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

DOI:10.1007/s00210-017-1436-6
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K. Y. Liow
K. Y. Liow
K. Y. Liow
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Sek C Chow at Monash University (Malaysia)
Sek C Chow
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Abstract and Figures

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.
Dose- and time-dependent decrease in intracellular GSH mediated by z-FA-CMK in Jurkat T cells. Intracellular GSH was determined in Jurkat T cells incubated with various concentrations of z-FA-CMK and analogues (z-FA-FMK, z-FA-DMK, and z-YVAD-CMK) for 6 h (a) or incubated with 12.5 μM z-FA-CMK for various time points where indicated (b). Intracellular GSH was determined using the MCB assay as described in the “Materials and methods.” The results are the means ± SEM from three independent experiments
Dose- and time-dependent decrease in intracellular GSH mediated by z-FA-CMK in Jurkat T cells. Intracellular GSH was determined in Jurkat T cells incubated with various concentrations of z-FA-CMK and analogues (z-FA-FMK, z-FA-DMK, and z-YVAD-CMK) for 6 h (a) or incubated with 12.5 μM z-FA-CMK for various time points where indicated (b). Intracellular GSH was determined using the MCB assay as described in the “Materials and methods.” The results are the means ± SEM from three independent experiments
… 
Dose- and time-dependent increase in ROS induced by z-FA-CMK in Jurkat T cells. ROS were determined in Jurkat T cells incubated with various concentrations of z-FA-CMK for 6 h (a) or incubated with 12.5 μM (b) or 50 μM (c) z-FA-CMK for various time points where indicated. ROS was determined using DHE as described in the “Materials and methods.” The results are the means ± SEM from three independent experiments. *p < 0.05, significantly increased compared with the untreated control cells
Dose- and time-dependent increase in ROS induced by z-FA-CMK in Jurkat T cells. ROS were determined in Jurkat T cells incubated with various concentrations of z-FA-CMK for 6 h (a) or incubated with 12.5 μM (b) or 50 μM (c) z-FA-CMK for various time points where indicated. ROS was determined using DHE as described in the “Materials and methods.” The results are the means ± SEM from three independent experiments. *p < 0.05, significantly increased compared with the untreated control cells
… 
The effect of NAC on z-FA-CMK-induced cell death in Jurkat T cells. Cells were treated with various concentrations of z-FA-CMK in the presence and absence of NAC (1 mM) for 6 h. The cell viability was determined using the MTS assay as outlined in the “Materials and methods.” The results are means ± SEM from three independent experiments
The effect of NAC on z-FA-CMK-induced cell death in Jurkat T cells. Cells were treated with various concentrations of z-FA-CMK in the presence and absence of NAC (1 mM) for 6 h. The cell viability was determined using the MTS assay as outlined in the “Materials and methods.” The results are means ± SEM from three independent experiments
… 
The effect of NAC on z-FA-CMK-mediated ROS increase in Jurkat T cells. Cells were treated with 12.5 and 50 μM z-FA-CMK in the presence and absence of 1 mM NAC. Following 6 h of treatment, the intracellular level of ROS was determined using DHE. The results are means ± SEM from three independent experiments. *p < 0.05, significantly increased or decreased compared to 12.5 μM z-FA-CMK alone. #p < 0.05, significantly increased or decreased compared with 50 μM z-FA-CMK alone
The effect of NAC on z-FA-CMK-mediated ROS increase in Jurkat T cells. Cells were treated with 12.5 and 50 μM z-FA-CMK in the presence and absence of 1 mM NAC. Following 6 h of treatment, the intracellular level of ROS was determined using DHE. The results are means ± SEM from three independent experiments. *p < 0.05, significantly increased or decreased compared to 12.5 μM z-FA-CMK alone. #p < 0.05, significantly increased or decreased compared with 50 μM z-FA-CMK alone
… 
The effect of BSO on intracellular GSH, ROS, and cell viability in Jurkat T cells. Cells were incubated with various concentrations of BSO for 24 h. Following treatments, intracellular GSH level (a), ROS (b), and cell viability (c) were determined using assays as outlined in the “Materials and methods.” The results are the means ± SEM from three independent experiments
+4
The effect of BSO on intracellular GSH, ROS, and cell viability in Jurkat T cells. Cells were incubated with various concentrations of BSO for 24 h. Following treatments, intracellular GSH level (a), ROS (b), and cell viability (c) were determined using assays as outlined in the “Materials and methods.” The results are the means ± SEM from three independent experiments
… 
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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) 391:71–82
https://doi.org/10.1007/s00210-017-1436-6
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-chloromethylketone (z-FA-CMK) was found to be toxic and readily induce cell death in the human T cell line, Jurkat, whereas two other analogues benzyloxycarbonyl-phenylalanine-alanine-fluoromethylketone (z-FA-FMK) and benzyloxycarbonyl-phenylalanine-alanine-diazomethylketone (z-FA-DMK) were not toxic. The toxicity of z-FA-CMK requires not only the CMK group, but also the presence of alanine in the P1 position and the benzyloxycarbonyl group at the N-terminal. Dose-response studies showed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. In z-FA-CMK-induced apoptosis, both initiator caspases (-8 and -9) and effector caspases (-3, -6 and -7) were processed to their respective subunits in Jurkat T cells. However, only the pro-form of the initiator caspases were reduced in z-FA-CMK-induced necrosis and no respective subunits were apparent. The caspase inihibitor benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) inhibits apoptosis and caspase processing in Jurkat T cells treated with low concentration of z-FA-CMK but has no effect on z-FA-CMK-induced necrosis and the loss of initiator caspases. This suggests that the loss of initiator caspases in Jurkat T cells during z-FA-CMK-induced necrosis is not a caspase-dependent process. Taken together, we have demonstrated that z-FA-CMK is toxic to Jurkat T cells and induce apoptosis at low concentrations while at higher concentrations the cells die of necrosis.
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Glutathione Synthesis Is Not Involved in Protection by N-Acetylcysteine Against UVB-Induced Systemic Immunosuppression in Mice
Article
  • Jul 1998
Abstract— Irradiation of the skin with ultraviolet-B (UVB) radiation causes a local and systemic suppression of T-cell-medi-ated immune responses. Recently, N-acetylcysteine (NAC) was found to protect against UVB-induced immunosuppression and several other types of UV damage. The protective effects appeared to be based on the ability of NAC to increase glutathione (GSH) levels by promoting GSH synthesis. In this study, it was investigated whether topical application of NAC was still effective against UVB-induced suppression of contact hypersensitivity if GSH synthesis was blocked. Mice were pretreated with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, which succeeded in blocking the increase in epidermal GSH after topical application of NAC. Whereas the non-BSO-treated animals showed an increase to around 155% of the control GSH level for all NAC doses tested, only a slight (nonsignificant) increase in epidermal GSH was observed in the BSO-treated animals. Surprisingly, the protective efficacy of NAC against UVB-induced immunosuppression was not affected by the BSO pretreatment. No significant difference between the protective efficacy of NAC in the two groups was observed. Apparently, the antioxidant effect of NAC itself was sufficient to provide protection against UVB-immunosuppression, independent of GSH synthesis.
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