Mitochondrion-targeted antioxidant SkQ1 prevents rapid animal death caused by highly diverse shocks.

The response to stress involves the activation of pathways leading either to protection from the stress origin, eventually resulting in development of stress resistance, or activation of the rapid death of the organism. Here we hypothesize that mitochondrial reactive oxygen species (mtROS) play a key role in stress-induced programmed death of the organism, which we called "phenoptosis" in 1997. We demonstrate that the synthetic mitochondria-targeted antioxidant SkQ1 (which specifically abolishes mtROS) prevents rapid death of mice caused by four mechanistically very different shocks: (a) bacterial lipopolysaccharide (LPS) shock, (b) shock in response to intravenous mitochondrial injection, (c) cold shock, and (d) toxic shock caused by the penetrating cation C12TPP. Importantly, under all these stresses mortality was associated with a strong elevation of the levels of pro-inflammatory cytokines and administration of SkQ1 was able to switch off the cytokine storms. Since the main effect of SkQ1 is the neutralization of mtROS, this study provides evidence for the role of mtROS in the activation of innate immune responses mediating stress-induced death of the organism. We propose that SkQ1 may be used clinically to support patients in critical conditions, such as septic shock, extensive trauma, cooling, and severe infection by bacteria or viruses.

[Electrophysiological and psychophysical studies in assessment of visual functions in patients with hereditary optic neuropathy]. / Elektrofiziologicheskie i psikhofizicheskie issledovaniya v otsenke zritel'nykh funktsii pri nasledstvennoi opticheskoi neiropatii.

PURPOSE: To study the capabilities of electrophysiological and psychophysical examination methods for assessment of the functional state of ganglion cells, retina and optic nerve in patients with hereditary optic neuropathy (HON). MATERIAL AND METHODS: The study included 60 patients (118 eyes) with a genetically confirmed diagnosis of HON. All study patients underwent visual field test (VFT), spectral optical coherence tomography (OCT), flash and pattern visual evoked potentials (VEP) (Flash-VEP, FVEP; Pattern-VEP, PVEP), photopic electroretinography with photonegative response (PhNR) registration and the color vision test. In 24 patients (46 eyes), these parameters were assessed before the start of treatment and one year later. The treatment involved the mitochondria-targeted antioxidant SkQ1 - plastoquinonyl-decyl-triphenylphosphonium bromide (PDTP) in the form of eye drops. RESULTS: The main PVEP components for 1.0° and 0.3° were registered in 20% and in 14% of patient eyes with HON and high visual functions, respectively. After one year of PDTP use, a significant decrease in P100 peak latency was found only in the group with disease duration of ≤1.5 years as of the time of treatment start (p<0.05). Significant differences were observed in the PhNR amplitude (p<0.004) between patients of the main and the control groups, as well as in the PhNR amplitude between patients with visual acuity of ≤0.1 and ≥0.13 (p<0.01). Patients with high visual functions were found to have a correlation between the PhNR amplitude, GCC thickness and the global loss index (GLV). CONCLUSION: Along with VFT, OCT and color vision tests, electrophysiological studies are one of the main methods of examining patients with HON. After one year of PDTP use, there was a significant decrease in the FVEP P2 peak latency in the group with a disease duration of ≤1.5 years as of the time of treatment start. The PhNR amplitude in patients with high visual functions was found to correlate with structural changes in the ganglion cell layer and the retinal nerve fiber layer.

Design, Synthesis, and Some Aspects of the Biological Activity of Mitochondria-Targeted Antioxidants.

This review summarizes for the first time data on the design and synthesis of biologically active compounds of a new generation - mitochondria-targeted antioxidants, which are natural (or synthetic) p-benzoquinones conjugated via a lipophilic linker with (triphenyl)phosphonium or ammonium cations with delocalized charge. It also describes the synthesis of mitochondria-targeted antioxidants - uncouplers of oxidative phosphorylation - based on fluorescent dyes.

Programmed Aging of Mammals: Proof of Concept and Prospects of Biochemical Approaches for Anti-aging Therapy.

