 60 min) significantly ameliorated HS-induced mucosal inflammation and apoptotic cell death in the ileum, as judged by significant decreases in gene expression of TNF-alpha, iNOS, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1, myeloperoxidase activity, the number of infiltrated neutrophils, DNA fragmentation by in situ oligo ligation assay, and activated caspase-3 expression, and by increases in gene expression of IL-10 and Bcl-2. In contrast, treatment with tin mesoporphyrin, a specific inhibitor of HO activity, abolished the beneficial effect of GLN pretreatment. These findings indicate that GLN pretreatment significantly ameliorated tissue injury in the ileum after HS by inducing HO-1. Glutamine treatment may thus protect mucosal cells from HS-induced oxidative damage via the anti-inflammatory and antiapoptotic properties of HO-1.
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http://dx.doi.org/10.1097/SHK.0b013e318177823aDOI Listing
January 2009

Alpha-lipoic acid suppresses 6-hydroxydopamine-induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1.

Brain Res 2008 Apr 12;1206:1-12. Epub 2008 Feb 12.

Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikatacho, Okayama 700-8558, Japan.

We previously reported that the generation of reactive oxygen species (ROS) is the initial event in cell death induced by 6-hydroxydopamine (6-OHDA), an experimental model of Parkinsonism. Since recent studies suggested the important role of antioxidant activity of alpha-lipoic acid (LA) in the suppression of apoptosis of various types, we studied the effect on 6-OHDA-induced apoptosis of PC12 cells. Biochemical analysis revealed that LA suppressed the 6-OHDA-induced ROS generation, increase of caspase-like activity and chromatin condensation. The suppression of 6-OHDA-induced apoptosis by LA required pre-incubation of PC12 cells with LA for 12-24 h. LA increased the intracellular levels of heme oxygenase-1 (HO-1) and glutathione (GSH) and stimulated the expression of GSH synthesis-related genes such as cystine/glutamate antiporter and gamma-glutamylcysteine synthetase (gamma-GCS). However, Sn-mesoporphyrin IX, an inhibitor of HO-1, did not attenuate the LA-induced suppression of apoptosis. In contrast, buthionine sulfoximine, an inhibitor of gamma-GCS, attenuated the LA-induced suppression of ROS generation and chromatin condensation. In addition, a transcription factor Nrf2, which regulates the expression of antioxidant enzymes such as gamma-GCS, translocated to the nucleus by LA. These results suggested that LA suppressed the 6-OHDA induced-apoptosis by the increase in cellular glutathione through stimulation of the GSH synthesis system but not by the expression of HO-1.
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http://dx.doi.org/10.1016/j.brainres.2008.01.081DOI Listing
April 2008

[Metabolism of heme and novel drug design].

Authors:
Reiko Akagi

Nihon Yakurigaku Zasshi 2007 Oct;130(4):247

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October 2007

[Gene defects identified in porphyrias and their participation in onset of this disease].

Nihon Yakurigaku Zasshi 2007 Oct;130(4):266-9

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October 2007

Protective role of heme oxygenase 1 in the intestinal tissue injury in hemorrhagic shock in rats.

Shock 2008 Feb;29(2):252-61

Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan.

Heme oxygenase (HO) 1 is inducible by a variety of oxidative stress and is thought to play an important role in the protection of tissues from oxidative injuries. Because hemorrhagic shock (HS) is an oxidative stress that results in tissue injury, we examined in this study the role of HO-1 induction in intestinal tissue injuries in a rat model of HS. The levels of HO-1 were significantly increased after HS both at transcriptional and protein levels in mucosal epithelial cells in the duodenum, jejunum, and colon, whereas their expression in the ileum was hardly detectable and not increased at all by the treatment. In contrast, HS-induced mucosal inflammation and apoptotic cell death in the duodenum, jejunum, and colon were far less than those observed in ileum as judged by the levels of expression of TNF-alpha, iNOS, activated caspase 3, and Bcl-2. Of note, inhibition of HO activity by tin-mesoporphyrin resulted in an aggravation of HS-induced tissue inflammation and apoptotic cell death. These findings indicate that HO-1 expression in the intestine is regulated in a highly site-specific manner after HS, and that HO-1 induction plays a fundamental role in protecting mucosal cells of the intestine from oxidative damages induced by HS.
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http://dx.doi.org/10.1097/shk.0b013e3180cab913DOI Listing
February 2008

[Diversity of physiological functions mediated by heme metabolism].

