Molecular mechanism of satratoxin H-induced apoptosis in PC12 cells

Abstract

The purpose of this study was to elucidate the molecular mechanism of satratoxin H, and the protective effect of antioxidants on satratoxin H-induced apoptosis in PC12 cells. Treatment with satratoxin H decreased cell viability in a concentration- and time-dependency measured by MTT assay with the IC₅₀ of approximately 50 nM. We evaluated the type of cell death. Hoechst 33342 assay showed a significant increase in apoptosis after 24 h-incubation of satratoxin H.DNA fragmentation by agarose gel electrophoresis confirmed that DNA ladder was entirely found after 1 day-incubation in serum-free medium, and then the band began fade suggesting that apoptosis might change to secondary necrosis. In contrast, the cells in serum-containing medium showed DNA ladder after 3 day-incubation. The result was supported by flow cytometry using propidium iodide that apoptotic cell were significantly augmented after 24 h- and 48 h-incubation in serum-free and in serum-containing medium, respectively. The results suggested that incubation of cells in serum-free medium was more sensitive to satratoxin H. The molecular mechanism of satratoxin H-induced apoptosis was therefore examined under the condition in serum- free medium. Treatment with satratoxin H showed a significant increase in the ROS level (6 h) and MDA content (36 h) detected by flow cytometry using DCFH-DA and by TBARS, respectively. It was implied that satratoxin H-induced apoptosis might be through the generation of ROS. Since lipid peroxidation occurred during the decrement of apoptosis, lipid peroxides may further induce necrosis. MAPKs, a well-known downstream of ROS, were determined by Western blot analysis. Treatment with satratoxin H increased the phosphorylation of p38 MAPK as well as JNK at 3-6 h and ERK 1/2 at 0.5-6 h. The activation of MAPKs was confirmed by using MAPKs inhibitors. Preincubation with SB203580, a p38 MAPK inhibitor or SP600125, a JNK inhibitor for 1 h following by 48 h-incubation of satratoxin H significantly increased cell viability. The results indicated that p38 MAPK and JNK pathways were involved in satratoxin H-induced apoptosis. We demonstrated whether antioxidants could protect satratoxin H-treated cells. The coincubation of the antioxidants GSH, NAC, or trolox with satratoxin H resulted in no significant change the percentage of cell viability, the percentage of apoptosis, and the production of lipid peroxides. Meanwhile GSH (1 mM) significantly reduced the generation of ROS after 6 h- coincubation. It was suggested that ROS may play a role in satratoxin H-induced apoptosis and GSH exhibited low potential in protection of satratoxin H- induced apoptosis in PC12 cells. In conclusion, satratoxin H- induced apoptosis was mediated through the generation of ROS coincident with the activation of p38 MAPK and JNK. These events led to the production of lipid peroxides, which may involve in satratoxin H- induced necrosis. However, antioxidants did not protect satratoxin H- induced the ROS generation and production of lipid peroxides.