(i) In 2015-2017 we compared possible reasons for longevity of two mammalian highly social species, i.e. naked mole rats and humans. We proposed that in both cases longevity is a result of neoteny, prolongation of youth by deceleration of late ontogeny (Skulachev, V. P. (2015) Abst. 11th Conf. on Mitochondrial Physiology (MiP2015), Lucni Bouda, Czech Republic, pp. 64-66; Skulachev, V. P., Holtze, S., Vyssokikh, M. Y., Bakeeva, L. E., Skulachev, M. V., Markov, A. V., Hildebrandt, T. B., and Sadovnichii, V. A. (2017) Physiol. Rev., 97, 699-720). Both naked mole rats and humans strongly decreased the pressure of natural selection, although in two different manners. Naked mole rats preferred an "aristocratic" pathway when reproduction (and, hence, involvement in evolution) is monopolized by the queen and her several husbands. Huge number of subordinates who have no right to take part in reproduction and hence in evolution serves the small queen's family. Humans used an alternative, "democratic" pathway, namely technical progress facilitating adaptation to the changing environmental conditions. This pathway is open to all humankind. (ii) As a result, aging as a mechanism increasing evolvability by means of facilitation of natural selection became unnecessary for naked mole rats and humans due to strong attenuation of this selection. This is apparently why aging became a counterproductive atavism for these two species and was strongly shifted to late ages. This shift is direct evidence of the hypothesis that aging is programmed, being the last step of late ontogeny. (iii) Further deceleration of aging for humans by means of neoteny is unrealistic since the development of neoteny probably takes million years. (iv) However, if biological aging is a program, an alternative and much simpler way to avoid it seems possible. We mean inhibition of an essential step of this program. (v) At present, the most probable scheme of the aging program assumes that it is a mechanism of slow poisoning of an organism by reactive oxygen species produced by mitochondria. If this is the case, a mitochondria-targeted antioxidant might be an inhibitor of the aging program. During the last 12 years, such an antioxidant, namely SkQ1, was synthesized and studied in detail in our group. It consists of plastoquinone and decyltriphenylphosphonium (a penetrating cation responsible for electrophoretic accumulation of SkQ1 in mitochondria). It was shown that long-term treatment with SkQ1 increased the lifespan of plants, fungi, invertebrates, fish, and mammals. Moreover, SkQ1 is effective in the therapy of various age-related diseases. It was also shown that a single SkQ1 injection could save life in certain models of sudden death of animals. (vi) A tentative scheme is proposed considering aging as a process of chronic phenoptosis, which eventually results in initiation of acute phenoptosis and death. This scheme also suggests that under certain conditions chronic phenoptosis can be neutralized by an anti-aging program that is activated by food restriction regarded by an organism as a signal of starvation. As for acute phenoptosis, it is apparently controlled by receptors responsible for measuring key parameters of homeostasis. The first experimental indications have been already obtained indicating that both chronic and acute phenoptosis are suppressed by SkQ1.

Mitochondrial Genome and Longevity.

The mitochondrial genome provides not only respiratory chain function, but it also ensures the impact of mitochondria on nearly all crucial metabolic processes. It is well known that mitochondria regulate aging and lifespan. However, until now there were no direct experimental data concerning the influence of various mitochondrial DNA variants on lifespan of animals with identical nuclear genome. In a recent paper of J. A. Enríquez and coworkers (Latorre-Pellicer, A., et al. (2016) Nature, 535, 561-565), it was shown that mice carrying nuclear DNA from one strain and mitochondrial DNA from another had longer median lifespan and retarded development of various aging traits. This review critically analyzes that paper and considers some aspects of the crosstalk between the nuclear and mitochondrial genomes. We also discuss new perspectives of gerontology in the light of the discovery made by Enríquez's group.

Influence of 10-(6'-Plastoquinonyl)decyltriphenylphosphonium (SkQ1) on Oxidative Status in Rats with Protamine Sulfate-Induced Hyperglycemia.

