REPOSITORY > RESULTS

Abstract

Cell death is an inevitable consequence of cellular life. In the past decades, researchers
characterized a set of genetically encoded mechanisms for targeted elimination of superfluous,
irreversibly damaged and harmful cells. Oxidative stress is also related to cell death by affecting
a wide variety of cellular components. In our study, we studied the impact of oxidative stress
produced by four different cell death models: menadione-triggered necrosis, staurosporineinduced
intrinsic apoptosis, and TNF (tumour necrosis factor)-stimulated extrinsic apoptosis and
necroptosis. For that purpose, four different ROS (reactive oxygen species) scavengers, N-acetyl-
L-cysteine (NAC), α-tocopherol (αTOC) and two superoxide dismutase mimetics, n(III)tetrakis
(4-benzoic acid) porphyrin chloride (MnTBAP), and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-
oxyl (Tempol; TE) were used. Our results show that the menadione-triggered necrosis was
completely prevented by NAC, however, the ROS targeting was found to have a marginal effect
on the other cell death modalities. Moreover, Tempol was able to substantially prevent TNFinduced
extrinsic apoptosis and partially decreased TNF-induced necroptosis. Other antioxidants
did not demonstrate any significant cell death prevention.
Furthermore, we investigated the lysosomal membrane permeabilization (LMP) which is
mediated by cathepsins that can trigger a variety of cell death modalities. The synthetic
lysosomotropic detergent L-Leucyl-leucine methyl ester (LLOMe), which causes cathepsin C
activity and lysosomal leaking, leading to cell death, was chosen as a model molecule for this
study. We observed that while the incubation of cell with LLOMe leads to an early increase in
lysosomal pH, followed by LMP and release of hydrolases, the selective inhibitor of cathepsin C,
GlyPheDMK, can prevent all these processes. In contrast, the broad spectrum cathepsin inhibitor
E64d efficiently prevented cell death, but only partially lysosomal pH increase. This mechanism
was confirmed using different types of primary cells deficient for the major cytosolic inhibitor of
cathepsins, stefin B (PyMT W.T. and PyMT StfB -/-), and cells double deficient for cathepsins B
and L (MEF W.T. and MEF DKO). According to the results we propose that cathepsin C is
required for the exit of lysosomal cathepsins into the cytosol, where the latter then trigger caspasedependent
cell death in the next step.
Doctoral dissertation presents new findings in both in the field of cell death and the role of
ROS in cell death and in the field of the mechanism of action of lysosomotropic detergent LLOMe
in cells. Understanding of these mechanisms is cruical in cell death regulation, since it might
affect for designing treatments.