REPOSITORY > RESULTS

Abstract

Apoptosis is an innate mechanism by which a multicellular organisms eliminates
unwanted, damaged and potentially harmful cells. During apoptosis inflammation of the
surrounding tissues is prevented. Defects in an apoptotic signaling can lead to numerous
pathological conditions. Apoptosis is mediated through two main pathways, the extrinsic
or the death receptor pathway and the intrinsic or mitochondrial pathway. In the intrinsic
pathway proapoptotic signals trigger release of cytochrome c from the mitochondria into
the cytosol, where it bounds with Apaf-1 protein and forms apoptosome complex and
subsequent activation of caspase 9. In the extrinsic pathway proapoptotic molecules bind
to the death receptors on the cell surface resulting in a formation of death-inducing
signaling complex (DISC) in which capases 8 and 10 are activated. In the end of both
pathways effector caspases 3 and 7 are activated.
Caspases are a family of evolutionary conserved cystein proteases mediating initiation
and execution of apoptosis. But there is increasing evidence that alternative proteolytic
enzymes as lysosomal proteases (cathepsins) can initiate or propagate proapototic signals.
As a result of lysosomal membrane permabilization cathepsins are released into the
cytosol. Once in the cytosol they can activate apoptotic pathway through cleveage of
proapoptotic molecule Bid or inactivation of antiapoptotic Bcl-2 proteins and with
cleavage of XIAP cathepsins have effect also in later stages of apoptosis.
The mechanism of lysosomal membrane destabilization in death receptor pathway is
still not well understood. It was reported that lysosomal rapture was an early event and it
preceded mitochondrial membrane destabilization. However, recent studies with Fas and
TNFa induced apoptosis have shown that lysosomes were destabilized after mitochondria.
In our experiments with mouse embryonic fibroblasts (MEF) we showed that caspases
are crucial in TRAIL induced apoptosis and that destabilization of mitochondria precede
destabilization of lysosomes. Effect of cathepsin B was evaluated using cathepsin B
knockout mice. Our result show absence of cathepsin B has no effect on either
mitochondrial or lysosomal membrane destabilization process in comparison to wild type
cells. However the decrease in apoptosis was observed. We conclude that cathepsin B
does not play a role in initial phases of apoptosis, but it may be a factor in later stages of
apoptosis in TRAIL induces apoptosis in MEFs. As a possible link between mitochondria
and lysosomes we identified reactive oxygen species (ROS). ROS are produced by
mitochondria and if mitochondria are damaged ROS can cause irreversible damage in the
cell. We also found out that release of cytochrome c depends on cardiolipin oxidation and
ROS importance was confirmed with iron chelator DFO in scavenger Tempol.
In the second part we evaluated involvement of cathepsins in apoptosis in human cell
lines. Using cathepsins inhibitors E-64d and CA-074Me we could not prevent TRAIL
induced apoptosis. Although mitochondria and lysosomes were damaged in the process
and the active cathepsins are released into the cytosol, inhibiting cathepsins did not have
any effect on organelle stability or level of apoptosis.
Oxidative stress can cause lysosome membrane permeabilization and leakage of
cathepsins into the cytosol. Sirtuines, which play important role in subverted autophagy
leading to apoptosis, are among possible cathepsin targets. We showed in vitro cleavages
of sirtuines by cathepsins B, L and S.
Understanding of the molecular mechanisms of apoptosis is crucial in the application
of protease inhibitors in cancer treatment. On a model of TRAIL induced apoptosis in
mouse embryonic fibroblasts and several human cancer cell lines we here describe link
between mitochondria and lysosomes as well as involvement of lysosomes and
lysosomsal proteases, cathepsins, in the process.