An influence of 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) on oxidative status and activity of some antioxidant enzymes in the liver and blood serum from rats was examined during experimental hyperglycemia developed after injecting protamine sulfate. It was found that SkQ1 lowered glycemic level in rats treated with protamine sulfate. Moreover, it was also accompanied by restoration of the normal range of biochemiluminescence parameters indicating the rate of ongoing free radical processes, magnitude of primary products of lipid peroxidation such as diene conjugates, activity of aconitate hydratase, and level of citrate in rat liver and blood. Hence, it was demonstrated that activity of superoxide dismutase and catalase, increasing during hyperglycemia, was decreased after administering SkQ1. This might be related to the ability of SkQ1 to normalize free-radical homeostasis imbalanced during hyperglycemia.

New data on programmed aging - slow phenoptosis.

This review summarizes the latest data on biochemistry and physiology of living organisms. These data suggest that aging, i.e. coordinated age-dependent weakening of many vital functions leading to gradual increase in the probability of dying, is not common to all organisms. Some species have been described whose probability of death does not depend on age or even decreases with age, this being accompanied by constant or increasing fertility. In the case of the naked mole rat (a non-aging mammal), a mechanism has been identified that protects this animal from cancer and the most common age-related diseases. The high molecular weight polysaccharide hyaluronan, a linear polymer composed of multiple repeated disaccharide of glucuronic acid and glucosamine, plays the key role in this mechanism. Hyaluronan is accumulated in the intercellular spaces in the organs and tissues of the naked mole rat. This polysaccharide provides early contact inhibition of cell division (anti-cancer effect). In addition, hyaluronan prevents the development of certain types of apoptosis, in particular, those induced by reactive oxygen species (ROS) (geroprotective effect preventing ROS-induced decrease in cellularity in the organs and tissues of aging organisms). Extraordinary longevity of the naked mole rat (over 30 years, which is long for a rodent the size of a mouse) is connected to its eusocial lifestyle, when only the "queen" and its few "husbands" breed, while the huge army of non-breeding "subordinates" provide the "royal family" with protection from predators, food, and construction and maintenance of an underground labyrinth size of a football field. This way of life removes the pressure of natural selection from the "family" and makes aging - the program that is counterproductive for the individual but increases "evolvability" of its offspring - unnecessary. The example of the naked mole rat demonstrates the optional character of the aging program for the organism. Many facts indicating that aging can be regulated by an organism provide another argument in favor of optionality of aging. Cases have been described when aging as a program useful for the evolution of offspring but counterproductive for the parental individual slows under conditions that threaten the very existence of the individual. These conditions include food restriction (the threat of death from starvation), heavy muscular work, decrease or increase in the environmental temperature, small amounts of poisons (including ROS; here we speak about the paradoxical geroprotective effect of the low doses of prooxidants that inhibit apoptosis). On the other hand, aging can be inhibited (and maybe even cancelled) artificially. This can be done by turning off the genes encoding the proteins participating in the aging program, such as FAT10, p66shc, and some others. In addition, the gene of the antioxidant enzyme catalase can be addressed into mitochondria, where it will split mitochondrial hydrogen peroxide, the level of which increases with age. However, today the simplest way to slow down the aging program is the use of mitochondria-targeted low molecular weight antioxidant compounds of plastoquinonyl decyltriphenylphosphonium-type (SkQ1), which prolong the life of animals, plants, and fungi and inhibit the development of many age-related diseases and symptoms.

Receptor regulation of senile phenoptosis.

