Tag Archives: thymocytes

Effects of α-tocopherol and its anologues on rat thymocytes programmed death induced by protein kinase inhibitors

G. V. Petrova, N. V. Delemenchuk, G. V. Donchenko

Palladin Institute of Вiochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: petrova@biochem.kiev.ua

It is established that α-tocopherol (α-ТPh) shows cytoprotective effect at the induction of rats’ thymocytes apoptosis by endocellular protein kinase inhibitors – staurosporine and phorbol ether in high concentration, and also on necrosis of the cells caused by sphyngosine. The effect of α-ТPh on thymocytes death caused by protein phosphatase type 2А inhibitor ocadaic acid is much less expressed. The obtained data testify that the known ability of α-ТPh to the inhibition of PKC and to the activation of protein phosphatase type 2А is not the main mechanism of its cytoprotective action. Partial reproduction of α-ТPh effects by its analogue α-tocopheryl acetate which is not capable to enter in redox reactions, and the absence of influence on the studied processes of an antioxidant of N-acetyl-L-cysteine do not confirm the antioxidant mechanism of α-ТPh action in this case. The inhibition by α-ТPh of the release of cytochrome c in the cytosol of cells testifies to the implementation of its cytoprotective effect at the level of mitochondrial membranes. We assume the existence of the universal mechanism of α-ТPh cytoprotective action that does not depend on the nature of apoptogenes and realized on the general for the majority of them stage of the cells death induction. The prevention by α-ТPh of mitochondria dysfunction by stabilizing mitochondrial membranes and reduction of their permeabilization is supposed as that.

Photoactivated fullerene C(60) induces store-operated Ca(2+) entry and cytochrome c release in Jurkat cells

S. M. Grebinyk1, K. O. Palyvoda2, S. V. Prylutska1, I. I. Grynyuk1,
A. A. Samoylenko2, L. B. Drobot2, O. P. Matyshevska1

1Taras Shevchenko Kyiv National University, Ukraine;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: matysh@yahoo.com

The values of endoplasmic reticulum Ca2+-pool and store-operated Ca2+ entry (SOCE) were estimated in rat thymocytes and Jurkat cells loaded with indo-1 and treated with thapsigargin. It was shown that the relative value of SOCE in thymocytes was substantially lower than in Jurkat cells. Significant increase of SOCE in Jurkat cells preincubated with 10-5 M C60 and exposed to uv/visible light irradiation was detected at 1-3 h after exposure. At this time FCCP-induced Ca2+-release from mitochondria was shown to be reduced, while cytochrome c level into the cytoplasm of Jurkat cells, detected by Western blot analysis, to be increased. It is supposed that Ca2+ flux remodulation induced by photoexcited fullerene C60 in Jurkat cells might be involved in the initiation of signalling events leading to cell apoptosis.

Generation of active oxygen forms in rat tymocytes under action of hydrogen peroxide and fullerene C(60)

S. M. Grebinyk, I. I. Grynyuk, S. V. Prylutska, O. P. Matyshevska

Taras Shevchenko Kyiv National University, Ukraine;
e-mail: grebnik_z@yahoo.com

The dynamics of active oxygen forms (AOF) generation in rat thymocytes 50 min after treatment with 0.1 and 0.5 mM H2O2 was estimated with the use of fluorescent probe DCFDA. Both enhanced AOF generation, which was dependent on H2O2 concentration, and glutathione peroxidase and superoxide dismutase activation, followed by a decrease of thymocytes viability were demonstrated.
Preincubation of cells with 10-5 M fullerene C60 was shown not only to prevent H2O2 – induced AOF generation but to increase viability of H2O2-treated thymocytes at more prolonged time period. The data obtained indicate to fullerene C60 ability to prevent oxidative stress in thymocytes.

Modulation of cisplatin-induced reactive oxygen species production by fullerene C(60) in normal and transformed lymphoid cells

D. V. Franskevych, I. I. Grynyuk, S. V. Prylutska, O. P. Matyshevska

Taras Shevchenko National University of Kyiv, Ukraine;
е-mail: dashaqq@gmail.com

The early response of normal (Wistar rat thymocytes) and transformed (mice lymphoid leukemia L1210) cells to treatment with anticancer drug cisplatin or to combined treatment with cisplatin and carbon nanostructure fullerene C60 was studied. We demonstrated with fluorescent probes DCFH-DA and TMRE that cisplatin at concentration 1 μg/ml induced reactive oxygen species (ROS) production and decreased the value of mitochondrial membrane potential in both cell types. The combined treatment with cisplatin (1 μg/ml) and fullerene C60 (7.2 μg/ml) was shown to be followed by oppositely directed modulation of ROS production in thymocytes and L1210 cells. Cisplatin-induced ROS production was intensified in L1210 cells, while in thymocytes it was decreased. It is supposed that the different effects of combined treatment are associated with peculiarities of fullerene C60 accumulation and localization in normal and cancer cells.

