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Das A, Sankaralingam M. Are Zn(II) pincer complexes efficient apoptosis inducers? a deep insight into their activity against A549 lung cancer cells. Dalton Trans 2023; 52:14465-14476. [PMID: 37772631 DOI: 10.1039/d3dt02419a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/30/2023]
Abstract
To expand the array of chemotherapeutic drugs, earth-abundant metal complexes are found to be the future direction. In this regard, new zinc(II) complexes 1-3 of 8-aminoquinoline-based pincer ligands were synthesized, characterized and tested for their anticancer activity. The IC50 values of these complexes were estimated by an MTT assay to be 16.35-17.95 μM and 33.35-40 μM against A549 lung and MCF-7 breast cancer cells respectively. Among them, 3 was slightly better than the other complexes and, thus, subjected to detailed studies. Moreover, the ligand corresponding to 3 was less active against both the cell lines than the complex. Further, 3 showed no toxicity against normal fibroblast cell line L929, which instantly elevated the drug characteristic of our complex. An AO-EB staining assay revealed that 3 can induce apoptosis in A549, and it was quantified by flow cytometry as 22.77%. Moreover, the depolarization of the mitochondrial membrane potential determined by JC-1 staining indicated excess ROS production sites in the mitochondria, which was confirmed by carboxy-H2DCFDA staining. Interestingly, the present complexes show better activity than that of the standard drug cisplatin against A549 cells. Overall, the studies provided promising results that can be extended for clinical applications.
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Affiliation(s)
- Athulya Das
- Bioinspired & Biomimetic Inorganic Chemistry Laboratory, Department of Chemistry, National Institute of Technology Calicut, Kozhikode-673601, Kerala, India.
| | - Muniyandi Sankaralingam
- Bioinspired & Biomimetic Inorganic Chemistry Laboratory, Department of Chemistry, National Institute of Technology Calicut, Kozhikode-673601, Kerala, India.
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Isolated Mitochondria State after Myocardial Ischemia-Reperfusion Injury and Cardioprotection: Analysis by Flow Cytometry. Life (Basel) 2023; 13:life13030707. [PMID: 36983862 PMCID: PMC10053810 DOI: 10.3390/life13030707] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Revised: 02/22/2023] [Accepted: 02/27/2023] [Indexed: 03/08/2023] Open
Abstract
Rationale: Mitochondria are key organelles involved in cell survival and death during the acute phenomena of myocardial ischemia-reperfusion (i.e., myocardial infarction). To investigate the functions of isolated mitochondria such as calcium retention capacity, oxidative phosphorylation, and reactive oxygen species (ROS) production, already established methods are based on extramitochondrial measurements of the whole mitochondria population. Objective: The aim of this study was to develop a reliable and well-characterized method for multiparametric analysis of isolated single mitochondrion by flow cytometry (FC) in the context of myocardial infarction. The advantage of FC is the possibility to give a simultaneous analysis of morphological parameters (side and forward scatters: SSC and FSC) for each mitochondrion, combined with intramitochondrial measurements of several biological markers, such as ROS production or membrane potential (Δφm), using specific fluorescent probes. Methods and Results: For this study, a rat model of ischemia-reperfusion and a protective approach of post-conditioning using low reperfusion pressure was used. Thanks to the use of specific probes (NAO, MTR, TMRM, DilC1, and DHR123) combined with flow cytometry, we propose a method: (i) to identify mitochondrial populations of interest based on quality criteria (NAO/TMRM double staining); (ii) to monitor their morphological criteria, especially during swelling due to calcium overload; and (iii) to compare mitochondrial functions (membrane potential and ROS production) in different experimental groups. Applied to mitochondria from ischemic hearts, these measurements revealed that individual mitochondria are altered and that cardioprotection by low-pressure reperfusion reduces damage, as expected. Conclusions: Our results highlight FC as a reliable and sensitive method to investigate changes in mitochondrial functions and morphology in pathological conditions that disrupts their activity such as the case in ischemia-reperfusion. This methodological approach can be extended to other pathologies involving mitochondrial dysfunctions. Moreover, FC offers the possibility to work with very small amounts of isolated mitochondria, a factor that may limit the use of classical methods.
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Shirmard LR, Shabani M, Moghadam AA, Zamani N, Ghanbari H, Salimi A. Protective Effect of Curcumin, Chrysin and Thymoquinone Injection on Trastuzumab-Induced Cardiotoxicity via Mitochondrial Protection. Cardiovasc Toxicol 2022; 22:663-675. [PMID: 35567651 DOI: 10.1007/s12012-022-09750-w] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/06/2022] [Accepted: 04/30/2022] [Indexed: 11/03/2022]
Abstract
Mitochondrial dysfunction may lead to cardiomyocyte death in trastuzumab (TZM)-induced cardiotoxicity. Accordingly, this study was designed to evaluate the mitochondrial protective effects of curcumin, chrysin and thymoquinone alone in TZM-induced cardiotoxicity in the rats. Forty-eight male adult Wistar rats were divided into eight groups: control group (normal saline), TZM group (2.5 mg/kg I.P. injection, daily), TZM + curcumin group (10 mg/kg, I.P. injection, daily), TZM + chrysin (10 mg/kg, I.P. injection, daily), TZM + thymoquinone (0.5 mg/kg, I.P. injection, daily), curcumin group (10 mg/kg, I.P. injection, daily), chrysin group (10 mg/kg, I.P. injection, daily) and thymoquinone group (10 mg/kg, I.P. injection, daily). Blood and tissue were collected on day 11 and used for assessment of creatine phosphokinase, lactate dehydrogenase (LDH), troponin, malondialdehyde (MDA) amount, glutathione levels and mitochondrial toxicity parameters. TZM increased mitochondrial impairments (reactive oxygen species formation, mitochondrial swelling, mitochondrial membrane potential collapse and decline in succinate dehydrogenase activity) and histopathological alterations (hypertrophy, enlarged cell, disarrangement, myocytes degeneration, infiltration of fat in some areas, hemorrhage and focal vascular thrombosis) in rat heart. As well as TZM produced a significant increase in the level of CK, LDH, troponin, MDA, glutathione disulfide. In most experiments, the co-injection of curcumin, chrysin and thymoquinone with TZM restored the level of CK, LDH, troponin, MDA, GSH, mitochondrial impairments and histopathological alterations. The study revealed the cardioprotective effects of curcumin, chrysin and thymoquinone against TZM-induced cardiotoxicity which could be attributed to their antioxidant and mitochondrial protection activities.
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Affiliation(s)
- Leila Rezaie Shirmard
- Department of Pharmaceutics, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran
| | - Mohammad Shabani
- Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, P.O. Box: 56189-53141, Ardabil, Iran
| | - Amin Ashena Moghadam
- Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, P.O. Box: 56189-53141, Ardabil, Iran
| | - Nasim Zamani
- Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, P.O. Box: 56189-53141, Ardabil, Iran
| | - Hadi Ghanbari
- Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, P.O. Box: 56189-53141, Ardabil, Iran
| | - Ahmad Salimi
- Department of Pharmacology and Toxicology, School of Pharmacy, Ardabil University of Medical Sciences, P.O. Box: 56189-53141, Ardabil, Iran. .,Traditional Medicine and Hydrotherapy Research Center, Ardabil University of Medical Sciences, Ardabil, Iran.
