Showing papers by "Balaraman Kalyanaraman published in 2010"

Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity

Abstract: Otto Warburg's theory on the origins of cancer postulates that tumor cells have defects in mitochondrial oxidative phosphorylation and therefore rely on high levels of aerobic glycolysis as the major source of ATP to fuel cellular proliferation (the Warburg effect). This is in contrast to normal cells, which primarily utilize oxidative phosphorylation for growth and survival. Here we report that the major function of glucose metabolism for Kras-induced anchorage-independent growth, a hallmark of transformed cells, is to support the pentose phosphate pathway. The major function of glycolytic ATP is to support growth under hypoxic conditions. Glutamine conversion into the tricarboxylic acid cycle intermediate alpha-ketoglutarate through glutaminase and alanine aminotransferase is essential for Kras-induced anchorage-independent growth. Mitochondrial metabolism allows for the generation of reactive oxygen species (ROS) which are required for Kras-induced anchorage-independent growth through regulation of the ERK MAPK signaling pathway. We show that the major source of ROS generation required for anchorage-independent growth is the Qo site of mitochondrial complex III. Furthermore, disruption of mitochondrial function by loss of the mitochondrial transcription factor A (TFAM) gene reduced tumorigenesis in an oncogenic Kras-driven mouse model of lung cancer. These results demonstrate that mitochondrial metabolism and mitochondrial ROS generation are essential for Kras-induced cell proliferation and tumorigenesis.

Prevention of diabetic nephropathy in Ins2(+/)⁻(AkitaJ) mice by the mitochondria-targeted therapy MitoQ.

Abstract: Mitochondrial production of ROS (reactive oxygen species) is thought to be associated with the cellular damage resulting from chronic exposure to high glucose in long-term diabetic patients. We hypothesized that a mitochondria-targeted antioxidant would prevent kidney damage in the Ins2+/−AkitaJ mouse model (Akita mice) of Type 1 diabetes. To test this we orally administered a mitochondria-targeted ubiquinone (MitoQ) over a 12-week period and assessed tubular and glomerular function. Fibrosis and pro-fibrotic signalling pathways were determined by immunohistochemical analysis, and mitochondria were isolated from the kidney for functional assessment. MitoQ treatment improved tubular and glomerular function in the Ins2+/−AkitaJ mice. MitoQ did not have a significant effect on plasma creatinine levels, but decreased urinary albumin levels to the same level as non-diabetic controls. Consistent with previous studies, renal mitochondrial function showed no significant change between any of the diabetic or wild-type groups. Importantly, interstitial fibrosis and glomerular damage were significantly reduced in the treated animals. The pro-fibrotic transcription factors phospho-Smad2/3 and β-catenin showed a nuclear accumulation in the Ins2+/−AkitaJ mice, which was prevented by MitoQ treatment. These results support the hypothesis that mitochondrially targeted therapies may be beneficial in the treatment of diabetic nephropathy. They also highlight a relatively unexplored aspect of mitochondrial ROS signalling in the control of fibrosis.

Role of mitochondrial reactive oxygen species in osteoclast differentiation

Abstract: Previously we showed that hypoxia-induced mitochondrial respiratory stress in RAW 264.7 macrophages and other cells caused activation of retrograde signaling (also known as mitochondrial respiratory stress signaling) and the appearance of tartrate-resistant acid phosphatase (TRAP)-positive cells. In the present study, we used N-acetyl cysteine and ascorbate (general antioxidants) and MitoQ, a mitochondria-specific antioxidant, to investigate the role of intracellular reactive oxygen species (ROS) in osteoclast differentiation. Our results show that hypoxia-mediated mitochondrial dysfunction, as tested by disruption of mitochondrial transmembrane potential, was suppressed by MitoQ as well as by the other antioxidants. These agents also suppressed the activation of mitochondrial retrograde signaling. Interestingly, in terms of molar concentrations, MitoQ was more than 1000-fold more effective than general antioxidants in suppressing the receptor activator of nuclear factor-B ligand-induced differentiation of RAW 264.7 cells into multinucleated and TRAP-positive osteoclasts. We propose that mitochondrial function and intramitochondrial ROS play important roles in osteoclastogenesis.

Reactive oxygen species control senescence-associated matrix metalloproteinase-1 through c-Jun-N-terminal kinase.

