Figure 2.

Increased Cytosolic Ca²⁺ Levels Induced by TR Result in Thiol Oxidation and Mitochondrial Permeabilization Associated with Cell Death. (A) Mitochondrial membrane potential (∆Ψ) of K562 cells incubated with TR (7.5, 10, and 15 µM) for 24 h was evaluated by flow cytometry using the JC-1 dye. Uncoupler CCCP was used as a positive control. (B) Quantification of the percentage of the dissipation of ΔΨ considering all replicates. Data are expressed as percentages of dissipation (mean ± SD). * Statistically different from the control (p < 0.05). (C) Representative images of the temporal evaluation of effects of 15 µM TR on ∆Ψ and both cytosolic and mitochondrial Ca²⁺ levels. K562 cells were loaded with rhod-2/AM (red) and fluo-3/AM (green) for simultaneous Ca²⁺ measurement by confocal microscopy. (D) Effect of 2.5 mM EGTA or 10 µM BAPTA-AM on the cytotoxicity of 15 µM TR incubated for 24 h with K562 cells. Data are expressed as percentages of control, considered to be 100% (mean ± SD). * Statistically different from TR (p < 0.05). (E) Representative traces of the effect of 2.5 mM EGTA on the TR-induced ΔΨ dissipation in digitonin-permeabilized K562 cells loaded with rhodamine 123. (F) Control (open circles), 15 µM TR (filled circles), 2.5 mM EGTA plus 15 µM TR (filled squares), and 2.0 mM t-BOOH (filled squares) were added to K562 cell suspension, and changes in the DCF fluorescence were recorded in real time. (G) Oxidation of reduced thiol groups of proteins (gray bars) and glutathione (white bars) by 15 µM TR in K562 cells after 24 h of incubation. t-BOOH was used as a positive control. * Statistically different from the control (p < 0.05). (H) Effect of 1.0 mM DTT on the cytotoxicity of 15 µM TR in K562 cells after 24 h of incubation. (I) Effect of 2.5 mM EGTA on the oxidation of reduced thiol groups of mitochondrial proteins induced by 15 µM TR or 2.0 mM t-BOOH after 24 h of incubation. (J) Impairment of the establishment of ∆Ψ by 15 µM TR in succinate-energized digitonin-permeabilized K562 cells and the protective effect of 2.5 mM EGTA.