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Jakab Á, Emri T, Csillag K, Szabó A, Nagy F, Baranyai E, Sajtos Z, Géczi D, Antal K, Kovács R, Szabó K, Dombrádi V, Pócsi I. The Negative Effect of Protein Phosphatase Z1 Deletion on the Oxidative Stress Tolerance of Candida albicans Is Synergistic with Betamethasone Exposure. J Fungi (Basel) 2021; 7:jof7070540. [PMID: 34356919 PMCID: PMC8305657 DOI: 10.3390/jof7070540] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2021] [Revised: 06/30/2021] [Accepted: 07/02/2021] [Indexed: 12/28/2022] Open
Abstract
The glucocorticoid betamethasone (BM) has potent anti-inflammatory and immunosuppressive effects; however, it increases the susceptibility of patients to superficial Candida infections. Previously we found that this disadvantageous side effect can be counteracted by menadione sodium bisulfite (MSB) induced oxidative stress treatment. The fungus specific protein phosphatase Z1 (CaPpz1) has a pivotal role in oxidative stress response of Candida albicans and was proposed as a potential antifungal drug target. The aim of this study was to investigate the combined effects of CaPPZ1 gene deletion and MSB treatment in BM pre-treated C. albicans cultures. We found that the combined treatment increased redox imbalance, enhanced the specific activities of antioxidant enzymes, and reduced the growth in cappz1 mutant (KO) strain. RNASeq data demonstrated that the presence of BM markedly elevated the number of differentially expressed genes in the MSB treated KO cultures. Accumulation of reactive oxygen species, increased iron content and fatty acid oxidation, as well as the inhibiting ergosterol biosynthesis and RNA metabolic processes explain, at least in part, the fungistatic effect caused by the combined stress exposure. We suggest that the synergism between MSB treatment and CaPpz1 inhibition could be considered in developing of a novel combinatorial antifungal strategy accompanying steroid therapy.
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Affiliation(s)
- Ágnes Jakab
- Department of Molecular Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, 4032 Debrecen, Hungary; (T.E.); (K.C.); (A.S.); (D.G.); (I.P.)
- Correspondence:
| | - Tamás Emri
- Department of Molecular Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, 4032 Debrecen, Hungary; (T.E.); (K.C.); (A.S.); (D.G.); (I.P.)
| | - Kinga Csillag
- Department of Molecular Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, 4032 Debrecen, Hungary; (T.E.); (K.C.); (A.S.); (D.G.); (I.P.)
| | - Anita Szabó
- Department of Molecular Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, 4032 Debrecen, Hungary; (T.E.); (K.C.); (A.S.); (D.G.); (I.P.)
| | - Fruzsina Nagy
- Department of Medical Microbiology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary; (F.N.); (R.K.)
| | - Edina Baranyai
- Agilent Atomic Spectroscopy Partner Laboratory, Department of Inorganic and Analytical Chemistry, University of Debrecen, 4032 Debrecen, Hungary; (E.B.); (Z.S.)
| | - Zsófi Sajtos
- Agilent Atomic Spectroscopy Partner Laboratory, Department of Inorganic and Analytical Chemistry, University of Debrecen, 4032 Debrecen, Hungary; (E.B.); (Z.S.)
| | - Dóra Géczi
- Department of Molecular Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, 4032 Debrecen, Hungary; (T.E.); (K.C.); (A.S.); (D.G.); (I.P.)
| | - Károly Antal
- Department of Zoology, Faculty of Sciences, Eszterházy Károly University, 3300 Eger, Hungary;
| | - Renátó Kovács
- Department of Medical Microbiology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary; (F.N.); (R.K.)
- Faculty of Pharmacy, University of Debrecen, 4032 Debrecen, Hungary
| | - Krisztina Szabó
- Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary; (K.S.); (V.D.)
| | - Viktor Dombrádi
- Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary; (K.S.); (V.D.)
| | - István Pócsi
- Department of Molecular Biotechnology and Microbiology, Faculty of Science and Technology, University of Debrecen, 4032 Debrecen, Hungary; (T.E.); (K.C.); (A.S.); (D.G.); (I.P.)
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Wen Y, Long Z, Huo F, Yin C. Photoexcited molecular probes for selective and revertible imaging of cellular reactive oxygen species. Org Chem Front 2021. [DOI: 10.1039/d0qo01260b] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Redox homeostasis is key to maintaining the normal physiological status of living cells.
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Affiliation(s)
- Ying Wen
- Institute of Molecular Science
- Shanxi University
- Taiyuan
- P. R. China
| | - Zhiqing Long
- Institute of Molecular Science
- Shanxi University
- Taiyuan
- P. R. China
| | - Fangjun Huo
- Research Institute of Applied Chemistry
- Shanxi University
- Taiyuan
- P. R. China
| | - Caixia Yin
- Institute of Molecular Science
- Shanxi University
- Taiyuan
- P. R. China
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Kim HK, Jeon J, Song IS, Heo HJ, Jeong SH, Long LT, Thu VT, Ko TH, Kim M, Kim N, Lee SR, Yang JS, Kang MS, Ahn JM, Cho JY, Ko KS, Rhee BD, Nilius B, Ha NC, Shimizu I, Minamino T, Cho KI, Park YS, Kim S, Han J. Tetrahydrobiopterin enhances mitochondrial biogenesis and cardiac contractility via stimulation of PGC1α signaling. Biochim Biophys Acta Mol Basis Dis 2019; 1865:165524. [PMID: 31381993 DOI: 10.1016/j.bbadis.2019.07.018] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 07/10/2019] [Accepted: 07/29/2019] [Indexed: 02/05/2023]
Abstract
Tetrahydrobiopterin (BH4) shows therapeutic potential as an endogenous target in cardiovascular diseases. Although it is involved in cardiovascular metabolism and mitochondrial biology, its mechanisms of action are unclear. We investigated how BH4 regulates cardiovascular metabolism using an unbiased multiple proteomics approach with a sepiapterin reductase knock-out (Spr-/-) mouse as a model of BH4 deficiency. Spr-/- mice exhibited a shortened life span, cardiac contractile dysfunction, and morphological changes. Multiple proteomics and systems-based data-integrative analyses showed that BH4 deficiency altered cardiac mitochondrial oxidative phosphorylation. Along with decreased transcription of major mitochondrial biogenesis regulatory genes, including Ppargc1a, Ppara, Esrra, and Tfam, Spr-/- mice exhibited lower mitochondrial mass and severe oxidative phosphorylation defects. Exogenous BH4 supplementation, but not nitric oxide supplementation or inhibition, rescued these cardiac and mitochondrial defects. BH4 supplementation also recovered mRNA and protein levels of PGC1α and its target proteins involved in mitochondrial biogenesis (mtTFA and ERRα), antioxidation (Prx3 and SOD2), and fatty acid utilization (CD36 and CPTI-M) in Spr-/- hearts. These results indicate that BH4-activated transcription of PGC1α regulates cardiac energy metabolism independently of nitric oxide and suggests that BH4 has therapeutic potential for cardiovascular diseases involving mitochondrial dysfunction.
