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.

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.

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.

Uncoupling effect of lauryl sulfate on mitochondria can be mediated by release of bound endogenous fatty acids.

The mechanism of uncoupling by lauryl sulfate (LS) has been studied. The very fact that uncoupling by low concentration of LS (a strong acid) resembles very much that by fatty acids (weak acids) was used as an argument against the fatty acid cycling scheme of uncoupling where protonated fatty acids operate as a protonophore. We have found that rat liver and heart muscle mitochondria can be uncoupled by low (70 microM) LS concentration in a fashion completely arrested by the ATP/ADP antiporter inhibitor carboxyatractylate (CAtr). On the other hand, uncoupling by two-fold higher LS concentration is not sensitive to CAtr. Addition of oleate desensitizes mitochondria to low LS so that addition of bovine serum albumin becomes necessary to recouple mitochondria. The data are accounted for assuming that low LS releases endogenous fatty acids from some mitochondrial depots, and these fatty acids are responsible for uncoupling. As to high LS, it causes a nonspecific (CAtr-insensitive) damage to the mitochondrial membrane.

[Cytotoxic action of polymorphonuclear leukocytes on tumor and normal cells during ascite tumor development in vitro and in vivo].

A vast number of studies, including the authors' own research, support the important role polymorphonuclear leukocytes (PMNL) in the development of ascite tumors. The method of luminol-dependent chemiluminescence (CL) was used to show the presence of two functionally different PMNL pools in a tumor-bearing organism: 1) "primed" PMNL, which circulate in the blood stream, and 2) "activated" PMNL, which are accumulated in the tumor zone and are capable of spontaneous CL. The purpose of the present investigation was to compare cytotoxic effects of primed and activated PMNL on tumor cells (ascite Ehrlich carcinoma (AEC), ascite Zajdel hepatoma) upon co-cultivation, as well as on normal cells of the organism, erythrocytes in vitro and in vivo. Upon stimulation with phorbol myristate acetate (PMA), PMNL effectively damaged AEC cells within the first 24 h until PMNL apoptosis occurred. Upon further co-cultivation, the tumor cells grew in number, which suggest the participation of PMNL in tumor protection. When stimulated with PMNL, pools suppressed tumor growth in vitro, since in this case the cytotoxicity was due to both reactive oxygen species and proteolytic enzymes. As it has been shown earlier by the authors, the functional potential of PMNL increases many times during tumor growth, and we suggested that not only tumor but also normal cells could be damaged. In this connection, we have studied the cytotoxic effect of primed and activated PMNL on rat erythrocytes in vitro on their co-cultivation. On stimulation with PMA, the rate of lysis of erythrocytes by primed PMNL increase many times compared to the norm. The fMLP-stimulated cytotoxity was 1.5-2.0 times higher than in the norm. Activated PMNL without stimulation are capable of producing only a partial lysis of erythrocytes (5-7 %). In order to assess the cytotoxic action of PMNL on erythrocytes in vivo, the hemoglobin content in erythrocytes and blood plasm of rats was measured in the course of tumor growth. The hemoglobin content in erythocytes during growth tumor decreased from 135 +/- 10 to 85 +/- 5 g/l, whereas in the blood plasm the hemoglobin content gradually increased by almost two times. The results enable us to suggest that one of death causes of tumor-bearing organisms may be the cytotoxic action of PMNL on normal cells of the organism caused by hyperproduction of ROS.

Generation of reactive oxygen species by polymorphonuclear leukocytes and its modulation by calcium ions during tumor growth.

The generation of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNL) and the role of Ca2+ in regulating their activity during Zajdela hepatoma growth in the animal peritoneal cavity were studied. We found a marked increase in the ROS-generating activity of PMNL in circulating blood, the result of increases in both the specific activity of leukocytes and total number of PMNL in circulating blood. The ROS-generating activity of PMNL was substantially activated by Ca2+ ions and a calcium ionophore (ionomycin), but this effect virtually completely disappeared during tumor growth. Perhaps the high ROS-generating activity of PMNL and the lack of the sensitivity to extracellular Ca2+ during tumor growth in the organism are due to an accumulation of intracellular Ca2+ ions.

[Generation of reactive forms of oxygen by polymorphonuclear leukocytes during hepatoma growth in the peritoneal cavity of animals]. / Generatsiia reaktivnykh form kisloroda polimorfnoiadernymi leikotsiamiv protsesse razvitiia gepatomy v briushnoi polosti zhivotnykh.

In the present work, an attempt was made to analyse generation of reactive oxygen species (ROS) by polymorphonuclear leucocytes (PMN) in the course of tumour growth, using chemiluminescence (CL). A multiple increase in the capacity of polymorphonuclear leucocytes of generating active forms of oxygen in the course of tumor growth was discovered. Two causes of this process were found. 1) the increase in specific activity of leucocytes; 2) the increase in the total quantity of PMN circulating in the blood. Leucocytes were also found in the ascite liquid. PMN leucocytes were shown to participate in the antitumor defence of the organism.

[The effect of catecholamines on the luminol-dependent chemiluminescence of rat polymorphonuclear leukocytes]. / Vliianie katekholaminov na liuminolzavisimuiu khemiliuminestsentsiiu polimorfnoiadernykh leikotsitov krysy.

The effects of catecholamines on reactive oxygen species (ROS) generation by polymorphonuclear leucocytes were studied using chemiluminescence (CL). It has been found that catecholamines in 10(-9)-10(-10) M concentration range are able to increase reactive oxygen species generation. Within the micromolar concentration range (10(-6)-10(-5) M) cetacholamines (CA) strongly suppress CL intensity and ROS production. The efficiency of inhibitory action of CA: noradrenaline > adrenaline > dopamine > L-DOPA correspond to their redox properties. A conclusion is made that peroxidase is one of the main targets of CA effect.