Abstract: The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox-responsive molecular signals that drive senescence-associated (SA) matrix metalloproteinase-1 (MMP-1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady-state (H2O2) 3.5-fold (13.7–48.6 pM) relative to young cells. Restricting H2O2 production through low O2 exposure or by antioxidant treatments prevented SA increases in MMP-1 expression. The H2O2-dependent control of SA MMP-1 is attributed to sustained JNK activation and c-jun recruitment to the MMP-1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK-4) and inhibitory phosphatase (MKP-1), respectively. Enforced MKP-1 expression negates SA increases in JNK phosphorylation and MMP-1 production. Overall, these studies define redox-sensitive signaling networks regulating SA MMP-1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover. J. Cell. Physiol. 225: 52–62, 2010. © 2010 Wiley-Liss, Inc.

Lipid peroxyl radicals mediate tyrosine dimerization and nitration in membranes

Abstract: Protein tyrosine dimerization and nitration by biologically relevant oxidants usually depend on the intermediate formation of tyrosyl radical ((*)Tyr) In the case of tyrosine oxidation in proteins associated with hydrophobic biocompartments, the participation of unsaturated fatty acids in the process must be considered since they typically constitute preferential targets for the initial oxidative attack Thus, we postulate that lipid-derived radicals mediate the one-electron oxidation of tyrosine to (*)Tyr, which can afterward react with another (*)Tyr or with nitrogen dioxide ((*)NO(2)) to yield 3,3'-dityrosine or 3-nitrotyrosine within the hydrophobic structure, respectively To test this hypothesis, we have studied tyrosine oxidation in saturated and unsaturated fatty acid-containing phosphatidylcholine (PC) liposomes with an incorporated hydrophobic tyrosine analogue BTBE (N-t-BOC l-tyrosine tert-butyl ester) and its relationship with lipid peroxidation promoted by three oxidation systems, namely, peroxynitrite, hemin, and 2,2'-azobis (2-amidinopropane) hydrochloride In all cases, significant tyrosine (BTBE) oxidation was seen in unsaturated PC liposomes, in a way that was largely decreased at low oxygen concentrations Tyrosine oxidation levels paralleled those of lipid peroxidation (ie, malondialdehyde and lipid hydroperoxides), lipid-derived radicals and BTBE phenoxyl radicals were simultaneously detected by electron spin resonance spin trapping, supporting an association between the two processes Indeed, alpha-tocopherol, a known reactant with lipid peroxyl radicals (LOO(*)), inhibited both tyrosine oxidation and lipid peroxidation induced by all three oxidation systems Moreover, oxidant-stimulated liposomal oxygen consumption was dose dependently inhibited by BTBE but not by its phenylalanine analogue, BPBE (N-t-BOC l-phenylalanine tert-butyl ester), providing direct evidence for the reaction between LOO(*) and the phenol moiety in BTBE, with an estimated second-order rate constant of 48 x 10(3) M(-1) s(-1) In summary, the data presented herein demonstrate that LOO(*) mediates tyrosine oxidation processes in hydrophobic biocompartments and provide a new mechanistic insight to understand protein oxidation and nitration in lipoproteins and biomembranes

Mitochondrial targeting of the electrophilic lipid 15-deoxy-Delta12,14-prostaglandin J2 increases apoptotic efficacy via redox cell signalling mechanisms.

Abstract: Prototypical electrophiles such as the lipid 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) are well recognized for their therapeutic potential. Electrophiles modify signalling proteins in both the cytosol and mitochondrion, which results in diverse cellular responses, including cytoprotective effects and, at high doses, cell death. These findings led us to the hypothesis that targeting electrophiles to specific compartments in the cell could fine-tune their biological effects. To examine this, we synthesized a novel mitochondrially targeted analogue of 15d-PGJ 2 (mito-15d-PGJ 2 ) and tested its effects on redox cell signalling. Mito-15d-PGJ 2 caused profound defects in mitochondrial bioenergetics and mitochondrial membrane depolarization when compared with 15d-PGJ 2 . We also found that mito-15d-PGJ 2 modified different members of the electrophile-responsive proteome, was more potent at initiating intrinsic apoptotic cell death and was less effective than 15d-PGJ 2 at up-regulating the expression of HO-1 (haem oxygenase-1) and glutathione. These results demonstrate the feasibility of modulating the biological effects of electrophiles by targeting the pharmacophore to mitochondria.

Mitochondrially targeted antioxidants for the treatment of cardiovascular diseases.