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Affiliation(s)
- Hyoung Kyu Kim
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Jouhyun Jeon
- Department of Life Science, POSTECH, Pohang 37673, Republic of Korea
| | - In-Sung Song
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Hae Jin Heo
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Seung Hun Jeong
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Le Thanh Long
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Vu Thi Thu
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Tae Hee Ko
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Min Kim
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Nari Kim
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Sung Ryul Lee
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Jae-Seong Yang
- Department of Life Science, POSTECH, Pohang 37673, Republic of Korea
| | - Mi Seon Kang
- Department of Pathology, Inje University, Busan 47392, Republic of Korea
| | - Jung-Mo Ahn
- Department of Veterinary Biochemistry, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea
| | - Je-Yoel Cho
- Department of Veterinary Biochemistry, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea
| | - Kyung Soo Ko
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Byoung Doo Rhee
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea
| | - Bernd Nilius
- KU Leuven, Department of Cellular and Molecular Medicine, Leuven 3000, Belgium
| | - Nam-Chul Ha
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea
| | - Ippei Shimizu
- Department of Cardiovascular Biology and Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan
| | - Tohru Minamino
- Department of Cardiovascular Biology and Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan
| | - Kyoung Im Cho
- Division of Cardiology, Department of Internal Medicine, College of Medicine Kosin, University Busan, Republic of Korea
| | - Young Shik Park
- School of Biotechnology and Biomedical Science, Inje University, Kimhae 50834, Republic of Korea.
| | - Sanguk Kim
- Department of Life Science, POSTECH, Pohang 37673, Republic of Korea.
| | - Jin Han
- Cardiovascular and Metabolic Disease Center, Inje University, Busan 47392, Republic of Korea.
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Can K, Menzfeld C, Rinne L, Rehling P, Kügler S, Golubiani G, Dudek J, Müller M. Neuronal Redox-Imbalance in Rett Syndrome Affects Mitochondria as Well as Cytosol, and Is Accompanied by Intensified Mitochondrial O 2 Consumption and ROS Release. Front Physiol 2019; 10:479. [PMID: 31114506 PMCID: PMC6503037 DOI: 10.3389/fphys.2019.00479] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2018] [Accepted: 04/05/2019] [Indexed: 12/31/2022] Open
Abstract
Rett syndrome (RTT), an X chromosome-linked neurodevelopmental disorder affecting almost exclusively females, is associated with various mitochondrial alterations. Mitochondria are swollen, show altered respiratory rates, and their inner membrane is leaking protons. To advance the understanding of these disturbances and clarify their link to redox impairment and oxidative stress, we assessed mitochondrial respiration in defined brain regions and cardiac tissue of male wildtype (WT) and MeCP2-deficient (Mecp2-/y ) mice. Also, we quantified for the first time neuronal redox-balance with subcellular resolution in cytosol and mitochondrial matrix. Quantitative roGFP1 redox imaging revealed more oxidized conditions in the cytosol of Mecp2-/y hippocampal neurons than in WT neurons. Furthermore, cytosol and mitochondria of Mecp2-/y neurons showed exaggerated redox-responses to hypoxia and cell-endogenous reactive oxygen species (ROS) formation. Biochemical analyzes exclude disease-related increases in mitochondrial mass in Mecp2-/y hippocampus and cortex. Protein levels of complex I core constituents were slightly lower in Mecp2-/y hippocampus and cortex than in WT; those of complex V were lower in Mecp2-/y cortex. Respiratory supercomplex-formation did not differ among genotypes. Yet, supplied with the complex II substrate succinate, mitochondria of Mecp2-/y cortex and hippocampus consumed more O2 than WT. Furthermore, mitochondria from Mecp2-/y hippocampus and cortex mediated an enhanced oxidative burden. In conclusion, we further advanced the molecular understanding of mitochondrial dysfunction in RTT. Intensified mitochondrial O2 consumption, increased mitochondrial ROS generation and disturbed redox balance in mitochondria and cytosol may represent a causal chain, which provokes dysregulated proteins, oxidative tissue damage, and contributes to neuronal network dysfunction in RTT.
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Affiliation(s)
- Karolina Can
- Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Georg-August-University Göttingen, Göttingen, Germany
- Zentrum Physiologie und Pathophysiologie, Institut für Neuro- und Sinnesphysiologie, Universitätsmedizin Göttingen, Georg-August-Universität Göttingen, Göttingen, Germany
| | - Christiane Menzfeld
- Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Georg-August-University Göttingen, Göttingen, Germany
- Zentrum Physiologie und Pathophysiologie, Institut für Neuro- und Sinnesphysiologie, Universitätsmedizin Göttingen, Georg-August-Universität Göttingen, Göttingen, Germany
| | - Lena Rinne
- Zentrum Physiologie und Pathophysiologie, Institut für Neuro- und Sinnesphysiologie, Universitätsmedizin Göttingen, Georg-August-Universität Göttingen, Göttingen, Germany
| | - Peter Rehling
- Zentrum Biochemie und Molekulare Zellbiologie, Institut für Zellbiochemie, Universitätsmedizin Göttingen, Georg-August-Universität Göttingen, Göttingen, Germany
| | - Sebastian Kügler
- Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Georg-August-University Göttingen, Göttingen, Germany
- Klinik für Neurologie, Universitätsmedizin Göttingen, Georg-August-Universität Göttingen, Göttingen, Germany
| | - Gocha Golubiani
- Zentrum Physiologie und Pathophysiologie, Institut für Neuro- und Sinnesphysiologie, Universitätsmedizin Göttingen, Georg-August-Universität Göttingen, Göttingen, Germany
- Institute of Chemical Biology, Ilia State University, Tbilisi, Georgia
| | - Jan Dudek
- Zentrum Biochemie und Molekulare Zellbiologie, Institut für Zellbiochemie, Universitätsmedizin Göttingen, Georg-August-Universität Göttingen, Göttingen, Germany
| | - Michael Müller
- Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Georg-August-University Göttingen, Göttingen, Germany
- Zentrum Physiologie und Pathophysiologie, Institut für Neuro- und Sinnesphysiologie, Universitätsmedizin Göttingen, Georg-August-Universität Göttingen, Göttingen, Germany
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Liu Y, Zhi D, Wang X, Fei D, Zhang Z, Wu Z, Li Y, Chen P, Li H. Kushui Rose (R. Setate x R. Rugosa) decoction exerts antitumor effects in C. elegans by downregulating Ras/MAPK pathway and resisting oxidative stress. Int J Mol Med 2018; 42:1411-1417. [PMID: 29956725 PMCID: PMC6089776 DOI: 10.3892/ijmm.2018.3738] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2016] [Accepted: 01/25/2018] [Indexed: 12/31/2022] Open
Abstract
Kushui rose (R. Setate x R. Rugosa) (KR) is a traditional Chinese medicine proven to be a potent antioxidant, and used for thousands of years. Approximately 30% of all human cancers relevant to mutational activated Ras, and over-activated Ras are accompanied by increased accumulation of reactive oxygen species (ROS). Thus, one way of developing anticancer drugs is to reduce ROS accumulation. Therefore, KR was predicted to have potential to combat over-activated Ras-related cancer. C. elegans with let‑60(gf)/ras mutant, which exhibited tumor-like symptoms of the multivulva phenotype, was employed to determine the effect of KR on Ras/MAPK pathway. Other strains of worms and H2DCF-DA dye were also applied to study the antioxidant stress capacity of KR. This study was aimed to determine whether KR has a potential effect on combat over-activated Ras-related cancer through resistance to oxidative stress. Our results showed that Kushui rose decoction (KRD) has potent antioxidant activity in vitro, and can inhibit over-activated Ras in vivo. Further, KRD significantly suppressed over-activated Ras/MAPK pathway by regulating oxidative stress-related proteins, such as forkhead transcription factor (DAF-16), glutathione S-transferase-4 (GST-4), superoxide dismutases (SODs) and heat shock protein-16.2 (HSP-16.2). However, essential oil and hydrosol of KR had no effect on over-activated Ras. Thus these results reminded us that people usually soak rose in hot water to prepare 'rose tea' as an effective way for health care. Thus, KRD was demonstrated to be a potential drug candidate for combating over-activated Ras-related cancer as an antioxidant.