Authors:
Reiko Akagi

Seikagaku 2007 May;79(5):441-5

Faculty of Health and Welfare Science, Okayama Prefectural University, Japan.

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May 2007

Upregulation of heme oxygenase-1 by degranulation in rat basophilic leukemia cells.

Biol Pharm Bull 2007 Mar;30(3):443-6

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University.

Heme oxygenase (HO)-1, which is a rate-limiting enzyme involved in the catabolism of heme, is upregulated by a variety of stresses including oxidative stresses and inflammatory cytokines, in many cell types. Recent studies have suggested that upregulation of HO-1 might provide cytoprotection and immunomodulatory functions in addition to its obvious role in heme metabolism. In this study, we examined whether HO-1 was upregulated following degranulation in mast cells that initiate vigorous immunity reactions. To trigger degranulation, rat basophilic leukemia (RBL)-2H3 cells were passively sensitized using an antiserum collected from ovalbumin (OA) immunized-Brown Norway rats, and the cells were stimulated by treatment with OA. Degranulation was confirmed by measuring the release of beta-hexosaminidase. HO-1 mRNA and presence of HO-1 protein were detected using Northern blot and Western blot analyses, respectively. The effect of the antioxidant N-acetyl-L-cysteine (NAC) on HO-1 expression was also tested. HO-1 mRNA transiently increased at 1--2 h after RBL-2H3 cells were stimulated to degranulate. Its mRNA increases were dependent on the extent of degranulation. Following the upregulation of HO-1 mRNA, HO-1 protein was also increased. We also detected intracellular production of reactive oxygen species following degranulation in RBL-2H3 cells. NAC attenuated the HO-1 expression in a dose-dependent manner. This is the first report to reveal induction of both HO-1 mRNA and protein by degranulation in RBL-2H3 cells. We showed that NAC inhibited HO-1 upregulation. These results suggest that oxidative stress in activated RBL-2H3 cells results in the upregulation of HO-1.
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http://dx.doi.org/10.1248/bpb.30.443DOI Listing
March 2007

Epicatechin conjugated with fatty acid is a potent inhibitor of DNA polymerase and angiogenesis.

Life Sci 2007 Apr 2;80(17):1578-85. Epub 2007 Feb 2.

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan.

Anti-cancer and anti-angiogenesis effects of green tea catechins have been demonstrated. It has been found that chemical modification of tea catechins improves their biological activities. We examined the chemical modification of epicatechin enhanced anti-cancer and anti-angiogenic effects. Epicatechin conjugated with fatty acid (acyl-catechin) strongly inhibited DNA polymerase activity, HL-60 cancer cell growth and angiogenesis. Epicatechin conjugated with palmitic acid ((2R,3R)-3',4',5,7-tetrahydroxyflavan-3-yl hexadecanoate, epicatechin-C16) was the strongest inhibitor in DNA polymerase alpha, beta, lambda and angiogenesis assays. Epicatechin-C16 also suppressed human endothelial cell (HUVEC) tube formation on reconstituted basement membrane, suggesting that it affected not only DNA polymerase activity but also the signal transduction pathways needed for the tube formation in HUVECs. These results suggest that acylation of epicatechin is an effective chemical modification to improve the anti-cancer activity of epicatechin.
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http://dx.doi.org/10.1016/j.lfs.2007.01.049DOI Listing
April 2007

Heme oxygenase-1 inhibits cytokine production by activated mast cells.

Biochem Biophys Res Commun 2007 Mar 10;354(2):485-90. Epub 2007 Jan 10.

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Nishihama-bouji, Tokushima-shi, Tokushima 770-8514, Japan.