Abstract: Oxidative stress resulting from imbalance between Reactive Oxygen Species (ROS) generation and antioxidant mechanisms is important in the pathogenesis of cardiovascular diseases such as atherosclerosis, ischemic heart disease, heart failure, stroke, hypertension and diabetes. Paradoxically, antioxidant therapy such as vitamin E has not been shown on large randomized clinical trials to favorably affect clinical outcomes. Since mitochondria are involved not only with bioenergetics but also with oxidative damage through ROS generation and cell signaling leading to apoptosis, antioxidants targeted at the mitochondria are appealing novel agents to attenuate oxidative stress. In particular, antioxidants conjugated with triphenylphosphonium cation such as mitoquinone, mitovitamin E and mitophenyltertbutyline achieve concentrations in the mitochondrial matrix several-fold greater than those achieved in the cytosol because of the high negative membrane potential of the inner mitochondrial membrane. We review preliminary experiments and also some patents on cell and animal models of cardiovascular diseases where mitochondrially targeted antioxidants have been used and were shown to reduce ROS production and the effects of oxidative stress due to ROS, apoptosis and improve cardiac function. Although ongoing human clinical studies involve only non-cardiovascular applications at this time, preclinical studies show promise for eventual human trials for cardiovascular diseases.

Detoxifying carcinogenic polyhalogenated quinones by hydroxamic acids via an unusual double Lossen rearrangement mechanism

Abstract: Hydroxamic acids, which are best-known for their metal-chelating properties in biomedical research, have been found to effectively detoxify the carcinogenic polyhalogenated quinoid metabolites of pentachlorophenol and other persistent organic pollutants. However, the chemical mechanism underlying such detoxication is unclear. Here we show that benzohydroxamic acid (BHA) could dramatically accelerate the conversion of the highly toxic tetrachloro-1, 4-benzoquinone (p-chloranil) to the much less toxic 2,5-dichloro-3, 6-dihydroxy-1, 4-benzoquonine (chloranilic acid), with rate accelerations of up to 150,000-fold. In contrast, no enhancing effect was observed with O-methyl BHA. The major reaction product of BHA was isolated and identified as O-phenylcarbamyl benzohydroxamate. On the basis of these data and oxygen-18 isotope-labeling studies, we proposed that suicidal nucleophilic attack coupled with an unexpected double Lossen rearrangement reaction was responsible for this remarkable acceleration of the detoxication reaction. This is the first report of an unusually mild and facile Lossen-type rearrangement, which could take place under normal physiological conditions in two consecutive steps. Our findings may have broad biological and environmental implications for future research on hydroxamic acids and polyhalogenated quinoid carcinogens, which are two important classes of compounds of major biomedical and environmental interest.

Tyrosine−Lipid Peroxide Adducts from Radical Termination: Para Coupling and Intramolecular Diels−Alder Cyclization

Abstract: Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR spectroscopy as well as by mass spectrometry (MS). The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic (13)C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl (13)C chemical shifts at ~198 ppm. All of the NMR HMBC and HSQC correlations support the structure assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found in either the primary or cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts, which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein cross-links via interprotein Michael adducts.

Oxidation of histidine residues in copper-zinc superoxide dismutase by bicarbonate-stimulated peroxidase and thiol oxidase activities: pulse EPR and NMR studies.

Abstract: In this work, we investigated the oxidative modification of histidine residues induced by peroxidase and thiol oxidase activities of bovine copper-zinc superoxide dismutase (Cu-ZnSOD) using NMR and pulse EPR spectroscopy. 1D NMR and 2D-NOESY were used to determine the oxidative damage at the Zn(II) and Cu(II) active sites as well as at distant histidines. Results indicate that during treatment of SOD with hydrogen peroxide (H(2)O(2)) or cysteine in the absence of bicarbonate anion (HCO(3)(-)), both exchangeable and nonexchangeable protons were affected. Both His-44 and His-46 in the Cu(II) active site were oxidized based on the disappearance of NOESY cross-peaks between CH and NH resonances of the imidazole rings. In the Zn(II) site, only His-69, which is closer to His-44, was oxidatively modified. However, addition of HCO(3)(-) protected the active site His residues. Instead, resonances assigned to the His-41 residue, 11 A away from the Cu(II) site, were completely abolished during both HCO(3)(-)-stimulated peroxidase activity and thiol oxidase activity in the presence of HCO(3)(-) . Additionally, ESEEM/HYSCORE and ENDOR studies of SOD treated with peroxide/Cys in the absence of HCO(3)(-) revealed that hyperfine couplings to the distal and directly coordinated nitrogens of the His-44 and His-46 ligands at the Cu(II) active site were modified. In the presence of HCO(3)(-), these modifications were absent. HCO(3)(-)-mediated, selective oxidative modification of histidines in SOD may be relevant to understanding the molecular mechanism of SOD peroxidase and thiol oxidase activities.

Neuroprotective compounds and their use

Abstract: Apocynin derivative compounds, active pharmaceutical ingredients, dosage forms, and methods of use thereof as neuroprotectants in the brain of mammals.