Here we present a concept that considers organism aging as an additional facultative function promoting evolution, but counterproductive for an individual. We hypothesize that aging can be inhibited or even arrested when full mobilization of all resources is needed for the survival of an individual. We believe that the organism makes such a decision based on the analysis of signals of special receptors that monitor a number of parameters of the internal and external environment. The amount of available food is one of these parameters. Food restriction is perceived by the organism as a signal of coming starvation; in response to it, the organism inhibits its counterproductive programs, in particular, aging. We hypothesize that the level of protein obtained with food is estimated based on blood concentration of one of the essential amino acids (methionine), of carbohydrates - via glucose level, and fats - based on the level of one of the free fatty acids. When the amount of available food is sufficient, these receptors transmit the signal allowing aging. In case of lack of food, this signal is cancelled, and as a result aging is inhibited, i.e. age-related weakening of physiological functions is inhibited, and lifespan increases (the well-known geroprotective effect of partial food restriction). In Caenorhabditis elegans, lowering of the ambient temperature has a similar effect. This geroprotective effect is removed by the knockout of one of the cold receptors, and replacement of the C. elegans receptor by a similar human receptor restores the ability of low temperature to increase the lifespan of the nematode. A chain of events linking the receptor with the aging mechanism has been discovered in mice - for one of the pain receptors in neurons, the nerve endings of which entwine pancreas ß-cells. Age-related activation of these receptors inhibits the work of insulin genes in ß-cells. Problems with insulin secretion lead to oxidative stress, chronic inflammation, and type II diabetes, which can be regarded as one of the forms of senile phenoptosis. In conclusion, we consider the role of some psychological factors in the regulation of the aging program.

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants.

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273-1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.

Lithium salts -- simple but magic.

For many decades pharmacological drugs based on lithium salts have been successfully used in psychiatry to treat bipolar disorder, and they remain the "gold standard" of pharmacological therapy of patients with this disease. At the same time, over recent years in experiments in vitro and in vivo a plethora of evidence has accumulated on a positive effect of lithium ions in other areas including their neuro-, cardio-, and nephroprotective properties, regulation of stem cells functions, regulation of inflammation, and others. Numerous studies have shown that the effect of lithium ions involves several mechanisms; however, one of its main targets in the implementation of most of the effects is glycogen synthase kinase 3ß, a key enzyme in various pathological and protective signaling pathways in cells. However, one of the main limitations of the use of lithium salts in clinics is their narrow therapeutic window, and the risk of toxic side effects. This review presents the diversity of effects of lithium ions on the organism emphasizing their potential clinical applications with minimal undesirable side effects. In the end, we present a schematic "Lithiometer", comparing the range of Li(+) concentrations that might be used for the treatment of acute pathologies with possible toxic effects of Li(+).

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.

Novel mitochondria-targeted compounds composed of natural constituents: conjugates of plant alkaloids berberine and palmatine with plastoquinone.

Novel mitochondria-targeted compounds composed entirely of natural constituents have been synthesized and tested in model lipid membranes, in isolated mitochondria, and in living human cells in culture. Berberine and palmatine, penetrating cations of plant origin, were conjugated by nonyloxycarbonylmethyl residue with the plant electron carrier and antioxidant plastoquinone. These conjugates (SkQBerb, SkQPalm) and their analogs lacking the plastoquinol moiety (C10Berb and C10Palm) penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in isolated mitochondria or in mitochondria in living human cells in culture. Reduced forms of SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations. In isolated mitochondria and in living cells, the berberine and palmatine moieties were not reduced, so antioxidant activity belonged exclusively to the plastoquinol moiety. In human fibroblasts, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide. At higher concentrations, conjugates of berberine and palmatine induced proton transport mediated by free fatty acids both in model and in mitochondrial membrane. In mitochondria this process was facilitated by the adenine nucleotide carrier. As an example of application of the novel mitochondria-targeted antioxidants SkQBerb and SkQPalm to studies of signal transduction, we discuss induction of cell cycle arrest, differentiation, and morphological normalization of some tumor cells. We suggest that production of oxygen radicals in mitochondria is necessary for growth factors-MAP-kinase signaling, which supports proliferation and transformed phenotype.

Mitochondrial-targeted plastoquinone derivatives. Effect on senescence and acute age-related pathologies.

