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Multiple sclerosis (MS) is a heterogeneous autoimmune disease of unknown etiology characterized by inflammation, demyelination, and axonal degeneration that affects both the white and gray matter of CNS. Recent large-scale epidemiological and genomic studies identified several genetic and environmental risk factors for the disease. Among them are environmental factors of infectious origin, possibly causing MS, which include Epstein-Barr virus infection, reactivation of some endogenous retrovirus groups, and infection by pathogenic bacteria (mycobacteria, Chlamydia pneumoniae, and Helicobacter pylori). However, the nature of the events leading to the activation of immune cells in MS is mostly unknown and there is no effective therapy against the disease. Amazingly, whatever the cause of the disease, signs of damage to the nerve tissue with MS lesions were the same as with infectious leprosy, while in the latter case nitrozooxidative stress was suggested as the main cause of the nerve damage. With this in mind and following the hypothesis that excessive production of mitochondrial reactive oxygen species critically contributes to MS pathogenesis, we studied the effect of mitochondria-targeted antioxidant SkQ1 in an in vitro MS model of the primary oligodendrocyte culture of the cerebellum, challenged with lipopolysaccharide (LPS). SkQ1 was found to accumulate in the mitochondria of oligodendrocytes and microglial cells, and it was also found to prevent LPS-induced inhibition of myelin production in oligodendrocytes. The results implicate that mitochondria-targeted antioxidants could be promising candidates as components of a combined therapy for MS and related neurological disorders.

Radioprotection appeared to be an important problem of today due to atom energetic development and utilization of radiation material in the industry, science and medicine. It has been shown that mitochondrial targeted antioxidant SkQR1 could attenuate radiation injury of human erythroleukemia K562 cells. Pretreatment with SkQR1 before irradiation decreased DNA double strand breaks formation, diminished the number of chromosomal aberrations and suppressed delayed ROS production. Prevention of oxidative stress and normalization of mitochondrial function by mitochondria-targeted antioxidants may be a potential therapeutic strategy not only against immediate consequences of radiation, but, either against its late consequences such as genomic instability. SkQR1 did not protect against radiation-induced damage the K562 subline with high level of multidrug resistance (MDR) due to SkQR1 extrusion with Pgp 170 MDR pump. We suggest that mitochondria-targeted antioxidants might be used for selective protection of normal cells against radiation-induced damage without interference with radiotherapy of MDR-positive tumors.

We show here that mitochondria-targeted antioxidant composed of plastoquinone conjugated through hydrocarbon linker with cationic rhodamine 19 (SkQR1) protected against nuclear DNA damage induced by gamma radiation in K562 erythroleukemia cells. We also demonstrate that SkQR1 prevented the early (1 h postirradiation) accumulation of phosphorylated histone H2AX (γ-H2AX) an indicator of DNA double-strand break formation, as well as the radiation-induced increase in chromosomal aberrations. These data suggested that nuclear DNA damage induced by gamma radiation may be mediated by mitochondrial reactive oxygen species (ROS) production. We show that SkQR1 suppressed delayed accumulation of ROS 32 h after irradiation probably by inhibiting mitochondrial ROS-induced ROS release mechanisms. This suggests that mitochondria-targeted antioxidants may protect cells from the late consequences of radiation exposure related to delayed oxidative stress. We have previously reported that SkQRl is the substrate of multidrug resistance pump P-glycoproten (Pgp 170) and selectively protects Pgp 170-negative cells against oxidative stress. In line with this finding, we demonstrate here that SkQR1 did not protect Pgp170-positive K562 subline against DNA damage induced by gamma radiation. The selective radioprotection of normal Pgp 170-negative cells by mitochondria-targeted antioxidants could be a promising strategy to increase the efficiency of radiotherapy for multidrug-resistant tumors.

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.

Mitochondria-targeted antioxidants of the SkQ family that accumulate in energized mitochondria protect cells from oxidative stress by increasing the level of reduced glutathione and decreasing cell damage induced by hydrogen peroxide. The exposure of various human transformed cell lines to SkQR1, a fluorescent member of the SkQ family, showed that SkQR1 was pumped out of the chemotherapy resistant cells by P-glycoprotein, one of the main transport proteins that determines multidrug resistance typical for many neoplastic cells. It was also shown that SkQR1 pumping is neutralized by P-glycoprotein inhibitors (verapamil and pluronic L61). In experiments on K-562 cells, it was found that the subline sensitive to chemotherapy is protected by SkQR1 from apoptosis induced by hydrogen peroxide. The protection of resistant subline cells is only evident after the inhibition of P-glycoprotein.

