Figure 2

ESA triggers ROS generation in mitochondria, which is blocked by antioxidants. (a) Mitochondrial ROS and intracellular ROS were measured using fluorescent probes, MitoSOX and BeSSo-AM, respectively. Images in PC12 cells were obtained from ESA-treated cells for 5 h, and the fluorescent intensity of each image (three fields per sample were obtained) was quantified using the MetaMorph software. ROS was generated in mitochondria, which was suppressed by Toc (2 μg/ml) and U0126 (5 μM). In FBD-102b cells, the time course of fluorescent intensity was investigated. Data were obtained from two independent experiments. (b) Toc and tocotrienol blocked ESA (2 μg/ml) apoptosis at a concentration of 0.2 μg/ml or below, whereas 20 μg/ml Toc-Ac was needed to prevent ESA-mediated apoptosis. Methyl ester of ESA (ESA-Me) did not induce cell death. (c) Antioxidants blocked ESA-mediated apoptosis. BHT, EGCG, GSP, Quercetin, sesamine, sesaminol and NAC were tested. BHT blocked ESA-mediated apoptosis at a physiological concentration (12.5 μM). For other antioxidants, higher concentrations were needed to prevent ESA-mediated apoptosis (50 μM). Data were obtained from at least two independent experiments in triplicates. Values are the means±S.D. (n=6, 9; *P<0.05 to ESA-treated cells). (d) ATP level was decreased in a dose-dependent manner upon ESA (2 μg/ml) stimulation. Nec1 (50 μM), U0126 (5 μM) and Toc (2 μg/ml) recovered the decrease in ATP level in ESA-treated cells. ATP levels were measured 5 h after ESA stimulation (left and middle panels). ATP depletion was time-dependent, and the reduction in ATP occurred 4 h after ESA stimulation (right panel). (e) Mitochondrial membrane potential was decreased to 50% as compared with control (time=0 min) upon ESA stimulation. The potential did not disappear until the final stage of cell death. CCCP (50 μM) disappeared the potential. (f) Intracellular GSH levels were measured when cells were treated with or without BSO (10 or 50 μM) and NAC (1 mM). PC12 cells were cultured in the presence of BSO for 48 h, and then ESA (2 μg/ml) was added to the cells pretreated with buffer or NAC. Cell viability and GSH levels were measured 4 h after ESA addition. ESA did not affect intracellular GSH levels. Decrease in GSH by BSO did not trigger cell death. Values are the means±S.D. *P<0.05 (n=3, 6 in d, f).