REPOSITORY > RESULTS

Abstract

Apoptosis is an evolutionary conserved form of programmed cell death by which eukaryotic organisms eliminate potentially dangerous, superfluous and damaged cells. Caspases are a family of cysteine proteases which have a key role in the initiation and execution of apoptosis. In addition, upon lysosomal membrane permeabilization (LMP) proteases are released into the cytosol; however their mechanism of action is not well understood.
Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. The aim of our work was to understand the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event. In addition, apoptosis was not prevented by preincubation of cells with either catD inhibitor, PepA-P or cysteine cathepsin inhibitor, E64d, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species (ROS). For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B. CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of stefin B, as a response to TNF, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFαtreatment.
In the second part, we aimed to explain the role of catD in menadione-induced intrinsic apoptotic pathway in U937 human monocytic cells. It was previously shown that menadione is a ROS inducer and was able to induce apoptosis, which was concentration dependent. Again, the impact of cathepsins in this apoptotic pathway is not fully understood. For the first time, we show that menadione-induced apoptosis is accompanied by cleavage of the proapoptotic protein Bid, thus involving cysteine cathepsins. In addition, we show that initiator caspases 8 and 9 are activated, a process that culminates with PARP cleavage. Apoptosis was not prevented by preincubation of cells with either PepA-P or E64d, meaning that neither cathepsin D nor cysteine cathepsins are crucial in this pathway. Similarly as in the case of TNF-induced apoptosis, we observed the cleavage of stefin B as a consequence of menadion-triggered apoptosis, which was prevented by preincubation of cells with z-VAD-FMK and partially with PepA-P.