O. Y. Ivanova

Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants--the "Skulachev-ion" derivatives.

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1–2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of “bad” mitochondria in cell physiology is discussed.

Novel mitochondria-targeted antioxidants: plastoquinone conjugated with cationic plant alkaloids berberine and palmatine.

ABSTRACTPurposeTo develop effective mitochondria-targeted antioxidants composed entirely of natural constituents.MethodsNovel mitochondria-targeted antioxidants were synthesized containing plant electron carrier and antioxidant plastoquinone conjugated by nonyloxycarbonylmethyl residue with berberine or palmatine, penetrating cations of plant origin. These compounds, SkQBerb and SkQPalm, were tested in model planar phospholipid membranes and micelles, liposomes, isolated mitochondria and living cells.ResultsSkQBerb and SkQPalm penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in mitochondria isolated or in living human cells in culture. Reduced forms of SkQBerb and SkQPalm as well as C10Berb and C10Palm (SkQBerb and SkQPalm analogs lacking plastoquinol moiety) revealed radical scavenging activity in lipid micelles and liposomes, while oxidized forms were inactive. In isolated mitochondria and in living cells, berberine and palmatine moieties were not reduced, so antioxidant activity of C10Berb and C10Palm was not detected. SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations; their prooxidant effect was observed at 1,000 times higher concentrations. In human cell cuture, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide.ConclusionThis is the first successful attempt to construct mitochondria-targeted antioxidants composed entirely of natural components, namely plastoquinone, nonyl, acetyl and berberine or palmatine residues.

Novel mitochondria-targeted antioxidants, “Skulachev-Ion” derivatives, accelerate dermal wound healing in animals

It is shown that the novel mitochondria-targeted antioxidant SkQ1, (10-(6′-plastoquinonyl) decyltriphenylphosphonium) stimulates healing of full-thickness dermal wounds in mice and rats. Treatment with nanomolar doses of SkQ1 in various formulations accelerated wound cleaning and suppressed neutrophil infiltration at the early (7 h) steps of inflammatory phase. SkQ1 stimulated formation of granulation tissue and increased the content of myofibroblasts in the beginning of regenerative phase of wound healing. Later this effect caused accumulation of collagen fibers. Local treatment with SkQ1 stimulated re-epithelization of the wound. Lifelong treatment of mice with SkQ1 supplemented with drinking water strongly stimulated skin wounds healing in old (28 months) animals. In an in vitro model of wound in human cell cultures, SkQ1 stimulated movement of epitheliocytes and fibroblasts into the “wound”. Myofibroblast differentiation of subcutaneous fibroblasts was stimulated by SkQ1. It is suggested that SkQ1 stimulates wound healing by suppression of the negative effects of oxidative stress in the wound and also by induction of differentiation. Restoration of regenerative processes in old animals is consistent with the “rejuvenation” effects of SkQ1, which prevents some gerontological diseases.

Scavenging of Reactive Oxygen Species in Mitochondria Induces Myofibroblast Differentiation

The goal of this study was to investigate the possible role of reactive oxygen species (ROS) in signaling, in modulation of the cytoskeleton, and in differentiation of fibroblasts. For this purpose, we have applied a novel mitochondria-targeted antioxidant: plastoquinone conjugated with decyltriphenylphosphonium (SkQ1). This antioxidant at nanomolar concentration prevented ROS accumulation and cell death induced by H(2)O(2) in fibroblasts. We found that scavenging of ROS produced by mitochondria activated the Rho/ROCK/LIMK signaling pathway that was followed by phosphorylation of cofilin and stabilization of actin stress fibers. The mitochondria-targeted antioxidant induced differentiation of human subcutaneous fibroblasts to myofibroblasts as revealed by expression of fibronectin isoform (EDA-FN) and smooth muscle actin (α-SMA). This effect was shown to be mediated by transforming growth factor β1 (TGFβ1), which was activated by matrix metalloprotease 9 (MMP9) in the culture medium. Scavenging of ROS stimulated secretion of MMP9 rather than its processing. The same effect was achieved by the nontargeted antioxidant Trolox at higher concentration, but the thiol antioxidant N-acetylcysteine (NAC) inhibited MMP activity and was not able to induce myofibroblast differentiation. The myofibroblast phenotype was supported due to autocrine TGFβ1-dependent stimulation after removal of SkQ1. It is concluded that ROS scavenging in mitochondria induces TGFβ1-dependent myofibroblast differentiation.

