Fluorescent Estimation of H2O2-Induced Changes in Cell Viability and Cellular Nonprotein Thiol Level of Dissociated Rat Thymocytes

Chikahisa, Lumi; Oyama, Yasuo; Okazaki, Eisuke; Noda, Katsuhiko

doi:10.1254/jjp.71.299

Cited by 135 publications

(111 citation statements)

References 11 publications

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“…To monitor cellular thiols, dual staining with CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) for thiols 24 and annexin V-PE for cell death were performed and analyzed by flow cytometry. Because CMF fluorescence is stable in the cells for at least 72 hours, we stained cells with CMFDA before annexin-PE.…”

Section: Determination Of Thiol Contents In Whole Cellsmentioning

confidence: 99%

Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism

Trachootham

Zhang

et al. 2008

Blood

180

179

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Chronic lymphocytic leukemia (CLL) is the most common adult leukemia, and resistance to fludarabine-based therapies is a major challenge in CLL treatment. Because CLL cells are known to have elevated levels of reactive oxygen species (ROS), we aimed to test a novel ROS-mediated strategy to eliminate fludarabine-resistant CLL cells based on this redox alteration. Using primary CLL cells and normal lymphocytes from patients (n ‫؍‬ 58) and healthy subjects (n ‫؍‬ 12), we showed that both fludarabineresistant and -sensitive CLL cells were highly sensitive to ␤-phenylethyl isothiocyanate (PEITC) with mean IC 50 values of 5.4 M and 5.1 M, respectively. Normal lymphocytes were significantly less sensitive to PEITC (IC 50 ‫؍‬ 27 M, P < .001). CLL cells exhibited intrinsically higher ROS level and lower cellular glutathione, which were shown to be the critical determinants of CLL sensitivity to PEITC. Exposure of CLL cells to PEITC induced severe glutathione depletion, ROS accumulation, and oxidation of mitochondrial cardiolipin leading to massive cell death. Such ROS stress also caused deglutathionylation of MCL1, followed by a rapid degradation of this cell survival molecule. Our study demonstrated that the natural compound PEITC is effective in eliminating fludarabine-resistant CLL cells through a redox-mediated mechanism with low toxicity to normal lymphocytes, and warrants further clinical evaluation.

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Section: Determination Of Thiol Contents In Whole Cellsmentioning

confidence: 99%

Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism

Trachootham

Zhang

et al. 2008

Blood

180

179

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“…Therefore, the effect of IT-354 on 5-CMF fluorescence was examined. The intensity of 5-CMF fluorescence in rat thymocytes and cellular glutathione content were very strongly correlated (correlation coefficient = 0.965) (Chikahisa et al, 1996). Treatment with 3 μM IT-354 attenuated 5-CMF fluorescence in some cells and augmented it in others.…”

Section: Changes In Oxonol and 5-cmf Fluorescence Induced By It-354mentioning

confidence: 82%

“…14124), Tokushima, Japan. The cell suspension was prepared as previously reported (Chikahisa et al, 1996;Matsui et al, 2010). In brief, thymus glands dissected from ether-anesthetized rats were sliced under cold conditions (2-4°C).…”

Section: Animals and Cell Preparationmentioning

confidence: 99%

“…The methods for measuring cellular and membrane parameters using a flow cytometer (CytoACE-150; JASCO, Tokyo, Japan) and fluorescent probes were similar to those previously described (Chikahisa et al, 1996). Various concentrations of IT-354 (1-10 mM in 2 μL DMSO) were added to cell suspensions (2 mL per test tube), which were incubated at 36-37°C.…”

Section: Fluorescence Measurements Of Cellular Parametersmentioning

confidence: 99%

“…Changes in intracellular Ca 2+ and Zn 2+ levels were estimated using 500 nM Fluo-3-AM (Kao et al, 1989) and 500 nM FluoZin-3-AM (Gee et al, 2002), respectively. 5-CMF-DA at a concentration of 1 μM was used to monitor changes in cellular non-protein thiol content, presumably glutathione (Chikahisa et al, 1996). The excitation wavelength for the fluorescent probes was 488 nM.…”

Section: Fluorescence Measurements Of Cellular Parametersmentioning

confidence: 99%

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Cytotoxic actions of N-(2,4,6-trichlorophenyl)maleimide (IT-354), an antifouling agent, in rat thymic lymphocytes

Fukunaga

Saito

Kurumi

et al. 2015

Fundam. Toxicol. Sci.