Plastoquinone, a very effective electron carrier and antioxidant of chloroplasts, was conjugated with decyltriphenylphosphonium to obtain a cation easily penetrating through membranes. This cation, called SkQ1, is specifically targeted to mitochondria by electrophoresis in the electric field formed by the mitochondrial respiratory chain. The respiratory chain also regenerates reduced SkQ1H(2) from its oxidized form that appears as a result of the antioxidant activity of SkQ1H(2). SkQ1H(2) prevents oxidation of cardiolipin, a mitochondrial phospholipid that is especially sensitive to attack by reactive oxygen species (ROS). In cell cultures, SkQ1 and its analog plastoquinonyl decylrhodamine 19 (SkQR1) arrest H(2)O(2)-induced apoptosis. When tested in vivo, SkQs (i) prolong the lifespan of fungi, crustaceans, insects, fish, and mice, (ii) suppress appearance of a large number of traits typical for age-related senescence (cataract, retinopathies, achromotrichia, osteoporosis, lordokyphosis, decline of the immune system, myeloid shift of blood cells, activation of apoptosis, induction of ß-galactosidase, phosphorylation of H2AX histones, etc.) and (iii) lower tissue damage and save the lives of young animals after treatments resulting in kidney ischemia, rhabdomyolysis, heart attack, arrhythmia, and stroke. We suggest that the SkQs reduce mitochondrial ROS and, as a consequence, inhibit mitochondria-mediated apoptosis, an obligatory step of execution of programs responsible for both senescence and fast "biochemical suicide" of an organism after a severe metabolic crisis.

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, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-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 Deltapsi 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 1.3x10(8) 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.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke).

Effects of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6'-plastoquinonyl) decylrhodamine 19 (SkQR1) on rat models of H2O2- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H2O2 or by ischemia/reperfusion. Higher SkQ1 (125-250 nmol/kg per day for 2-3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2-4 days, whereas one injection of SkQ1 or SkQR1 (1 micromol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 micromol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound.

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.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals.

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age-induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 microM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 5. SkQ1 prolongs lifespan and prevents development of traits of senescence.

Very low (nano- and subnanomolar) concentrations of 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1) were found to prolong lifespan of a fungus (Podospora anserina), a crustacean (Ceriodaphnia affinis), an insect (Drosophila melanogaster), and a mammal (mouse). In the latter case, median lifespan is doubled if animals live in a non-sterile vivarium. The lifespan increase is accompanied by rectangularization of the survival curves (an increase in survival is much larger at early than at late ages) and disappearance of typical traits of senescence or retardation of their development. Data summarized here and in the preceding papers of this series suggest that mitochondria-targeted antioxidant SkQ1 is competent in slowing down execution of an aging program responsible for development of age-related senescence.

New viral vector for efficient production of target proteins in plants.

A new potato virus X (PVX)-based viral vector for superproduction of target proteins in plants has been constructed. The triple gene block and coat protein gene of PVX were substituted by green fluorescent protein. This reduced viral vector was delivered into plant cells by agroinjection (injection of Agrobacterium tumefaciens cells, carrying viral vector cDNA within T-DNA, into plant leaves), and this approach allowed to dramatically reduce the size of the vector genome. The novel vector can be used for production of different proteins including pharmaceuticals in plants.

An internal ribosome entry site located upstream of the crucifer-infecting tobamovirus coat protein (CP) gene can be used for CP synthesis in vivo.

It was previously shown that, unlike the type member of the genus Tobamovirus (TMV U1), a crucifer-infecting tobamovirus (crTMV) contains a 148 nt internal ribosome entry site (IRES)(CP,148)(CR) upstream of the coat protein (CP) gene. Here, viral vectors with substitutions in the stem-loop (SL) region of CP subgenomic promoters (TMV U1-CP-GFP/SL-mut and crTMV-CP-GFP/SL-mut) were constructed and the levels of CP synthesis in agroinoculation experiments were compared. No CP-GFP (green fluorescent protein) synthesis was detected in Nicotiana benthamiana leaves inoculated with TMV U1-CP-GFP/SL-mut, whereas a small amount of CP-GFP synthesis was obtained in crTMV-CP-GFP/SL-mut-injected leaves. Northern blots proved that both promoters were inactive. It could be hypothesized that IRES-mediated early production of the CP by crTMV is needed for realization of its crucifer-infecting capacity.