Marginal blebbing during the early stages of TNF-induced apoptosis indicates alteration in actomyosin contractility

Dynamics of alterations of cell surface topography during TNF‐induced apoptosis of HeLa cells was examined by phase‐contrast videomicroscopy and immunomorphological analysis. The final stage of apoptosis accompanied by cell rounding and general blebbing of the cell surface became after 4–6 h of incubation but much earlier, after 1.5–3 h, essentially flattened lamellipodia at the active edges transformed into the small blebs that were continuously extended and retracted during the next 1–2 h. This phenomenon was called “marginal blebbing”. It took place before the cytochrome c release from mitochondria to cytosol. Marginal blebbing was inhibited by drugs that depolymerized actin microfilaments (cytochalasin, latrunculin) or decreased Rho‐kinase‐dependent contractility of actin—myosin cortex (H7, HA‐1077, Y27632). A pancaspase inhibitor, zVAD‐fmk, completely prevented marginal and general blebbing, and TNF‐induced apoptosis. DEVD‐fmk, a specific inhibitor of caspase‐3, inhibited both marginal and general blebbing but not cell rounding and death. Thus, marginal blebbing is an early microfilament‐dependent apoptotic event. It is suggested that it is initiated by minimal activation of caspase‐3 and the following local Rho‐kinase‐dependent stimulation of actin—myosin cortex contractility. Localization of marginal blebs at the active edge may be associated with special organization of cortex in that zone.

Novel Penetrating Cations for Targeting Mitochondria

Novel penetrating cations were used for the design of mitochondria-targeted compounds and tested in model lipid membranes, in isolated mitochondria and in living human cells in culture. Rhodamine-19, berberine and palmatine were conjugated by aliphatic linkers with plastoquinone possessing antioxidant activity. These conjugates (SkQR1,SkQBerb, SkQPalm) and their analogs lacking plastoquinol moiety (C12R1,C10Berb and C10Palm) penetrated bilayer phospholipid membrane in their cationic forms and accumulated in isolated mitochondria or in mitochondria of living cells due to membrane potential negative inside. Reduced forms of SkQR1, SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations. In human fibroblasts SkQR1, SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by hydrogen peroxide. SkQR1 was effective at subnanomolar concentrations while SkQberb, SkQPalm and SkQ1 (prototypic conjugate of plastoquinone with dodecyltriphenylphosphonium) were effective at 10-times higher concentrations. The aliphatic conjugates of berberine and palmatine (as well as the conjugates of triphenylphosphonium) induced proton transport mediated by free fatty acids (FA) both in the model and mitochondrial membrane. In mitochondria this process was facilitated by the adenine nucleotide carrier. In contrast to the other cationic conjugates, SkQR1 and C12R1 induced FA-independent proton conductivity due to protonation/deprotonation of the rhodamine residue. This property in combination with the antioxidant activity probably makes rhodamine conjugates highly effective in protection against oxidative stress. The novel cationic conjugates described here are promising candidates for drugs against various pathologies and aging as mitochondria-targeted antioxidants and selective mild uncouplers.