The sensitivity of cells with the various level of NAD(P)H:quinone oxidoreductase 1 to cytotoxic action of quinonimines and α-тоcopherol synthetic derivatives

G. V. Petrova1, А. V. Parshykov2

1Palladin Institute of Biochemistry, National Academy
of Sciences of Ukraine, Kyiv;
e-mail: petrova@biochem.kiev.ua
2Institute of Pharmacology and Toxocology, National Academy
of Medical Science of Ukraine, Kyiv

The effects of α-tocopherol with shortened to 6 carbon atoms side chain (α-Toc-C6), α-tocopherol succinate (α-TS) and quinonimine 2,6-dichlorophenolindophenol (DCPIP) on DT-diaphorase activity and viability of rat thymocytes, splenocytes and hepatocytes were investigated. It was shown that the lowest basal activity of the enzyme is inherent in splenocytes. In comparison to splenocytes, DT-diaphorase activity was 1.4 and 5 times higher in thymocytes and hepatocytes, respectively. It was found that the sensitivity of cells to the cytotoxic effect of DCPIP was inversely proportional to the basal level of DT-diaphorase activity and accompanied by its activation with subsequent inhibition at non-toxic and toxic concentrations, respectively. Hepatocytes were least sensitive to the cytotoxic effect of α-Toc-C6. In thymocytes and splenocytes α-Toc-C6 exerts inhibitory effects on DT-diaphorase, whereas in hepatocytes an increased activity of the enzyme was observed, which probably caused their high survival rate. Simultaneous induction of cytochrome P450 enzyme expression by α-Toc-C6 in hepatocytes is also possible. Cytotoxic effect of α-TS does not depend on the basal level of DT-diaphorase activity in cells, is not accompanied by its induction and it is most likely determined by the non-specific esterase activity.

Aldehydes participation in oxidative stress in rat thymocytes in vitro

K. О. Tokarchuk, О. V. Zaitseva

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: kate_tokarchuk@ukr.net

A variety of lipid radicals are formed under oxidative stress development. The further oxidation of these radicals leads to formation of numerous aldehydes. They can form postsynthetic modifications in proteins and nucleic acids that disrupt their functions. In the present study aldehydes role in the formation of oxidative stress parameters in rat thymocytes was investigated. Two models were used: iron-stimulated oxidative stress and exogenous aldehydes exposure to thymocytes.
For oxidative stress induction, thymocytes (2×106 cells/ml HBSS, рН 7.2) were exposed to different concentrations of FeSO4 (20, 30, 40 μМ) and ascorbic acid (100 μМ) for 6 h. It resulted in increase of levels of aldehydes 29 times (90 ± 6 nmol/107 cells), these changes led to increase of TBARS levels 4.4 times; the levels of protein CO groups 10 times, cell mitochondrial activity and low-molecular weight SH groups were decreased 1.5 and 2.3 times, respectively. Treatment with aldehydes acceptor dimedone (200 μМ) significantly decreased the levels of aldehydes 3.7 times, TBARS 1.6 times and protein CO groups 5 times. It was shown that the levels of cell mitochondrial activity increase 1.4 times and the levels of SH groups 1.8 times.
To compare the effects of aldehydes in induction of oxidative stress, thymocytes (2×106 cells/ml HBSS, рН 7.2) were exposed to 50-600 μМ formaldehyde (FA), 50-600 μМ glyoxal (GL), 50-600 μМ methylglyoxal (MGL), 1-15 μМ acrolein (АCR) for 6 h. TBARS levels were increased for FA 1.3 times and for other aldehydes  about 5-7 times. The levels of protein CO groups were increase for FA 3.7 times, for MGL 7 times, for GL 13 times, for ACR 22 times. Levels of SH groups were decreased for FA 1.5 times, for MGL 2.6 times, for GL 3 times, for ACR 9 times. A decrease of cell mitochondrial activity 1.5 times observe for all aldehydes. Obtained results prove the aldehydes participation in the formation of oxidative stress parameters and their capability to oxidative stress induction in the rat thymocytes.