Signal role for activation of caspase-3-like protease and burst of superoxide anions during Ce4+-induced apoptosis of cultured Taxus cuspidata cells

Abstract

The signal events of 1 mM Ce4+ (Ce(NH4)2(NO3)6)-induced apoptosis of cultured Taxus cuspidata cells were investigated. The percentage of apoptotic cells increased from 0.82% to 51.32% within 6 days. Caspase-3-like protease activity became notable during the second day of Ce4+-treatment, and the maximum activity was 5-fold higher than that of control cells at the fourth day. When the experiment system was pretreated with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) at 100 microM, caspase-3-like activity resulted in distinct inhibition by 70% and 77.3% after 3 and 4 days of induction. Furthermore, 100 microM Ac-DEVD-CHO partially reduced the apoptotic cells by 58.6% and 60.8% at day 4 and 5 respectively. Ce4+ induced superoxide anions (O2*-) transient burst, and the first peak appeared at around 3.7-4 h, the second appeared at about 7 h. Both O2*- burst and cell apoptosis were effectively suppressed by application of diphenyl iodonium (NADPH oxidase inhibitor). Inhibition of O2*- production attenuated caspase-3-like activation by 49% and 53.6% during day 3 and 4 respectively. In addition, a total of 15 protein spots changed in response to caspase-3-like protease activation were identified by two-dimensional gel electrophoresis. These results suggest that Ce4+ of 1 mM induces apoptosis in suspension cultures of T. cuspidata through O2*- burst as well as caspase-3-like protease activation. The burst of O2*- exerts its activity as an upstream of caspase-3-like activation. Our results also implicate that other signal pathways independent of an O2*- burst possibly participate in mediating caspase-3-like protease activation.