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Affiliation(s)
- Yan Liu
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
| | - Dejuan Zhi
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
| | - Xin Wang
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
- Gansu Tianrun Rose Research Institute of Lanzhou University, Lanzhou University, Lanzhou, Gansu 730000, P.R. China
| | - Dongqing Fei
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
| | - Zhanxin Zhang
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
| | - Zhengrong Wu
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
- Gansu Tianrun Rose Research Institute of Lanzhou University, Lanzhou University, Lanzhou, Gansu 730000, P.R. China
| | - Yang Li
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
- Gansu Tianrun Rose Research Institute of Lanzhou University, Lanzhou University, Lanzhou, Gansu 730000, P.R. China
| | - Peng Chen
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
- Gansu Tianrun Rose Research Institute of Lanzhou University, Lanzhou University, Lanzhou, Gansu 730000, P.R. China
| | - Hongyu Li
- Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, Gansu 730020
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Hubbard WB, Harwood CL, Geisler JG, Vekaria HJ, Sullivan PG. Mitochondrial uncoupling prodrug improves tissue sparing, cognitive outcome, and mitochondrial bioenergetics after traumatic brain injury in male mice. J Neurosci Res 2018; 96:1677-1688. [PMID: 30063076 DOI: 10.1002/jnr.24271] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2018] [Revised: 04/12/2018] [Accepted: 05/29/2018] [Indexed: 12/17/2022]
Abstract
Traumatic brain injury (TBI) results in cognitive impairment, which can be long-lasting after moderate to severe TBI. Currently, there are no FDA-approved therapeutics to treat the devastating consequences of TBI and improve recovery. This study utilizes a prodrug of 2,4-dinitrophenol, MP201, a mitochondrial uncoupler with extended elimination time, that was administered after TBI to target mitochondrial dysfunction, a hallmark of TBI. Using a model of cortical impact in male C57/BL6 mice, MP201 (80 mg/kg) was provided via oral gavage 2-hr post-injury and daily afterwards. At 25-hr post-injury, mice were euthanized and the acute rescue of mitochondrial bioenergetics was assessed demonstrating a significant improvement in both the ipsilateral cortex and ipsilateral hippocampus after treatment with MP201. Additionally, oxidative markers, 4-hydroxyneneal and protein carbonyls, were reduced compared to vehicle animals after MP201 administration. At 2-weeks post-injury, mice treated with MP201 post-injury (80 mg/kg; q.d.) displayed significantly increased cortical sparing (p = .0059; 38% lesion spared) and improved cognitive outcome (p = .0133) compared to vehicle-treated mice. Additionally, vehicle-treated mice had significantly lower (p = .0019) CA3 neuron count compared to sham while MP201-treated mice were not significantly different from sham levels. These results suggest that acute mitochondrial dysfunction can be targeted to impart neuroprotection from reactive oxygen species, but chronic administration may have an added benefit in recovery. This study highlights the potential for safe, effective therapy by MP201 to alleviate negative outcomes of TBI.
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Affiliation(s)
- W Brad Hubbard
- Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky College of Medicine, Lexington, Kentucky.,Department of Neuroscience, University of Kentucky, Lexington, Kentucky
| | - Christopher L Harwood
- Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky College of Medicine, Lexington, Kentucky
| | | | - Hemendra J Vekaria
- Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky College of Medicine, Lexington, Kentucky.,Department of Neuroscience, University of Kentucky, Lexington, Kentucky
| | - Patrick G Sullivan
- Spinal Cord and Brain Injury Research Center (SCoBIRC), University of Kentucky College of Medicine, Lexington, Kentucky.,Department of Neuroscience, University of Kentucky, Lexington, Kentucky.,Lexington VAMC, Lexington, Kentucky
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Li B, Guo L, Ku T, Chen M, Li G, Sang N. PM 2.5 exposure stimulates COX-2-mediated excitatory synaptic transmission via ROS-NF-κB pathway. CHEMOSPHERE 2018; 190:124-134. [PMID: 28987401 DOI: 10.1016/j.chemosphere.2017.09.098] [Citation(s) in RCA: 37] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/09/2017] [Revised: 09/14/2017] [Accepted: 09/20/2017] [Indexed: 06/07/2023]
Abstract
Long-term exposure to fine particulate matter (PM2.5) has been reported to be closely associated with the neuroinflammation and synaptic dysfunction, but the mechanisms underlying the process remain unclear. Cyclooxygenase-2 (COX-2) is a key player in neuroinflammation, and has been also implicated in the glutamatergic excitotoxicity and synaptic plasticity. Thus, we hypothesized that COX-2 was involved in PM2.5-promoted neuroinflammation and synaptic dysfunction. Our results revealed that PM2.5 elevated COX-2 expression in primary cultured hippocampal neurons and increased the amplitude of field excitatory postsynaptic potentials (fEPSPs) in hippocampal brain slices. And the administration of NS398 (a COX-2 inhibitor) prevented the increased fEPSPs. PM2.5 also induced intracellular reactive oxygen species (ROS) generation accompanied with glutathione (GSH) depletion and the loss of mitochondrial membrane potential (MMP), and the ROS inhibitor, N-acetyl-L-cystein (NAC) suppressed the COX-2 overexpression and the increased fEPSPs. Furthermore, the nuclear factor kappa B (NF-κB) was involved in ROS-induced COX-2 and fEPSP in response to PM2.5 exposure. These findings indicated that PM2.5 activated COX-2 expression and enhanced the synaptic transmission through ROS-NF-κB pathway, and provided possible biomarkers and specific interventions for PM2.5-induced neurological damage.
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Affiliation(s)
- Ben Li
- College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR China
| | - Lin Guo
- College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR China
| | - Tingting Ku
- College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR China
| | - Minjun Chen
- College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR China
| | - Guangke Li
- College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR China
| | - Nan Sang
- College of Environment and Resource, Research Center of Environment and Health, Shanxi University, Taiyuan, Shanxi 030006, PR China.
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Le Borgne F, Ravaut G, Bernard A, Demarquoy J. L-carnitine protects C2C12 cells against mitochondrial superoxide overproduction and cell death. World J Biol Chem 2017; 8:86-94. [PMID: 28289521 PMCID: PMC5329717 DOI: 10.4331/wjbc.v8.i1.86] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/04/2016] [Revised: 11/04/2016] [Accepted: 12/13/2016] [Indexed: 02/05/2023] Open
Abstract
AIM To identify and characterize the protective effect that L-carnitine exerted against an oxidative stress in C2C12 cells. METHODS Myoblastic C2C12 cells were treated with menadione, a vitamin K analog that engenders oxidative stress, and the protective effect of L-carnitine (a nutrient involved in fatty acid metabolism and the control of the oxidative process), was assessed by monitoring various parameters related to the oxidative stress, autophagy and cell death. RESULTS Associated with its physiological function, a muscle cell metabolism is highly dependent on oxygen and may produce reactive oxygen species (ROS), especially under pathological conditions. High levels of ROS are known to induce injuries in cell structure as they interact at many levels in cell function. In C2C12 cells, a treatment with menadione induced a loss of transmembrane mitochondrial potential, an increase in mitochondrial production of ROS; it also induces autophagy and was able to provoke cell death. Pre-treatment of the cells with L-carnitine reduced ROS production, diminished autophagy and protected C2C12 cells against menadione-induced deleterious effects. CONCLUSION In conclusion, L-carnitine limits the oxidative stress in these cells and prevents cell death.