Scatter factor induces segregation of multinuclear cells into several discrete motile domains

The effects of scatter factor, HGF/SF, on multinuclear MDCK epitheliocytes were examined. Multinuclear cells were obtained by blocking cytokinesis by low concentration of cytochalasin D; these large cells had discoid shape and did not move much on the substrate. Incubation of these cells with HGF/SF induced their profound reorganization: their cytoplasm was reversibly segregated into several individually moving motile flattened domains, termed lamelloplasts and connected with one another by cylindrical domains termed cables. One or several nuclei were present in many lamelloplasts, but some lamelloplasts were anuclear. Nuclei were absent from the cables. Lamelloplasts continuously formed actin-rich ruffles at their edges; their cytoplasm contained small actin bundles and numerous focal adhesions. In contrast, cable, had no ruffles or focal adhesions. Dense networks of vimentin and keratin intermediate filaments were present in lamelloplasts; bundles of filaments of both types were seen in the cables. Segregation was accompanied by redistribution of centrosomes from perinuclear zone into lamelloplasts. As a result each lamelloplast in segregated cell acquired individual complex of centrosome and radiating microtubules. The cables contained numerous parallel microtubules but never had centrosomes. This reorganization of microtubular system was essential for segregation as alterations of shape and actin cytoskeleton were prevented by microtubule specific drugs: colcemid and Taxol (paclitaxel). It is suggested that mechanism of segregation is based on activation of two types of opposite actin reorganization: formation of actin networks in lamelloplasts and their dismantlement in the cables. Spatial distribution of the domains in which these opposite types of reorganizations occur may be regulated by microtubular system. It is also suggested that mechanisms of HGF/SF-induced segregation may be closely related to the mechanisms of important physiological reorganizations of cells, such as polarization of pseudopodial activities in motile cells and cytokinesis.

Effect of microtubule-specific drugs upon spatial organization of extracellular matrix in fibroblastic cultures.

The aim of this investigation was to study the effects of microtubule‐specific drugs, taxol and colcemid, on the distribution of cell‐associated extracellular matrix in dense cultures of fibroblasts. Immunomorphological examination of human seven‐day cultures revealed a dense network of fibronectin and tenascin matrix filaments preferentially oriented in parallel with the long axes of cell bodies. Depolymerization of the microtubular system by colcemid and its disorganization by taxol led to rapid and drastic changes in the organization of matrix network: fibronectin and tenascin filaments became disordered and, in particular, lost any orientation. These data show that the microtubular system controls the morphological organization, not only of intracellular cytoskeletal systems, but also of extracellular matrix structures.

SkBQ - prooxidant addressed to mitochondria.

Oxidative stress and mitochondrial dysfunction are the key links in the chain of development of pathologies associated with the violation of cellular energy metabolism. Development of mitochondria-addressed compounds highly specific for chemical processes is one of the most promising ways to develop approaches to the treatment of inherited and age-related diseases with mitochondrial etiology. Correlation of structure and chemical activity of the test compounds from a class of lipophilic cations revealed the key role of substituents in the aromatic ring of 1,4-benzoquinones in the manifestation of high antioxidant properties. In this work, it is shown that a synthesized benzoquinone derivative conjugated in position 6 with membrane-penetrating cation of decyltriphenylphosphonium and with substituents at position 2, 3, and 5 (SkBQ) has much lower antioxidant and significantly higher prooxidant activity in comparison with similar derivatives of plasto- and toluquinone SkQ1 and SkQT1 in experiments on isolated mitochondria. At the same time, SkBQ, like SkQ1 and SkQT1, can be reduced by the respiratory chain in the center i of complex III and decrease the mitochondrial membrane potential. In cell cultures of human fibroblasts, it was revealed that SkBQ does not protect cells from apoptosis induced by hydrogen peroxide. Under the same conditions, SkQ1 and SkQT1 exhibit a powerful protective effect. Thus, SkBQ can be seen as a mitochondria-addressed prooxidant. The possibility of using SkBQ as an anticancer drug for the treatment of cancers such as prostate cancer whose cells are sensitive to mitochondrial reactive oxygen species is discussed.