Heme oxygenase-1 (HO-1) is thought to contribute to host defense reactions against various stresses. In addition, recent reports have suggested that HO-1 modulates immunocyte activation and functions. HO-1 suppresses mast cell degranulation, but whether HO-1 suppresses cytokine synthesis as well is not yet known. We examined whether rat HO-1 cDNA transfected rat basophilic leukemia (RBL)-2H3 cells have altered cytokine production in response to stimulation with anti-ovalbumin (OA) serum/OA compared to Mock transfected RBL-2H3 cells. HO-1 inhibited anti-OA serum/OA-induced IL-3 and TNF-alpha production. Inhibition of HO-1 activity by Zn (II) protoporphyrin IX, a specific HO-1 inhibitor, prevented the suppression of TNF-alpha production. The cytokine inhibition by HO-1 was associated with selective suppression of the DNA-binding activity of AP-1 transcription factors. The suppression of mast cell cytokine production by HO-1 may be an important aspect of the processes that lead to resolution of allergic inflammation.
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http://dx.doi.org/10.1016/j.bbrc.2006.12.228DOI Listing
March 2007

A neutrophil elastase inhibitor, sivelestat, ameliorates lung injury after hemorrhagic shock in rats.

Int J Mol Med 2007 Feb;19(2):237-43

Department of Anesthesiology and Resuscitology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.

Hemorrhagic shock followed by resuscitation (HSR) causes neutrophil sequestration in the lung which leads to acute lung injury (ALI). Neutrophil elastase (NE) is thought to play a pivotal role in the pathogenesis of ALI. This study investigated whether sivelestat, a specific NE inhibitor, can attenuate ALI induced by HSR in rats. Male Sprague-Dawley rats were subjected to hemorrhagic shock by withdrawing blood so as to maintain a mean arterial blood pressure of 30+/-5 mm Hg for 60 min followed by resuscitation with the shed blood. HSR-treated animals received a bolus injection of sivelestat (10 mg/kg) intravenously at the start of resuscitation followed by continuous infusion for 60 min (10 mg/kg/h) during the resuscitation phase, or the vehicle. Lung injury was assessed by pulmonary histology, lung wet-weight to dry-weight (W/D) ratio, myeloperoxidase (MPO) activity, gene expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS), DNA binding activity of nuclear factor (NF)-kappaB, and immunohistochemical analysis of intercellular adhesion molecule (ICAM)-1. HSR treatment induced lung injury, as demonstrated by pulmonary edema with infiltration of neutrophils, the increase in lung W/D ratio, MPO activity, gene expression of TNF-alpha and iNOS, and DNA-binding activity of NF-kappaB, and enhanced expression of ICAM-1. In contrast, sivelestat treatment significantly ameliorated the HSR-induced lung injury, as judged by the marked improvement in all these indices. These results indicate that sivelestat attenuated HSR-induced lung injury at least in part through an inhibition of the inflammatory signaling pathway, in addition to the direct inhibitory effect on NE.
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February 2007

Antiangiogenic activity of brown algae fucoxanthin and its deacetylated product, fucoxanthinol.

J Agric Food Chem 2006 Dec;54(26):9805-10

Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.

The antiangiogenic effects of fucoxanthin and a deacetylated product, fucoxanthinol, were examined. Fucoxanthin significantly suppressed HUVEC proliferation and tube formation at more than 10 microM, but it had no significant effect on HUVEC chemotaxis. The formation of blood vessel-like structures from CD31-positive cells was evaluated using embryonic stem cell-derived embryoid bodies. Fucoxanthin effectively suppressed the development of these structures at 10-20 microM, suggesting that it could suppress differentiation of endothelial progenitor cells into endothelial cells involving new blood vessel formation. Fucoxanthin and fucoxanthinol suppressed microvessel outgrowth in an ex vivo angiogenesis assay using a rat aortic ring, in a dose-dependent manner. These results imply that fucoxanthin having antiangiogenic activity might be useful in preventing angiogenesis-related diseases.
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http://dx.doi.org/10.1021/jf062204qDOI Listing
December 2006

Highly liver-specific heme oxygenase-1 induction by interleukin-11 prevents carbon tetrachloride-induced hepatotoxicity.

Int J Mol Med 2006 Oct;18(4):537-46

Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama 700-8558, Japan.