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Chiu CC, Liao YE, Yang LY, Wang JY, Tweedie D, Karnati HK, Greig NH, Wang JY. Neuroinflammation in animal models of traumatic brain injury. J Neurosci Methods 2016; 272:38-49. [PMID: 27382003 PMCID: PMC5201203 DOI: 10.1016/j.jneumeth.2016.06.018] [Citation(s) in RCA: 205] [Impact Index Per Article: 22.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Traumatic brain injury (TBI) is a leading cause of mortality and morbidity worldwide. Neuroinflammation is prominent in the short and long-term consequences of neuronal injuries that occur after TBI. Neuroinflammation involves the activation of glia, including microglia and astrocytes, to release inflammatory mediators within the brain, and the subsequent recruitment of peripheral immune cells. Various animal models of TBI have been developed that have proved valuable to elucidate the pathophysiology of the disorder and to assess the safety and efficacy of novel therapies prior to clinical trials. These models provide an excellent platform to delineate key injury mechanisms that associate with types of injury (concussion, contusion, and penetration injuries) that occur clinically for the investigation of mild, moderate, and severe forms of TBI. Additionally, TBI modeling in genetically engineered mice, in particular, has aided the identification of key molecules and pathways for putative injury mechanisms, as targets for development of novel therapies for human TBI. This Review details the evidence showing that neuroinflammation, characterized by the activation of microglia and astrocytes and elevated production of inflammatory mediators, is a critical process occurring in various TBI animal models, provides a broad overview of commonly used animal models of TBI, and overviews representative techniques to quantify markers of the brain inflammatory process. A better understanding of neuroinflammation could open therapeutic avenues for abrogation of secondary cell death and behavioral symptoms that may mediate the progression of TBI.
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Affiliation(s)
- Chong-Chi Chiu
- Department of General Surgery, Chi Mei Medical Center, Tainan and Liouying, Taiwan
| | - Yi-En Liao
- School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
| | - Ling-Yu Yang
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Jing-Ya Wang
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - David Tweedie
- Drug Design & Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA
| | - Hanuma K Karnati
- Drug Design & Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA
| | - Nigel H Greig
- Drug Design & Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA
| | - Jia-Yi Wang
- Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan; Department of Physiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
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Singh N, Hroudová J, Fišar Z. In Vitro Effects of Cognitives and Nootropics on Mitochondrial Respiration and Monoamine Oxidase Activity. Mol Neurobiol 2016; 54:5894-5904. [PMID: 27660276 DOI: 10.1007/s12035-016-0121-y] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2016] [Accepted: 09/12/2016] [Indexed: 12/26/2022]
Abstract
Impairment of mitochondrial metabolism, particularly the electron transport chain (ETC), as well as increased oxidative stress might play a significant role in pathogenesis of Alzheimer's disease (AD). Some effects of drugs used for symptomatic AD treatment may be related to their direct action on mitochondrial function. In vitro effects of pharmacologically different cognitives (galantamine, donepezil, rivastigmine, 7-MEOTA, memantine) and nootropic drugs (latrepirdine, piracetam) were investigated on selected mitochondrial parameters: activities of ETC complexes I, II + III, and IV, citrate synthase, monoamine oxidase (MAO), oxygen consumption rate, and hydrogen peroxide production of pig brain mitochondria. Complex I activity was decreased by galantamine, donepezil, and memantine; complex II + III activity was increased by galantamine. None of the tested drugs caused significant changes in the rate of mitochondrial oxygen consumption, even at high concentrations. Except galantamine, all tested drugs were selective MAO-A inhibitors. Latrepirdine, donepezil, and 7-MEOTA were found to be the most potent MAO-A inhibitors. Succinate-induced mitochondrial hydrogen peroxide production was not significantly affected by the drugs tested. The direct effect of cognitives and nootropics used in the treatment of AD on mitochondrial respiration is relatively small. The safest drugs in terms of disturbing mitochondrial function appear to be piracetam and rivastigmine. The MAO-A inhibition by cognitives and nootropics may also participate in mitochondrial neuroprotection. The results support the future research aimed at measuring the effects of currently used drugs or newly synthesized drugs on mitochondrial functioning in order to understand their mechanism of action.
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Affiliation(s)
- Namrata Singh
- Department of Psychiatry, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 11, 120 00, Prague 2, Czech Republic
| | - Jana Hroudová
- Department of Psychiatry, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 11, 120 00, Prague 2, Czech Republic.
| | - Zdeněk Fišar
- Department of Psychiatry, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 11, 120 00, Prague 2, Czech Republic
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Abstract
OBJECTIVE Hemorrhagic shock may contribute to acute kidney injury (AKI) by profoundly altering renal mitochondrial function. Resveratrol (RSV), a naturally occurring sirtuin 1 (SIRT1) activator, has been shown to promote mitochondrial function and reduce oxidative damage in a variety of aging-related disease states. We hypothesized that RSV treatment during resuscitation would ameliorate kidney mitochondrial dysfunction and decrease oxidative damage following hemorrhagic shock. METHODS Using a decompensated hemorrhagic shock model, male Long-Evans rats (n = 6 per group) were killed prior to hemorrhage (sham), at severe shock, and following either lactated Ringer's (LR) resuscitation or LR + RSV resuscitation (RSV: 30 mg/kg). At each time point, blood samples were assayed for arterial blood gases, lactate, blood urea nitrogen, and serum creatinine. Mitochondria were also isolated from kidney samples in order to assess individual electron transport complexes (complexes I, II, and IV) using high-resolution respirometry. Total mitochondria reactive oxygen species were measured using fluorometry, and lipid peroxidation was assessed by measuring 4-hydroxynonenal by Western blot. Quantitative polymerase chain reaction was used quantify mRNA from peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α) SIRT1, and proteins known to mitigate oxidative damage and promote mitochondrial biogenesis. RESULTS Resveratrol supplementation during resuscitation restored mitochondrial respiratory capacity and decreased mitochondrial reactive oxygen species and lipid peroxidation. Compared with standard LR resuscitation, RSV treatment significantly increased SIRT1 and PGC1-α expression and significantly increased both superoxide dismutase 2 and catalase expression. Although RSV was associated with decreased lactate production, pH, blood urea nitrogen, and serum creatinine values did not differ between resuscitation strategies. CONCLUSIONS Resuscitation with RSV significantly restored renal mitochondrial function and decreased oxidative damage following hemorrhagic shock.
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Shen WJ, Hsieh CY, Chen CL, Yang KC, Ma CT, Choi PC, Lin CF. A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses. Biochem Biophys Res Commun 2013. [DOI: 10.1016/j.bbrc.2012.11.037] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Marchissio MJ, Francés DEA, Carnovale CE, Marinelli RA. Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability. Toxicol Appl Pharmacol 2012; 264:246-54. [PMID: 22910329 DOI: 10.1016/j.taap.2012.08.005] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2012] [Revised: 07/06/2012] [Accepted: 08/06/2012] [Indexed: 12/20/2022]
Abstract
Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H(2)O(2) release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p<0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H(2)O(2) release, assessed by Amplex Red, was reduced by about 45% (p<0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+120%, p<0.05) and loss of mitochondrial membrane potential (-80%, p<0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H(2)O(2) release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.