Self Cite

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-Of antifoulants that are substitutes for organotin compounds such as tributyltin and triphenyltin, N-(2,4,6-trichlorophenyl)maleimide (IT-354) is listed as a much less toxic agent, although the available information concerning IT-354 toxicity is the results of acute toxicity tests in freshwater fish. In this study, the effects of IT-354 on rat thymic lymphocytes were examined using flow-cytometric techniques with appropriate fluorescent probes in order to estimate the effects of IT-354 on mammalian cells. Treatment of cells with 1-10 μM IT-354 for 1 hr did not increase the population of dead cells (cell lethality). However, 10 μM IT-354 significantly increased the population of living, annexin V-positive cells. Annexin V-positive, living cells are expected to be undergoing apoptosis. IT-354 at 3-10 μM significantly elevated intracellular Ca 2+ and Zn 2+ levels mainly by increasing Ca 2+ influx and intracellular Zn 2+ release. Furthermore, IT-354 significantly depolarized membranes and decreased cellular non-protein thiol content. Assessments using selected antifouling agents showed that the cellular actions of IT-354 are most likely similar to those of other commonly used antifouling agents. Therefore, the toxic potency of IT-354 on wild mammals is speculated to be similar to those of the other tested antifoulants.

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Direct current electrical fields induce apoptosis in oral mucosa cancer cells by NADPH oxidase‐derived reactive oxygen species

Wartenberg

Wirtz

Grob

et al. 2007

Bioelectromagnetics

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The presence of more than one dental alloy in the oral cavity often causes pathological galvanic currents and voltage resulting in superficial erosions of the oral mucosa and eventually in the emergence of oral cancer. In the present study the mechanisms of apoptosis of oral mucosa cancer cells in response to electromagnetic fields was investigated. Direct current (DC) electrical fields with field strengths between 2 and 16 V/m, applied for 24 h to UM-SCC-14-C oral mucosa cancer cells, dose-dependently resulted in decreased cell proliferation as evaluated by Ki-67 immunohistochemistry and upregulation of the cyclin-dependent kinase (CDK) inhibitors p21(cip1/waf1) and p27(kip1), which are associated with cell cycle arrest. Electrical field treatment (4 V/m, 24 h) increased apoptosis as evaluated by immunohistochemical analysis of cleaved caspase-3 and poly-(ADP-ribose)-polymerase-1 (PARP-1). Furthermore, robust reactive oxygen species (ROS) generation, increased expression of NADPH oxidase subunits as well as Hsp70 was observed. Electrical field treatment (4 V/m, 24 h) resulted in increased expression of Cu/Zn superoxide dismutase and decreased intracellular concentration of reduced glutathione (GSH), whereas the expression of catalase remained unchanged. Pre-treatment with the free radical scavenger N-acetyl cysteine (NAC) and the superoxide dismutase mimetic EUK-8 abolished caspase-3 and PARP-1 induction, suggesting that apoptosis in oral mucosa cancer cells is initated by ROS generation in response to DC electrical field treatment.

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Fluorescent Estimation of H2O2-Induced Changes in Cell Viability and Cellular Nonprotein Thiol Level of Dissociated Rat Thymocytes

Cited by 135 publications

References 11 publications

Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism

Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism

Cytotoxic actions of N-(2,4,6-trichlorophenyl)maleimide (IT-354), an antifouling agent, in rat thymic lymphocytes

Direct current electrical fields induce apoptosis in oral mucosa cancer cells by NADPH oxidase‐derived reactive oxygen species

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