A conjugate of plastoquinone with decylrhodamine 19 (SkQR1) selectively accumulates in mitochondria of normal and tumor cells. SkQR1 protected the cellular pool of reduced glutathione under oxidative stress. Overexpression of P-glycoprotein (Pgp 170) multidrug resistance pump strongly suppresses accumulation of SkQR1. The inhibitors of Pgp 170 stimulate accumulation of SkQR1 in various cell lines indicating that SkQR1 is a substrate of Pgp 170. The protective effect of SkQR1 against oxidative stress is diminished in the cells overexpressing Pgp 170. It is suggested that mitochondria-targeted antioxidants could selectively protect normal (Pgp 170-negative) cells against the toxic effect of anti-cancer treatments related to oxidative stress.

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.

We studied the interaction of two main cell types, epitheliocytes and fibroblasts, in a mixed culture. Heterotypic cells had a different cytoskeleton organization and expressed different cell adhesion molecules, cadherins. In spite of this, when the cells contacted in the mixed cultures, a heterophilic contact was formed and the actin cytoskeleton of an epitheliocyte at the site of contact was reorganized: the marginal actin bundle was decomposed and actin structures were formed in its place, that were typical for the fibroblast lamella. No changes were observed in the actin organization of the fibroblast. In architecture, the heterophilic adhesion contacts resembled the contacts between fibroblasts. Both heterophilic and homophilic contacts were transient, rather than constant structures. The formation of heterophilic contacts in mixed cultures can serve as a model of formation of a tissue system consisting of epithelium and mesenchyme.

Contact interactions between different cell types play a number of important roles in development, for example in cell sorting, tissue organization, and ordered migration of cells. The nature of such heterocellular interactions, in contrast to interactions between cells of the same type, remains largely unknown. In this report, we present experimental data examining the dynamics of heterocellular interactions between epitheliocytes and fibroblasts, which express different cadherin cell adhesion molecules and possess different actin cytoskeletal organizations. Our analysis revealed two striking features of heterocellular contact. First, the active free edge of an epitheliocyte reorganizes its actin cytoskeleton after making contact with a fibroblast. Upon contact with the leading edge of a fibroblast, epitheliocytes disassemble their marginal bundle of actin filaments and reassemble actin filaments into a geometric organization more typical of a fibroblast lamella. Second, epitheliocytes and fibroblasts form cell–cell adhesion structures that have an irregular organization and are associated with components of cell adhesion complexes. The structural organization of these adhesions is more closely related to the type of contacts formed between fibroblasts rather than to those between epitheliocytes. Heterotypic epithelio-fibroblastic contacts, like homotypic contacts between fibroblasts, are transient and do not lead to formation of stable contact interactions. We suggest that heterocellular contact interactions in culture may be regarded as models of how tissue systems consisting of epithelia and mesenchyme interact and become organized in vivo .

Co-cultured contacting heterotypic cell groups compete with one another for the attachment to the substratum. In the course of this competition one cell group can progressively push another from the substratum. Alterations to the distribution of cell-associated matrix structures in the course of contact competition were examined by immunomorphological methods in the paired cultures of two epithelial lines and those of fibroblasts and epithelia. It was found that matrix structures formed by the retreating cells withdrew from the substratum simultaneously with the cells. These results show that two forms of matrix structures should be distinguished: the usually immovable matrix upon which the cells crawl and the movable matrix that the cells carry with them when they move.

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

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.

Co-cultured epithelial cells of various lines formed mixed cohesive sheets. Contact interactions of different cells in the sheets were studied by phase contrast and interference reflection microscopy, time-lapse microcinematography, transmission and scanning electron microscopy. It was found that heterotypic cells in the combined sheets were locked together by the complexes of specialized contacts and were metabolically coupled, as shown by the dye transfer. At the same time heterotypic cells continued to extend pseudopods at the contacting lateral surfaces and to attach these pseudopodies to substratum. Due to competition of pseudopod attachments one group of cells in the sheet often progressively pushed another group, from the substratum. This competition of cells for substratum may provide an important mechanism for morphogenetic reorganisation of epithelial sheets and of other groups of interacting cells in vivo.

Stimulation of the DNA synthesis and mitoses in stationary cultures of mouse embryo fibroblast-like cells was induced by various agents such as ribonuclease, digitonin, fresh medium and commercial preparations of hyaluronidases. Time sequence of stimulation was similar in experiments with all these agents. Cells were activated to enter S phase from GI phase. The rise of the number of DNA-synthesizing cells was preceded by a latent period of about 8–12 hours with the maximal number of DNA-synthesizing cells being observed at 16–24 hours. Mitotic wave was observed after the wave of DNA synthesis. Stimulation of DNA synthesis and mitosis was not preceded by any significant decrease of an average cell density in the culture. The progeny of activated cells had no greater chance than other cells to be activated again when stimulation was repeated. It is concluded that similar proliferative reactions can be induced in stationary cultures by a variety of diverse agents. Possible role of cell surface changes in the induction of these reactions is discussed.