Figure 7

The relevance of catalase inactivation for apoptosis-inducing ROS/RNS signaling. (A) MKN-45 cells remained without pretreatment (control), received 8 U/ml exogenous catalase or were treated with CAP for 1 min, followed by 25 min of incubation and three cycles of washing. CAP-pretreated cells then remained either without inhibitors, or received 8 U/ml catalase, 100 µM of the NOX1 inhibitor AEBSF, 150 µM of the peroxidase inhibitor ABH, 50 mM of the HOCl scavenger taurine (TAU) or 25 µM of the peroxynitrite (ONOO⁻) decomposition catalyst FeTPPS. The determination of apoptotic cells after 3.5 h showed that apoptosis induction required treatment with CAP. The effect of CAP treatment was due to selective establishment of HOCl signaling and was completely inhibited by exogenous catalase. This allows to conclude on the functional role of catalase for the protection of tumor cells towards HOCl signaling. This is in line with catalase being the central target for CAP treatment. (B,C) MKN-45 cells were pretreated with CAP for 1 min, followed by 25 min incubation in the same medium and three cycles of washing steps. The cells remained without inhibitor or received the indicated inhibitors. All assays then received the indicated increasing concentrations of catalase and apoptosis induction was determined after 3.5 h. The result shows that CAP treatment reactivated specifically HOCl signaling, without initial contribution of ^⋅NO/ONOO⁻ signaling. With increasing concentrations of catalase, first HOCl signaling was efficiently inhibited before ^⋅NO/ONOO⁻ signaling was reactivated. Finally ^⋅NO/ONOO⁻signaling was inhibited by very high concentrations of exogenous catalase. Statistical analysis: A: Apoptosis induction after CAP treatment, as well as inhibition by all inhibitors except FeTPPS was highly significant (p < 0.001). B, C: Apoposis induction after CAP treatment and in the presence of 0–2, as well as 31–250 U/ml catalase was highly significant (p < 0.001). The inhibition by AEBSF and mannitol was highly significant at all catalase concentrations (p < 0.001). Inhibition by taurine and ABH in the low concentration range of catalase (0–2 U/ml), and by L-NAME and FeTPPS in the high concentration range (31–250 U/ml) was highly significant (p < 0.001). The shifting effect of taurine in the concentration range of 16 and 31 U/ml was highly significant (p < 0.001).