Grebinyk S. Production of reactive oxygen species and changing of free cytosolic Ca2+ concentration in transformed T-cells under action of photoexcited fullerene C60

The thesis is devoted to the study of reactive oxygen species (ROS) production and calcium homeostasis indexes in T-cells with normal (Wistar rat thymocytes) and transformed (leukemic МТ-4, Jurkat, L1210 lines) genome under the influence of carbon nanostructure fullerene C60 in control and after its photoexcitation. The differences in membratotropic effects of fullerene C60 in test cells were shown. Treatment of thymocytes with fullerene C60 (5?10-5M) caused inhibition of ecto-ATPase activity in plasma membrane, initial velocity (V0) of the enzymatic reaction was decreased while the time of reaching a half-maximum velocity rate (?) was increased. Treatment of MT-4 cells was not followed by changes in ecto-ATPase activity. Ca2+ permeability of endoplasmic reticulum (ER) membrane was evaluated in experiments with Indo-1 loaded cells incubated in Ca2+-free medium followed by the addition of C60. Fullerene C60 did not influence Ca2+-permeability of ER membrane in thymocytes, but in MT-4 cells caused a gradual increase of [Ca2+]i. The differences revealed appear to be dependent on the rate of fullerene C60 distribution in plasma membrane and of its penetration into the cytoplasm of normal and transformed cells. With the use of fluorescent probe DCF-DA the аntioxidant properties of fullerene C60 were studied in thymocytes, exposed to 0.1 mM H2O2 as an inducer of oxidative stress. It was shown that H2O2-dependent ROS production was inhibited when the cells were preincubated with 10-5M C60. The obtained data indicate on C60 ability to prevent oxidative stress in thymocytes. Effects of photoexcited fullerene C60 were evaluated in cells treated for 1h with 10-5M C60 and irradiated ( =320-600 nm) by mercury-vapor lamp (200 mW/cm2). It was shown that fullerene C60 exhibited cytotoxic effect against leukemic L1210 and Jurkat cells decreasing cell viability when combined with UV-visible irradiation. No phototoxic effect of fullerene C60 against thymocytes was detected. Exposure of test cells to UV-visible irradiation only was followed by insignificant increase of intracellular ROS formation. Under the combined action of fullerene C60 and irradiation the dynamic of ROS production in thymocytes was the same as that under irradiation, but in L1210 and Jurkat cells ROS production was further intensified and its level significantly exceeded the level of ROS in control. Thus, photoexcitation of fullerene C60 was shown to be accompanied by increased ROS production in leukemic cells. To elucidate the influence of photoexcited fullerene C60 on Ca2+ signaling system its influence on the concentration of free cytosolic Ca2+ was investigated in normal and leukemic cells. It was shown that C60 photoexcitation was followed by only transient [Ca2+]i elevation in thymocytes while in leukemic a significant and sustained increase of [Ca2+]i was detected. To determine the mechanisms of photoexcited fullerene C60 on Ca2+-homeostasis Ca2+-pool of endoplasmic reticulum and capacitive calcium entry into the cells were evaluated. Depletion of ER Ca2+-pool was сaused by addition of thapsigargin - highly effective inhibitor of ER Ca2+-ATPase. The relative value of capacitive Ca2+ entry was estimated as the difference between the values of [Ca2+]i before and after addition of 1 mM CaCl2 to the incubation medium of thapsigargin-treated cells. It was shown that the system of maintaining Ca2+ homeostasis in leukemic cells is remodulated. The value of endoplasmic reticulum Ca2+-pool was less and that of capacitive Ca2+ entry across plasma membrane into ER was significantly lower in L1210 and Jurkat cells than in thymocytes. It was demonstrated that combined action of fullerene C60 and irradiation is followed by substantial increase of capacitive Ca2+ entry into leukemic cells. This phenomena resulted in [Ca2+]i increase and excessive Ca2+-accumulation by mitochondria of leucemic cells. It was shown that following C60 photoexcitation mitochondria of Jurkat and L1210 cells lost their ability to retain Ca2+ accumulated after cation release from ER. With the use of potential-sensitive fluorescent probe TRME it was demonstrated that fullerene C60 reduced the value of mitochondrial membrane potential in leukemic cells. These data confirm the ability of fullerene C60 to penetrate into cells and to localize near or in the membranes of mitochondria. The membrane potential of mitochondria in Jurkat and L1210 was shown to be dissipated following C60 photoexcitation. The data obtained indicate the promising application of photoexcited fullerene C60 for influence on Ca2+-signaling system and for induction of Ca2+-dependent apoptosis in leukemic cells.