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Affiliation(s)
- Maria Julia Marchissio
- Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina
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Thu VT, Kim HK, Long LT, Lee SR, Hanh TM, Ko TH, Heo HJ, Kim N, Kim SH, Ko KS, Rhee BD, Han J. NecroX-5 prevents hypoxia/reoxygenation injury by inhibiting the mitochondrial calcium uniporter. Cardiovasc Res 2012; 94:342-50. [DOI: 10.1093/cvr/cvs122] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
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Umegaki T, Okimura Y, Fujita H, Yano H, Akiyama J, Inoue M, Utsumi K, Sasaki J. Flow cytometric analysis of ca-induced membrane permeability transition of isolated rat liver mitochondria. J Clin Biochem Nutr 2011; 42:35-44. [PMID: 18231628 PMCID: PMC2212344 DOI: 10.3164/jcbn.2008006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2007] [Accepted: 06/21/2007] [Indexed: 01/14/2023] Open
Abstract
The membrane permeability transition (MPT) of mitochondria plays an important role in the mechanism of apoptotic cell death in various cells. Classic type MPT is induced by Ca2+ in the presence of inorganic phosphate and respiratory substrate, and is characterized by various events including generation of reactive oxygen species (ROS), membrane depolarization, swelling, release of Ca2+ and high sensitivity to cyclosporine A. However, the sequence of these events and the effect of antioxidants on their events remain obscure. Flow cytometry is a convenient method to investigate the order of events among various functions occurring in MPT using a limited amount of mitochondria (200 µl of 0.02 mg protein/ml) without contamination by other organelles. Flow cytometric analysis revealed that Ca2+ sequentially induced ROS generation, depolarization, swelling and Ca2+ release in mitochondria by a cyclosporine A-inhibitable mechanism. These results were supported by the finding that Ca2+-induced MPT was inhibited by antioxidants, such as glutathione and N-acetylcysteine. It was also revealed that various inhibitors of Ca2+-induced phospholipase A2 suppressed all of the events associated with Ca2+-induced MPT. These results suggested that ROS generation and phospholipase A2 activation by Ca2+ underlie the mechanism of the initiation of MPT.
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Affiliation(s)
- Teruo Umegaki
- Department of Cytology & Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikatacho, Okayama 700-8558, Japan
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Yang W, Hekimi S. A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans. PLoS Biol 2010; 8:e1000556. [PMID: 21151885 PMCID: PMC2998438 DOI: 10.1371/journal.pbio.1000556] [Citation(s) in RCA: 455] [Impact Index Per Article: 30.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2010] [Accepted: 10/27/2010] [Indexed: 11/29/2022] Open
Abstract
The study of long-lived C. elegans mutants suggests that mitochondrial oxidants can actually help reduce aging by acting as stress signals, rather than acting solely as toxic molecules. The nuo-6 and isp-1 genes of C. elegans encode, respectively, subunits of complex I and III of the mitochondrial respiratory chain. Partial loss-of-function mutations in these genes decrease electron transport and greatly increase the longevity of C. elegans by a mechanism that is distinct from that induced by reducing their level of expression by RNAi. Electron transport is a major source of the superoxide anion (O⋅–), which in turn generates several types of toxic reactive oxygen species (ROS), and aging is accompanied by increased oxidative stress, which is an imbalance between the generation and detoxification of ROS. These observations have suggested that the longevity of such mitochondrial mutants might result from a reduction in ROS generation, which would be consistent with the mitochondrial oxidative stress theory of aging. It is difficult to measure ROS directly in living animals, and this has held back progress in determining their function in aging. Here we have adapted a technique of flow cytometry to directly measure ROS levels in isolated mitochondria to show that the generation of superoxide is elevated in the nuo-6 and isp-1 mitochondrial mutants, although overall ROS levels are not, and oxidative stress is low. Furthermore, we show that this elevation is necessary and sufficient to increase longevity, as it is abolished by the antioxidants NAC and vitamin C, and phenocopied by mild treatment with the prooxidant paraquat. Furthermore, the absence of effect of NAC and the additivity of the effect of paraquat on a variety of long- and short-lived mutants suggest that the pathway triggered by mitochondrial superoxide is distinct from previously studied mechanisms, including insulin signaling, dietary restriction, ubiquinone deficiency, the hypoxic response, and hormesis. These findings are not consistent with the mitochondrial oxidative stress theory of aging. Instead they show that increased superoxide generation acts as a signal in young mutant animals to trigger changes of gene expression that prevent or attenuate the effects of subsequent aging. We propose that superoxide is generated as a protective signal in response to molecular damage sustained during wild-type aging as well. This model provides a new explanation for the well-documented correlation between ROS and the aged phenotype as a gradual increase of molecular damage during aging would trigger a gradually stronger ROS response. An unequivocal demonstration that mitochondria are important for lifespan comes from studies with the nematode Caenorhabditis elegans. Mutations in mitochondrial proteins such as ISP-1 and NUO-6, which function directly in mitochondrial electron transport, lead to a dramatic increase in the lifespan of this organism. One theory proposes that toxicity of mitochondrial reactive oxygen species (ROS) is the cause of aging and predicts that the generation of the ROS superoxide should be low in these mutants. Here we have measured superoxide generation in these mutants and found that it is in fact elevated, rather than reduced. Furthermore, we found that this elevation is necessary and sufficient for longevity, as it is abolished by antioxidants and induced by mild treatment with oxidants. This suggests that superoxide can act as a signal triggering cellular changes that attenuate the effects of aging. This idea suggests a new model for the well-documented correlation between ROS and the aged phenotype. We propose that a gradual increase of molecular damage during aging triggers a concurrent, gradually intensifying, protective superoxide response.
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Affiliation(s)
- Wen Yang
- Department of Biology, McGill University, Montreal, Quebec, Canada
| | - Siegfried Hekimi
- Department of Biology, McGill University, Montreal, Quebec, Canada
- * E-mail:
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Glutathione peroxidase 1 protects mitochondria against hypoxia/reoxygenation damage in mouse hearts. Pflugers Arch 2010; 460:55-68. [PMID: 20306076 DOI: 10.1007/s00424-010-0811-7] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2010] [Revised: 02/16/2010] [Accepted: 02/18/2010] [Indexed: 10/19/2022]
Abstract
Glutathione peroxidase 1 (GPx1) plays an important role in preventing cardiac dysfunction following ischemia-reperfusion injury. However, its role in protecting cardiac mitochondria against reoxygenation-induced reactive oxygen species (ROS) generation in vivo is unclear. We examined the role of GPx1 in protecting cardiac mitochondria against hypoxia-reoxygenation (HR) damage by testing for alterations in cardiac mitochondrial function. We used a two-dimensional gel electrophoresis proteomics analysis to examine the effects of reoxygenation on cardiac protein in wild-type (GPx1(+/+)) and GPx1 knockout (GPx1(-/-)) mouse hearts. We identified 42 protein spots showing differential expression in the two groups. Sixteen of the proteins identified were located in mitochondria and were involved in a number of key metabolic pathways. To verify our proteomics findings functionally, we performed NADH autofluorescence measurements and ATP production assays. The reduced expression of oxidative phosphorylation proteins in GPx1(-/-) mice following HR treatment resulted in loss of the mitochondrial membrane potential and decreased mitochondrial respiration. Mitochondrial ROS production and oxidative mtDNA damage were increased markedly during reoxygenation in GPx1(-/-) hearts. We also found morphological abnormalities in cardiac mitochondria and myocytes in HR-treated GPx1(-/-). This is the first report of the role of GPx1 in protecting cardiac mitochondria against reoxygenation damage in vivo. These findings will help clarify the mechanisms of HR injury and will aid in the development of antioxidant therapies to prevent cardiac mitochondrial dysfunction associated with reoxygenation.
