Vera Dugina

β- and γ-cytoplasmic actins display distinct distribution and functional diversity

Using newly generated monoclonal antibodies, we have compared the distribution of β- and γ-cytoplasmic actin in fibroblastic and epithelial cells, in which they play crucial roles during various key cellular processes. Whereas β-actin is preferentially localized in stress fibers, circular bundles and at cell-cell contacts, suggesting a role in cell attachment and contraction, γ-actin displays a more versatile organization, according to cell activities. In moving cells, γ-actin is mainly organized as a meshwork in cortical and lamellipodial structures, suggesting a role in cell motility; in stationary cells, γ-actin is also recruited into stress fibers. β-actin-depleted cells become highly spread, display broad protrusions and reduce their stress-fiber content; by contrast, γ-actin-depleted cells acquire a contractile phenotype with thick actin bundles and shrinked lamellar and lamellipodial structures. Moreover, β- and γ-actin depleted fibroblasts exhibit distinct changes in motility compared with their controls, suggesting a specific role for each isoform in cell locomotion. Our results reveal new aspects of β- and γ-actin organization that support their functional diversity.

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.

The N-terminal Ac-EEED sequence plays a role in alpha-smooth-muscle actin incorporation into stress fibers.

We have previously shown that the N-terminal sequence AcEEED of α-smooth-muscle actin causes the loss of α-smooth-muscle actin from stress fibers and a decrease in cell contractility when introduced in myofibroblasts as a cell-penetrating fusion peptide. Here, we have investigated the function of this sequence on stress fiber organization in living cells, using enhanced green fluorescent protein (EGFP)-tagged α-smooth-muscle actin. The fusion peptide provokes the gradual disappearance of EGFP fluorescence of α-smooth-muscle actin from stress fibers and the formation of hitherto unknown rod-like structures. In addition to α-smooth-muscle actin, these structures contain cytoplasmic actins, gelsolin and cofilin but not other major actin-binding proteins. These rod-like structures are also visible in wild-type fibroblasts during normal cell spreading, suggesting that they represent a physiological step in the organization of α-smooth-muscle actin in stress fibers. Fluorescence-recovery-after-photobleaching experiments suggest that the fusion peptide reduces the dynamics of α-smooth-muscle actin and its incorporation in stress fibers. Here, we propose a new mechanism of how α-smooth-muscle actin is incorporated in stress fibers involving the sequence Ac-EEED.

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.

Actin isoforms and reorganization of adhesion junctions in epithelial-to-mesenchymal transition of cervical carcinoma cells

Malignant cell transformation requires changes in the ability of cells to migrate. The disruption of actin cytoskeleton and intercellular adhesions is an important component of the acquisition of invasive properties in epithelial malignancies. The invasive ability of carcinoma cells is associated with reduced expression of adhesion junction molecules and increased expression of mesenchymal markers, frequently referred to as epithelial-to-mesenchymal transition (EMT). Standard features of the EMT program in cancer cells include fibroblastic phenotype, downregulation of the epithelial marker E-cadherin, induction of Snail-family transcription factors, as well as expression of mesenchymal proteins. We compared the epithelial and mesenchymal marker profiles of nonmalignant HaCaT keratinocytes to the corresponding profiles of cervical carcinoma cell lines C-33A, SiHa, and CaSki. The characteristics of the EMT appeared to be more developed in SiHa and CaSki cervical cancer cells. Further activation of the EMT program in cancer cells was induced by epidermal growth factor. Decreased epithelial marker E-cadherin in CaSki cells was accompanied by increased mesenchymal markers N-cadherin and vimentin. Downregulated expression of E-cadherin in SiHa and CaSki cells was associated with increased expression of Snail transcription factor. Our goal was to study actin reorganization in the EMT process in cell cultures and in tissue. We found that β-cytoplasmic actin structures are disorganized in the cervical cancer cells. The expression of β-cytoplasmic actin was downregulated.

E-Cadherin repression increases amount of cancer stem cells in human A549 lung adenocarcinoma and stimulates tumor growth.

ABSTRACT Here we show that cancer stem cells amount in human lung adenocarcinoma cell line A549 depends on E-cadherin expression. In fact, downregulation of E-cadherin expression enhanced expression of pluripotent genes (c-MYC, NESTIN, OCT3/4 and SOX2) and enriched cell population with the cells possessing the properties of so-called ‘cancer stem cells’ via activation of Wnt/β-catenin signaling. Repression of E-cadherin also stimulated cell proliferation and migration in vitro, decreased cell amount essential for xenografts formation in nude mice, increased tumors vascularization and growth. On the other hand, E-cadherin upregulation caused opposite effects i.e. diminished the number of cancer stem cells, decreased xenograft vascularization and decelerated tumor growth. Therefore, agents restoring E-cadherin expression may be useful in anticancer therapy.

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells

Loss of γ-cytoplasmic actin induces epithelial-to-myofibroblast transition (EmyT), which depends on activation of SRF and its cofactor, MRTF, formin-mediated actin polymerization, and activated Rho GTPase. This demonstrates a unique role of γ-cytoplasmic actin in regulating the epithelial phenotype and the suppression of EmyT.

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.