Domnina Lv

Mitochondria-Targeted Plastoquinone Derivatives as Tools to Interrupt Execution of the Aging Program. 1. Cationic Plastoquinone Derivatives: Synthesis and in vitro Studies*

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the “window” between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH·. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C1/2 values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1·10−11 and 8·10−13 M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Δψ values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000: 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3·108 times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

Thread-grain transition of mitochondrial reticulum as a step of mitoptosis and apoptosis

Association of mitochondrial population to a mitochondrial reticulum is typical of many types of the healthy cells. This allows the cell to organize a united intracellular power-transmitting system. However, such an association can create some difficulties for the cell when a part of the reticulum is damaged or when mitochondria should migrate from one cell region to another. It is shown that in these cases decomposition of extended mitochondria to small roundish organelles takes place (the thread-grain transition). As an intermediate step of this process, formation of bead-like mitochondria occurs when several swollen parts of the mitochondrial filament are interconnected with thin thread-like mitochondrial structures. A hypothesis is put forward that the thread-grain transition is used as a mechanism to isolate a damaged part of the mitochondrial system from its intact parts. If the injury is not repaired, spherical mitochondrion originated from the damaged part of the reticulum is assumed to convert to a small ultracondensed and presumably dead mitochondrion (this process is called ‘mitoptosis’). Then the dead mitochondrion is engulfed by an autophagosome. Sometimes, an ultracondensed mitoplast co-exists with a normal mitoplast, both of them being surrounded by a common outer mitochondrial membrane. During apoptosis, massive thread-grain transition is observed which, according to Youle et al. (S. Frank et al., Dev Cell 1: 515, 2002), is mediated by a dynamin-related protein and represents an obligatory step of the mitochondria-mediated apoptosis. We found that there is a lag phase between addition of an apoptogenic agent and the thread-grain transition. When started, the transition occurs very fast. It is also found that this event precedes complete de-energization of mitochondria and cytochrome c release to cytosol. When formed, small mitochondria migrate to (and in certain rare cases even into) the nucleus. It is suggested that small mitochondria may serve as a transportable form of organelles (‘cargo boats’ transporting some apoptotic proteins to their nuclear targets).

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53-/- mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53-/- xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53-/- and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53-/- tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

In search of novel highly active mitochondria-targeted antioxidants: thymoquinone and its cationic derivatives.

Since the times of the Bible, an extract of black cumin seeds was used as a medicine to treat many human pathologies. Thymoquinone (2‐demethylplastoquinone derivative) was identified as an active antioxidant component of this extract. Recently, it was shown that conjugates of plastoquinone and penetrating cations are potent mitochondria‐targeted antioxidants effective in treating a large number of age‐related pathologies. This review summarizes new data on the antioxidant and some other properties of membrane‐penetrating cationic compounds where 2‐demethylplastoquinone substitutes for plastoquinone. It was found that such a substitution significantly increases a window between anti‐ and prooxidant concentrations of the conjugates. Like the original plastoquinone derivatives, the novel compounds are easily reduced by the respiratory chain, penetrate through model and natural membranes, specifically accumulate in mitochondria in an electrophoretic fashion, and strongly inhibit H2O2‐induced apoptosis at pico‐ and nanomolar concentrations in cell cultures. At present, cationic demethylplastoquinone derivatives appear to be the most promising mitochondria‐targeted drugs of the quinone series.

Long-distance apoptotic killing of cells is mediated by hydrogen peroxide in a mitochondrial ROS-dependent fashion

Long-distance apoptotic killing of cells is mediated by hydrogen peroxide in a mitochondrial ROS-dependent fashion

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6′-ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in “organoptosis”, i.e. disappearance of organs during ontogenesis.

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.

Effects of the inhibitors of dynamics of cytoskeletal structures on the development of apoptosis induced by the tumor necrosis factor.

Changes in cytoskeletal structures have been investigated during apoptosis of epithelial HeLa cells induced by tumor necrosis factor-α (TNF-α). Shape and surface cell activity were investigated by time-lapse video microscopy, and changes of the cytoskeletal structure were studied by immune fluorescent microscopy. Addition of TNF-α to HeLa cell culture caused early disruption of the actin cytoskeleton and vinculin-containing focal contacts, keratin filaments, and microtubules. Rounding of cells, general blebbing, and nuclear fragmentation were observed at the terminal apoptotic stages. Actomyosin complex inhibitors, H7 and HA1077, suppressed blebbing (but not cell rounding) and activated the development of apoptosis. The latter suggests that in contrast to blebbing the general rounding does not depend on increased contractility of actomyosin cortex. These cytoskeletal inhibitors accelerated the development of apoptosis of HeLa cells and increased sensitivity of HeLa-Bcl-2 cells (transfected with DNA encoding antiapoptotic protein Bcl-2) to TNF-induced apoptosis. Damage of cytoskeletal structures significantly attenuated antiapoptotic activity of Bcl-2 in the HeLa-Bcl-2 cells. It is suggested that the stimulation of apoptosis by cytoskeletal inhibitors may be attributed to the altered distribution of cell organelles, especially, mitochondria.