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Riegel RE, Valvassori SS, Elias G, Réus GZ, Steckert AV, de Souza B, Petronilho F, Gavioli EC, Dal-Pizzol F, Quevedo J. Animal model of mania induced by ouabain: Evidence of oxidative stress in submitochondrial particles of the rat brain. Neurochem Int 2009; 55:491-5. [DOI: 10.1016/j.neuint.2009.05.003] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2009] [Revised: 04/22/2009] [Accepted: 05/04/2009] [Indexed: 01/13/2023]
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Shen E, Li Y, Li Y, Shan L, Zhu H, Feng Q, Arnold JMO, Peng T. Rac1 is required for cardiomyocyte apoptosis during hyperglycemia. Diabetes 2009; 58:2386-95. [PMID: 19592621 PMCID: PMC2750234 DOI: 10.2337/db08-0617] [Citation(s) in RCA: 151] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
OBJECTIVE Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia. RESEARCH DESIGN AND METHODS Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose. RESULTS The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91(phox) or p47(phox), or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function. CONCLUSIONS Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes.
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Affiliation(s)
- E. Shen
- Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medicine, University of Western Ontario, London, Ontario, Canada
| | - Yanwen Li
- Department of Microbiology, Imperial College London, London, U.K
| | - Ying Li
- Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medicine, University of Western Ontario, London, Ontario, Canada
| | - Limei Shan
- Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medicine, University of Western Ontario, London, Ontario, Canada
| | - Huaqing Zhu
- Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medicine, University of Western Ontario, London, Ontario, Canada
| | - Qingping Feng
- Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medicine, University of Western Ontario, London, Ontario, Canada
- Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
| | - J. Malcolm O. Arnold
- Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medicine, University of Western Ontario, London, Ontario, Canada
- Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada
| | - Tianqing Peng
- Critical Illness Research, Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
- Department of Medicine, University of Western Ontario, London, Ontario, Canada
- Department of Pathology, University of Western Ontario, London, Ontario, Canada
- Corresponding author: Tianqing Peng,
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Mitchell CS, Lee RH. Pathology dynamics predict spinal cord injury therapeutic success. J Neurotrauma 2009; 25:1483-97. [PMID: 19125684 DOI: 10.1089/neu.2008.0658] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Secondary injury, the complex cascade of cellular events following spinal cord injury (SCI), is a major source of post-insult neuron death. Experimental work has focused on the details of individual factors or mechanisms that contribute to secondary injury, but little is known about the interactions among factors leading to the overall pathology dynamics that underlie its propagation. Prior hypotheses suggest that the pathology is dominated by interactions, with therapeutic success lying in combinations of neuroprotective treatments. In this study, we provide the first comprehensive, system-level characterization of the entire secondary injury process using a novel relational model methodology that aggregates the findings of approximately 250 experimental studies. Our quantitative examination of the overall pathology dynamics suggests that, while the pathology is initially dominated by "fire-like", rate-dependent interactions, it quickly switches to a "flood-like", accumulation-dependent process with contributing factors being largely independent. Our evaluation of approximately 20,000 potential single and combinatorial treatments indicates this flood-like pathology results in few highly influential factors at clinically realistic treatment time frames, with multi-factor treatments being merely additive rather than synergistic in reducing neuron death. Our findings give new fundamental insight into the understanding of the secondary injury pathology as a whole, provide direction for alternative therapeutic strategies, and suggest that ultimate success in treating SCI lies in the pursuit of pathology dynamics in addition to individually involved factors.
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Affiliation(s)
- Cassie S Mitchell
- Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA
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Yang J, Ma L, Zhang Y, Fang F, Li L. Flow cytometric identification of two different rhodamine-123-stained mitochondrial populations in maize leaves. PROTOPLASMA 2007; 231:249-252. [PMID: 17922268 DOI: 10.1007/s00709-007-0259-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/01/2006] [Accepted: 01/06/2007] [Indexed: 05/25/2023]
Abstract
Flow cytometric analysis of mitochondria isolated from maize leaves revealed two distinct rhodamine-123-stained fluorescence populations distinguishable by their main fluorescence channel. Further microscopic observation of mitochondria stained with Janus Green B and rhodamine-123 revealed the occurrence of differently sized mitochondrial particles. It was shown by pulsed-field gel electrophoresis that the DNA from the isolated mitochondria ranged in size from 45 to 100 kb. These results suggest that different types of mitochondria with different physiological status, mass, and genomic DNA size probably coexist and carry out different physiological functions throughout the whole process of maize leaf growth and development.
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Affiliation(s)
- Jinling Yang
- Key Laboratory for Plant Developmental Biology of the Ministry of Education, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.
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Chen J, Adikari M, Pallai R, Parekh HK, Simpkins H. Dihydrodiol dehydrogenases regulate the generation of reactive oxygen species and the development of cisplatin resistance in human ovarian carcinoma cells. Cancer Chemother Pharmacol 2007; 61:979-87. [PMID: 17661040 PMCID: PMC2846169 DOI: 10.1007/s00280-007-0554-0] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2007] [Accepted: 06/26/2007] [Indexed: 11/28/2022]
Abstract
We have previously demonstrated that overexpression of dihydrodiol dehydrogenase isoform 1 (DDH1) or DDH2 leads to the induction of drug resistance to platinum based drugs in human ovarian, lung, cervical and germ cell tumor cell lines. DDH belongs to a family of aldoketo reductases that are involved in the detoxification of several endogenous and exogenous substrates. DDH1 and DDH2 in particular have been shown to be involved in the detoxification (activation?) of polycyclic aromatic hydrocarbons (PAH). Based on the involvement of DDH in the detoxification of electrophilic PAH intermediates, the effect of DDH on the production of reactive oxygen species (ROS) in a cisplatin-sensitive and -resistant human ovarian carcinoma cell line was investigated in the current study. In addition to the overexpression of DDH1 and DDH2, increased expression of DDH3 was demonstrated in the cisplatin-resistant 2008/C13* cells, compared to the parental 2008 cells. However, as assessed by RT-PCR, neither cell line expressed DDH4. The 2008/C13* cells were eightfold resistant to cisplatin, and transfection experiments utilizing cisplatin-sensitive 2008 cells suggest that this could be mediated by overexpression of either DDH1, DDH2, or DDH3. The 2008/C13* cells had lower basal intracellular ROS level as compared to the 2008 cells and ROS production was decreased in the recombinant 2008 cells with forced, constitutive overexpression of either, DDH1, DDH2, or DDH3. Transfection of siRNA against DDH1 or DDH2 in the cisplatin-resistant 2008/C13* cells not only significantly decreased their cisplatin-resistance index (as assayed by MTT and colony formation assay) but also led to an increase in the basal levels of ROS production (although transfection of siRNA against DDH3 resulted in cell death). The 2008/C13* cells were found to be cross-resistant to the cytotoxic effects of hydrogen peroxide and tert-butyl hydroperoxide and knockdown of either DDH1 or DDH2 expression (using siRNA) resulted in sensitization of the resistant cells to these agents. These results support the conclusion that the increased levels of DDH in the 2008/C13* cells are directly responsible for the reduced production of ROS and that this may play a role in the development of cisplatin resistance.