Heme oxygenase (HO)-1, the rate-limiting enzyme in heme catabolism, can be induced in response to various oxidative stimuli, and its induction is thought to be critical in the cellular defense against oxidative tissue injuries. Carbon tetrachloride (CCl(4)) treatment of rats causes lipid peroxidation of cell membranes and produces massive hepatic injury. We previously demonstrated that HO-1 induction following CCl(4) treatment is an essential part of the cellular defense against the CCl(4)-inducible toxic changes. As recombinant human interleukin-11 (rhIL-11) has been shown to induce HO-1 in cultured hepatoma cells, we examined the effect of rhIL-11 in vivo in rats on the CCl(4)-induced tissue injury. rhIL-11 treatment of animals by itself markedly induced HO-1 mRNA and its functional protein principally in the liver. rhIL-11 treatment (150 microg/kg) of the CCl(4)-administered (1 ml/kg) animals led to a further increase in HO-1 mRNA, while it markedly suppressed CCl(4)-induced serum alanine transaminase, hepatic malondialdehyde formation, tumor necrosis factor-alpha mRNA, nitric oxide synthase mRNA, nuclear factor-kappaB DNA-binding activity, as well as inflammatory changes of hepatocytes. In contrast, inhibition of HO activity by tin-mesoporphyrin, a competitive specific inhibitor of HO, entirely abolished the cytoprotective effect of rhIL-11. These findings thus demonstrate that rhIL-11 confers significant protection against CCl(4)-induced hepatic injury by virtue of its liver-specific HO-1 induction.
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October 2006

Heme oxygenase-1 protects gastric mucosal cells against non-steroidal anti-inflammatory drugs.

J Biol Chem 2006 Nov 31;281(44):33422-32. Epub 2006 Aug 31.

Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan.

Gastric mucosal cell death by non-steroidal anti-inflammatory drugs (NSAIDs) is suggested to be involved in NSAID-induced gastric lesions. Therefore, cellular factors that suppress this cell death are important for protection of the gastric mucosa from NSAIDs. Heme oxygenase-1 (HO-1) is up-regulated by various stressors and protects cells against stressors. Here, we have examined up-regulation of HO-1 by NSAIDs and the contribution of HO-1 to the protection of gastric mucosal cells against NSAIDs both in vitro and in vivo. In cultured gastric mucosal cells, all NSAIDs tested up-regulated HO-1. In rats, orally administered indomethacin up-regulated HO-1, induced apoptosis, and produced lesions at gastric mucosa. An inhibitor of HO-stimulated NSAID-induced apoptosis in vitro and in vivo and also stimulated NSAID-produced gastric lesions, suggesting that NSAID-induced up-regulation of HO-1 protects the gastric mucosa from NSAID-induced gastric lesions by inhibiting NSAID-induced apoptosis. Indomethacin activated the HO-1 promoter and caused nuclear accumulation of NF-E2-related factor 2 (Nrf2), a transcription factor for the HO-1 gene. Examination of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and experiments with its inhibitor strongly suggest that the nuclear accumulation of Nrf2 and resulting up-regulation of HO-1 by NSAIDs is mediated through NSAID-dependent activation (phosphorylation) of p38 MAPK. This is the first report showing the protective role of HO-1 against irritant-induced gastric lesions.
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http://dx.doi.org/10.1074/jbc.M602074200DOI Listing
November 2006

Catechin conjugated with fatty acid inhibits DNA polymerase and angiogenesis.

DNA Cell Biol 2006 Feb;25(2):95-103

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan.

Catechins in green tea have anticancer and antiangiogenesis activities, with epigallocatechin-3-gallate (EGCG) being the most potent antiangiogenic tea catechin. This study examined whether chemical modification of catechin enhanced anticancer and antiangiogenic effects. Catechin, conjugated with fatty acid (acyl-catechin), strongly inhibited DNA polymerase, HL-60 cancer cell growth, and angiogenesis. Catechin conjugated with stearic acid [(2R,3S)-3',4',5,7-tetrahydroxyflavan-3-yl octadecanoate; catechin-C18] was the strongest inhibitor in DNA polymerase alpha and beta and angiogenesis assays. Catechin-C18 also suppressed human endothelial cell (HUVEC) tube formation on the reconstituted basement membrane, suggesting that it affected not only DNA polymerases but also signal transduction pathways in HUVECs. These data indicate that acyl-catechins target both DNA polymerases and angiogenesis as anticancer agents. These results suggest that acylation of catechin is an effective chemical modification to improve the anticancer activity of catechin.
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http://dx.doi.org/10.1089/dna.2006.25.95DOI Listing
February 2006
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