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The cathepsin B inhibitor z-FA-CMK induces cell death in leukemic T cells via oxidative stress

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The cathepsin B inhibitor benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was recently found to induce apoptosis at low concentrations in Jurkat T cells, while at higher concentrations, the cells die of necrosis. In the present study, we showed that z-FA-CMK readily depletes intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) generation. The toxicity of z-FA-CMK in Jurkat T cells was completely abrogated by N-acetylcysteine (NAC), suggesting that the toxicity mediated by z-FA-CMK is due to oxidative stress. We found that l-buthionine sulfoximine (BSO) which depletes intracellular GSH through the inhibition of GSH biosynthesis in Jurkat T cells did not promote ROS increase or induce cell death. However, NAC was still able to block z-FA-CMK toxicity in Jurkat T cells in the presence of BSO, indicating that the protective effect of NAC does not involve GSH biosynthesis. This is further corroborated by the protective effect of the non-metabolically active d-cysteine on z-FA-CMK toxicity. Furthermore, in BSO-treated cells, z-FA-CMK-induced ROS increased which remains unchanged, suggesting that the depletion of GSH and increase in ROS generation mediated by z-FA-CMK may be two separate events. Collectively, our results demonstrated that z-FA-CMK toxicity is mediated by oxidative stress through the increase in ROS generation.
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ORIGINAL ARTICLE
The cathepsin B inhibitor z-FA-CMK induces cell death
in leukemic T cells via oxidative stress
K. Y. Liow
1
&Sek C. Chow
1
Received: 2 May 2017 /Accepted: 20 October 2017 / Published online: 31 October 2017
#Springer-Verlag GmbH Germany 2017
Abstract The cathepsin B inhibitor benzyloxycarbonyl-
phenylalanine-alanine-chloromethyl ketone (z-FA-CMK)
was recently found to induce apoptosis at low concentrations
in Jurkat Tcells, while at higher concentrations, the cells die of
necrosis. In the present study, we showed that z-FA-CMK
readily depletes intracellular glutathione (GSH) with a con-
comitant increase in reactive oxygen species (ROS) genera-
tion. The toxicity of z-FA-CMK in Jurkat T cells was
completely abrogated by N-acetylcysteine (NAC), suggesting
that the toxicity mediated by z-FA-CMK is due to oxidative
stress. We found that L-buthionine sulfoximine (BSO) which
depletes intracellular GSH through the inhibition of GSH bio-
synthesis in Jurkat T cells did not promote ROS increase or
induce cell death. However, NAC was still able to block z-FA-
CMK toxicity in Jurkat T cells in the presence of BSO, indi-
cating that the protective effect of NAC does not involve GSH
biosynthesis. This is further corroborated by the protective
effect of the non-metabolically active D-cysteine on z-FA-
CMK toxicity. Furthermore, in BSO-treated cells, z-FA-
CMK-induced ROS increased which remains unchanged,
suggesting that the depletion of GSH and increase in ROS
generation mediated by z-FA-CMK may be two separate
events. Collectively, our results demonstrated that z-FA-
CMK toxicity is mediated by oxidative stress through the
increase in ROS generation.
Keywords Z-FA-CMK .Tcells .Glutathione .Reactive
oxygen species .Oxidative stress .N-Acetylcysteine .
L-Cysteine .D-Cysteine .L-Buthionine sulfoximine
Introduction
Peptidyl chloromethyl ketones (CMKs) are among the first
active site-directed irreversible protease inhibitors developed
to block serine and cysteine proteases (Schoellmann and Shaw
1962). They have since become valuable tools for the eluci-
dation of enzyme function, structure, and reaction mecha-
nisms in normal physiology as well as in pathological condi-
tions (Powers et al. 2002;Paceetal.2013). Although widely
used, many studies have now shown that peptidyl CMKs pos-
sess numerous off-target effects besides blocking serine pro-
teases (Weis et al. 1995; Gillibert et al. 2005; Perez-G et al.
2008;Haetal.2009). This presumably is due to the highly
reactive electrophile CMK which lacks specificity and alkyl-
ates non-target molecules or cellular nucleophiles indiscrimi-
nately in cells (Rauber et al. 1986). Despite lacking in speci-
ficity, many peptidyl CMKs were useful biochemical and
pharmacological tools in characterizing the role of serine pro-
teases in animal disease models (Powers et al. 2002).
To overcome the lack of specificity in peptidyl CMKs,
analogous inhibitor structures were developed with different
leaving groups to replace the chlorine atom. Some of these
include bromomethyl and iodomethyl ketones which turn out
to be more reactive and less stable in aqueous solution
(Rasnick 1985;Demuth1990;Powersetal.2002). Efforts to
replace the reactive chlorine atom led to the eventual synthesis
of peptidyl fluoromethyl ketones (FMKs) (Rasnick 1985).
*Sek C. Chow
chow.sek.chuen@monash.edu
1
School of Science, Monash University Malaysia, Jalan Lagoon
Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia
Naunyn-Schmiedeberg's Arch Pharmacol (2018) 391:7182
https://doi.org/10.1007/s00210-017-1436-6
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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