Photocytotoxic effect of C60 fullerene against L1210 leukemic cells is accompanied by enhanced nitric oxide production and p38 MAPK activation.

AIM: To estimate the combined action of C60 fullerene and light irradiation on viability of L1210 leukemic cells, nitric oxide (NO) generation, p38 mitogen-activated protein kinase (MAPK) activity and cell cycle distribution. METHODS: Cell viability was assessed by MTT test. Light-emitting diode lamp (λ = 410-700 nm, 2.45 J/cm(2) ) was used for C60 fullerene photoexcitation. Nitrite level and NO-synthase activity were measured by Griess reaction and by conversion of L-arginine to L-citrulline, respectively. p38 MAPK activity was assessed by Western blot analysis. Cell cycle distribution was determined by flow cytometry. RESULTS: It was shown that light irradiation of C60 fullerene-treated L1210 cells was accompanied by 55% decrease of their viability at 48 h of culture. Nitrite level measured as an index of reactive NO generation was increased at the early period after C60 fullerene photoexcitation due to activation of both constitutive and inducible NO-synthase isoforms. The simultaneous activation of p38 MAPK was detected. Accumulation of L1210 cells in sub-G1 phase of cell cycle was observed after C60 fullerene photoexcitation. CONCLUSION: Photoexcited C60 fullerene exerts cytotoxic effect, at least in part, through triggering production of reactive NO species and activation of p38 kinase apoptotic pathways in L1210 leukemic cells.

Enhanced cytotoxicity of photoexcited fullerene C60 and cisplatin combination against drug-resistant leukemic cells.

AIM: To evaluate the viability of leukemic cells sensitive (L1210S) and resistant (L1210R) to cisplatin, ROS production and free cytosolic Ca(2+) concentration under treatment with cisplatin or its combination with photoexcited fullerene C60. METHODS: Cell viability was assessed by the MTT reduction assay. Light-emitting diode lamp (2.45 J/cm(2)) was used for photoexcitation of intracellular accumulated fullerene C60. Free cytosolic calcium concentration ([Ca(2+)]i) and ROS production in cells were estimated with the use of fluorescent probes Indo-1 and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. RESULTS: It is shown that viability of L1210R cells wasn't changed under treatment with cisplatin in concentration range 0.1-10 µg/ml. 50% and 30% decrease of L1210S cells were observed after 24 h of incubation with cisplatin at concentrations 5 and 1 µg/ml, respectively. Intensification of extranuclear cytotoxic effects (ROS production and [Ca(2+)]i increase) after treatment with 1 µg/ml was detected in L1210S, but not in L1210R cells. The most strongly pronounced increase of ROS production and [Ca(2+)]i in both L1210 cell lines was revealed in dynamics after combined treatment with cisplatin (1 µg/ml) and photoexcited fullerene C60 (10(-5) M) and was followed by decreased viability of not only L1210S, but of L1210R cells as well.. CONCLUSION: Combined treatment with photoexcited C60 and cisplatin allowed to decrease effective concentration of cisplatin against parental L1210 cells and to increase sensibility of resistant cells to the drug.

COMPUTER PREDICTION OF BIOLOGICAL ACTIVITY OF DIMETHYL-N-(BENZOYL)AMIDOPHOSPHATE AND DIMETHYL-N-(PHENYLSULFONYL)AMIDOPHOSPHATE, EVALUATION OF THEIR CYTOTOXIC ACTIVITY AGAINST LEUKEMIC CELLS IN VITRO.

