The effect of mitochondria-targeted antioxidants
on normal and transformed cells in culture

E.V. Titova, O.Y. Ivanova, E.N. Popova, O.Y. Pletjushkina, V.B. Dugina, V.P. Skulachev
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University

The reorganization of actin microfilaments plays a crucial role in oncogenic transformation and leads to distortion of cell polarization and motility. One of possible explanation of actin network system disorders is activation of oncogenic signaling pathways which determine cell polarization, directional motility and substrate adhesion by changing in gene expression of actin binding proteins such as myosin, gelsolin, tropomyosin. In this study we investigated the effect of antioxidants specifically addressed to mitochondria (SkQ1 and their analogs) on normal (MRC5) and transformed (MRC5-V1 and MRC5-V2) human pulmonary fibroblasts and on human fibrosarcoma HT1080 cells.
We have detected an important cell shape alteration during incubation of normal and transformed fibroblasts with mitochondria-targeted antioxidants (20-40 nM from 5 hours to 3-7 days): cells became highly spread, actin microfilaments bundles were formed. We have evaluated immunomorphologically that SkQ1 and their analogs led to formation of thick and parallel actin bundles (stress fibers containing beta actin, myosin II, alpha actinin, gelsolin, alpha smooth muscle actin) in MRC5-V1 and MRC5-V2 cultures. The effect of N-acetyl-cysteine (NAC) and Trolox on MRC5-V1 was similar to SkQ1, but working concentrations of this antioxidants were much higher: 5mM for NAC and 100μM for Trolox. Phenotype reversion by SkQ1 treatment was observed also with human fibrosarcoma HT1080 cell line.
We have revealed immunomorphologically and morphometrically that mitochondria-targeted antioxidants led to focal adhesion (FA) elongation and further reorganization with forming of mature FAs in transformed fibroblasts. Western blot analysis showed the increase of FA protein vinculin in MRC5-V1 after mitochondria-targeted antioxidants (20-40 nM, 7 days), NAC (5 mM, 7 days) and trolox (100 μM, 7 days) incubation.
We have showed the effect of SkQR1 on human fibrosarcoma cells proliferation. SkQR1 treatment (20 nM, 24 h) led to mitotic-entry delay in HT1080 cells after 18 h of serum stimulation.
Mitochondria-targeted antioxidants induced phenotypic reversion in our experimental model. The results of our present work are in concerning with the effect of antioxidants on ras-transformed cells investigated earlier (Alexandrova et. al, 2006; Popova et al., 2006).

Homo Sapiens Liberatus Workshop, Moscow State University, May 2010