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Affiliation(s)
- Jianli Chen
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Room 206, OMS, 3400 N. Broad Street, Philadelphia, PA 19140, USA
| | - Mahesha Adikari
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Room 206, OMS, 3400 N. Broad Street, Philadelphia, PA 19140, USA
| | - Rajash Pallai
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Room 206, OMS, 3400 N. Broad Street, Philadelphia, PA 19140, USA
| | - Hemant K. Parekh
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Room 206, OMS, 3400 N. Broad Street, Philadelphia, PA 19140, USA, Fels Institute of Cancer Research and Molecular Biology, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19140, USA
| | - Henry Simpkins
- Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Room 206, OMS, 3400 N. Broad Street, Philadelphia, PA 19140, USA, Fels Institute of Cancer Research and Molecular Biology, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19140, USA
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Osna NA, White RL, Todero S, McVicker BL, Thiele GM, Clemens DL, Tuma DJ, Donohue TM. Ethanol-induced oxidative stress suppresses generation of peptides for antigen presentation by hepatoma cells. Hepatology 2007; 45:53-61. [PMID: 17187415 DOI: 10.1002/hep.21442] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
UNLABELLED Processing of peptides for antigen presentation is catalyzed by antigen-trimming enzymes, including the proteasome and leucine aminopeptidase. Oxidative stress suppresses proteasome function. We hypothesized that in liver cells, processing of antigenic peptides is altered by ethanol metabolism. To address this issue, soluble extracts of ethanol-metabolizing VL-17A cells treated with 100 mM ethanol or left untreated were incubated with C-extended or N-extended 18-27 HBV core peptides. Peptide cleavage was measured by recovery after HPLC. Ethanol exposure to VL-17A cells increased CYP2E1 and decreased proteasome peptidase activities. The latter effect was prevented by treatment of cells with inhibitors, 4-methylpyrazole and diallyl sulfide. Ethanol treatment of VL-17A cells also reduced the activity of leucine aminopeptidase (LAP). Consequently, cleavage of both C-extended and N-extended peptides by cytosolic extracts was suppressed by pretreatment of cells with ethanol. Treatment of cells with interferon gamma, which enhances proteasome activity, did not reverse the effects of ethanol. Ethanol exerted similar effects on WIFB cells, indicating that its effects are not unique to one cell type. CONCLUSION Ethanol metabolism suppresses activities of antigen-trimming enzymes, thereby decreasing the cleavage of C-extended and N-extended peptides. This defect may potentially result in decreased MHC class I-restricted antigen presentation on virally infected liver cells.
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Affiliation(s)
- Natalia A Osna
- Liver Study Unit, The Omaha Veterans Affairs (VA) Medical Center, Omaha, NE 68105, USA.
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Gong Y, Sohn H, Xue L, Firestone GL, Bjeldanes LF. 3,3'-Diindolylmethane is a novel mitochondrial H(+)-ATP synthase inhibitor that can induce p21(Cip1/Waf1) expression by induction of oxidative stress in human breast cancer cells. Cancer Res 2006; 66:4880-7. [PMID: 16651444 DOI: 10.1158/0008-5472.can-05-4162] [Citation(s) in RCA: 107] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis in multiple organs. 3,3'-Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising antitumor agent. Previous studies in our laboratory showed that 3,3'-diindolylmethane induced a G(1) cell cycle arrest in human breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that 3,3'-diindolylmethane is a strong mitochondrial H(+)-ATPase inhibitor (IC(50) approximately 20 micromol/L). 3,3'-Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular ATP level, and significantly stimulated mitochondrial reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH(2)-terminal kinase. Using specific kinase inhibitors (SB203580 and SP600125), we showed the central role of p38 and c-Jun NH(2)-terminal kinase (JNK) pathways in 3,3'-diindolylmethane-induced p21 mRNA transcription. In addition, antioxidants significantly attenuated 3,3'-diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3'-diindolylmethane-induced p21 overexpression, we showed that 3,3'-diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient rho(0) MCF-7 cells, in which 3,3'-diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3'-diindolylmethane-induced p21 up-regulation in human breast cancer cells.
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Affiliation(s)
- Yixuan Gong
- Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720, USA
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Frey BN, Valvassori SS, Gomes KM, Martins MR, Dal-Pizzol F, Kapczinski F, Quevedo J. Increased oxidative stress in submitochondrial particles after chronic amphetamine exposure. Brain Res 2006; 1097:224-9. [PMID: 16730669 DOI: 10.1016/j.brainres.2006.04.076] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2006] [Revised: 04/12/2006] [Accepted: 04/17/2006] [Indexed: 01/04/2023]
Abstract
Previous studies have suggested that reactive oxygen species (ROS) production may play a role in the pathophysiology of many neuropsychiatric disorders, such as bipolar disorder (BD) and schizophrenia (SCZ). In addition, there is an emerging body of data indicating that BD and SCZ may be associated with mitochondrial dysfunction. We studied the effects of acute and chronic d-amphetamine on ROS production in submitochondrial particles of rat brain. Male Wistar rats were divided in two experimental groups: acute and chronic treatment. In the acute treatment, rats received one single IP injection of d-amphetamine (1, 2 or 4 mg/kg) or saline (control group). In the chronic treatment, rats received one daily IP injection of d-amphetamine (1, 2 or 4 mg/kg) or saline for 7 days. Locomotor activity was assessed with the open field task, and thiobarbituric acid reactive substances (TBARS) and superoxide production were measured in submitochondrial particles of the prefrontal cortex and hippocampus. Both acute and chronic amphetamine treatment increased locomotor behavior. Chronic amphetamine exposure induced a 3- to 6-fold increase of TBARS and a 1.5- to 2-fold increase of superoxide production in submitochondrial particles of prefrontal cortex and hippocampus (P < 0.05). No effects on superoxide or TBARS were observed with acute treatment. These findings suggest that amphetamine-induced mitochondrial ROS generation may be a useful model to investigate the hypothesis of altered brain energy metabolism associated with BD and SCZ. Further studies assessing the effects of mood stabilizers and antipsychotics in preventing mitochondrial oxidative stress are necessary.
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Affiliation(s)
- Benício N Frey
- Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, 90035-003 Porto Alegre, RS, Brazil
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Abstract
The oxidation of proteins may play an important role in the pathogenesis of chronic inflammatory lung diseases, and may contribute to lung damage. However, the extent of oxidation and the distribution among proteins are not known for most pediatric lung diseases. In this work, protein oxidation was assessed as protein carbonyls. Bronchoalveolar lavages (BAL) from children with chronic lung diseases were investigated by dot-blot assay for content and for pattern of distribution of oxidized proteins by two-dimensional (2D) electrophoresis and Western blotting. Significantly higher levels of protein oxidation than in healthy controls were determined in groups of patients with interstitial lung disease, gastro-esophageal reflux disease, and pulmonary alveolar proteinosis. The proteins most sensitive to oxidation were serum albumin, surfactant protein A, and alpha1-antitrypsin. Our data show increased oxidative stress in lungs of children with chronic pulmonary diseases, with significant interindividual variations. The extent of protein oxidation was proportional to the count of neutrophilic granulocytes in BAL fluid. These findings strongly support the concept that an abundance of reactive oxygen species produced during neutrophilic inflammation may be a deleterious factor, leading to pulmonary damage in these patients.
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Affiliation(s)
- V Starosta
- Lung Research Group, Children's Hospital, Ludwig Maximilians University, Munich, Germany
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