Structural analogues of ß-diketones--dimethyl-N-(benzoyl)amidophosphate (HCP) and dimethyl-N-(phenylsulfonyl)amidophosphate (HSP) were synthesized and identified by the methods of IR, 1H and 31P NMR spectroscopy. Screening of biological activity and calculation of physicochemical parameters of HCP and HSP compounds were done with the use of PASS and ACD/Labs computer programs. A wide range of biological activity of synthesized compounds, antitumor activity in particular, has been found. Calculations of the bioavailability criteria indicate that the investigated compounds have no deviations from Lipinski's rules. HCP compound is characterized by a high lipophilicity at physiological pH as compared to HSP. It was found that cytotoxic effect of the studied compounds on the leukemic L1210 cells was of time- and dose-dependent character. HCP is characterized by more pronounced and early cytotoxic effects as compared to HSP. It was shown that 2.5 mM HCP increased ROS production 3 times in the early period of incubation, and decreased cell viability by 40% after 48 h, and by 66%--after 72 h. Based on the computer calculation and undertaken research, HCP was selected for target chemical modifications and enhancement of its antitumor effect.

Photoactivated fullerene C60 induces store-operated Ca2 entry and cytochrome c release in Jurkat cells.

The values of endoplasmic reticulum Ca(2+)-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 Ca(2+)-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.

Photoinduced cytotoxic effect of fullerenes C60 on transformed T-lymphocytes.

AIM: To estimate the viability of normal and transformed T-lymphocytes after UV/Vis irradiation in the presence of pristine fullerenes C(60). METHODS: Thymocytes were isolated from Wistar rats' thymus. Murine leukemia L1210 and human lymphoma Jurkat cells were used in this study. Mercury-vapor lamp was used for fullerenes C(60) photoexcitation. Cytotoxicity was etermined by MTT assay. Changes in cell morphology were monitored by phase-contrast light microscopy. RESULTS: fullerenes C(60) exhibit cytotoxic effect against transformed T-lymphocytes when combined with UV/Vis irradiation (320-600 nm). Photoinduced effect was enhanced with the increasing of irradiation time period and C(60) concentration, cell death was registered after 24 hours incubation. Fullerenes C(60) photocytotoxicity against normal T-lymphocytes (thymocytes) was not observed. CONCLUSION: The present study suggests that pristine fullerenes C(60) have the potential to be an effective photosensitizer and exhibit cytotoxic effect on transformed T-cells in vitro .

Apoptosis photoinduction by C60 fullerene in human leukemic T cells.

The viability of normal (Wistar rat thymocytes) and transformed (human leukemia Jurkat cells) T cells after UV/Vis irradiation in the presence of pristine C60 fullerene was studied. The data obtained have shown that C60 fullerene exhibits cytotoxic effect against transformed T lymphocytes when combined with UV/Vis irradiation using mercury-vapor lamp (320-600 nm). C60 fullerene photocytotoxicity was not detected in thymocytes. C60-dependent photoinduced apoptosis of Jurkat cells was confirmed by DNA fragmentation and caspase-3 activation. No substantial increase of caspase-3 activation was observed in thymocytes treated with C60 fullerene plus irradiation, while antileukemic agent cytosine arabinoside was shown to induce caspase-3 activation both in Jurkat cells and thymocytes. The data obtained may be useful for development of photosensitizers for photodynamic therapy with selective action on leukemia cells.

Effect of the visible light irradiation of fullerene-containing composites on the ROS generation and the viability of tumor cells.

AIM: To study the effect of fullerene-containing composites, irradiated by visible light, on the radical oxygen species (ROS) generation in thymocytes, ascitic cells from Erlich's tumor and leukemia cells L1210; to investigate viability of these cells in the presence of fullerene-containing composites under irradiation conditions. MATERIALS AND METHODS: The viability of cells was evaluated by staining with 0.4% solution of the trypan blue; ROS were detected with the use of electron paramagnetic resonance (EPR) spectroscopy and spin traps; solutions of fullerene-containing composites were irradiated with mercury-vapor lamp. RESULTS: We demonstrated that under irradiation conditions fullerene-containing composites increase the rate of ROS generation and decrease the number of viable tumor cells. CONCLUSIONS: The obtained data allow to consider the fullerene-containing composites as potential agents for photodynamic therapy.