The aminopeptidase inhibitor, z-L-CMK, is toxic and induces cell death in Jurkat T cells through oxidative stress.

The leucine aminopeptidase inhibitor, benzyloxycarbonyl-leucine-chloromethylketone (z-L-CMK), was found to be toxic and readily induce cell death in Jurkat T cells. Dose-response studies show that lower concentration of z-L-CMK induced apoptosis in Jurkat T cells whereas higher concentration causes necrosis. In z-L-CMK-induced apoptosis, both the initiator caspases (-8 and -9) and effector caspases (-3 and -6) were processed to their respective subunits. However, the caspases remained intact in z-L-CMK-induced necrosis. The caspase inhibitor, z-VAD-FMK inhibited z-L-CMK-mediated apoptosis and caspase processing but has no effect on z-L-CMK-induced necrosis in Jurkat T cells. The high mobility group protein B1 (HMGB1) protein was found to be released into the culture medium by the necrotic cells and not the apoptotic cells. These results indicate that the necrotic cell death mediated by z-L-CMK at high concentrations is via classical necrosis rather than secondary necrosis. We also demonstrated that cell death mediated by z-L-CMK was associated with oxidative stress via the depletion of intracellular glutathione (GSH) and increase in reactive oxygen species (ROS), which was blocked by N-acetyl cysteine. Taken together, the results demonstrated that z-L-CMK is toxic to Jurkat T cells and induces apoptosis at low concentrations, while at higher concentrations the cells die of necrosis. The toxic side effects in Jurkat T cells mediated by z-L-CMK are associated with oxidative stress via the depletion of GSH and accumulation of ROS.

Keywords: Aminopeptidase; apoptosis; oxidative stress; necrosis

Peptidyl-halomethyl-ketones were designed specifically as active site-directed irreversible protease inhibitors. They are very useful pharmacological and biochemical tools for the elucidation of molecular structure, mechanism of reaction and biological functions of serine and cysteine proteases (Angliker et al. [1]; Krantz et al. [17]; Powers et al. [24]). The peptidyl moiety of the peptidyl-halomethyl-ketones serves as the affinity group, which complement the S1 and S2 sites of the target protease (Liow and Chow [18]). The halomethyl-ketone moiety, which contains the halide group, functions as the reactive entity that labels the enzyme. This reaction involves the expulsion of the halide group to form an irreversible thiomethylketone with the cysteine or serine residues at the active site of the enzyme (Powers et al. [24]).

Some of the earliest peptidyl chloromethyl ketone (CMK) serine protease inhibitors developed include tosyl-phenylalanine chloromethyl ketone (TPCK) and tosyl-lysine chloromethyl ketone (TLCK) which were initially used to block the activity of chymotrypsin and trypsin, respectively (Schoellmann [32]). Accumulating evidence over the years have shown that both TPCK and TLCK possess numerous side effects beside blocking serine proteases (Weis et al. [39]; Kim et al. [16]; Gillibert et al. [9]; Gillibert et al. [10]; Perez-G et al. [23]; Ha et al. [11]). The lack of specificity in these inhibitors maybe attributed to the highly reactive CMK moiety, which results in nucleophilic displacement and irreversible alkylation of nontarget molecules indiscriminately in cells (Rauber et al. [27]; Shaw et al. [33]). More recently, we reported that the cathepsin B inhibitor, z-FA-CMK, was toxic and readily induce cell death in human primary T cells as well as the leukemic Jurkat T cells (Liow and Chow [18]; Rajah and Chow [26]). We also observed that benzyloxycarbonyl-alanine-chloromethylketone (z-A-CMK), an analog of z-FA-CMK, exhibit similar toxicity in Jurkat T cells (Liow and Chow [18]). Peptidyl-CMK with alanine in the P1 position (A-CMK) was originally developed to block leucine aminopeptidase, one of the many aminopeptidases that are involved in protein maturation, degradation, and regulation of hormonal and nonhormonal peptides (Birch et al. [2]). Another potent inhibitor designed to block this aminopeptidase is L-CMK with leucine at the P1 position, and both were also effective inhibitors for amino acyl-tRNA synthetases (Birch et al. [2]).

In view of their similar mechanism of action on amino peptidases and amino acyl tRNA synthetases (Birch et al. [2]), we examined whether z-L-CMK possesses any toxic side effects in Jurkat T cells as seen with z-A-CMK (Liow and Chow [18]). Similar to z-A-CMK, we found z-L-CMK toxic and readily induced cell death in a time- and dose-dependent manner in Jurkat T cells. Cells treated with lower concentrations of z-L-CMK were found to undergo apoptosis whereas the cells die of necrosis when exposed to higher concentrations of the peptidyl CMK. The caspase-inhibitor, z-VAD-FMK, inhibits apoptotic cell death and caspase processing induced by low concentration of z-L-CMK in Jurkat T cells, but has no effect on necrosis mediated by high concentration of the peptidyl-CMK. The release of high mobility group protein B1 (HMGB1) into the culture medium by necrotic cells induced by high concentrations of z-L-CMK suggests that the cells die via necrosis instead of secondary necrosis. We also observed that z-L-CMK treatment leads to depletion of intracellular glutathione (GSH) and accumulation of ROS in Jurkat T cells and that cell death induced by z-L-CMK was abrogated by NAC. Collectively, these results suggest that z-L-CMK toxicity is mediated via oxidative stress.

Benzyloxycarbonyl-valine-alanine-aspartic acid-(O-methyl)-fluoromehylketone (z-VAD-FMK) and benzyloxycarbonyl-leucine chloromethylketone (z-L-CMK) were purchased from Bachem (Bubendorf, Switzerland). Rabbit antibodies to caspase-3 and caspase-2, and goat antibodies to caspase-8, were obtained from Santa Cruz Biotechnology (Dallas, TX). Rabbit polyclonal antibodies to caspase-6 and caspase-9 were from Cell Signaling Technology (Danvers, MA). Mouse monoclonal antibody to PARP-1 was from Enzo Life Sciences (Farmingdale, NY). RPMI 1640 and FCS were from Gibco (Grand Island, NY), and Hoechst 33358 and tetramethylrhodamine ethyl ester (TMRE) were from Molecular Probes (Eugene, OR). Chelex®100 resin was from BioRad (Hercules, CA). N-acetyl cysteine (NAC), monochlorobimane (MCB), and dihydroethidium (DHE) were obtained from Sigma Aldrich (St. Louis, MO).

The human leukemic T lymphocyte cell line, Jurkat, clone E6-1 was obtained from ATCC (Rockville, MD). Cell cultures were maintained in logarithmic growth phase with RPMI-1640 supplemented with 10% FCS and 2 mM L-glutamine in a humidified incubator at 37 °C with 5% CO2 in air. For treatments, Jurkat T cells (1 × 106 cells/ml) were incubated with various concentrations of z-L-CMK where indicated at 37 °C in an incubator with 5% CO2 in air. At time points where indicated, the treated cells were assessed for cell viability, apoptosis, or prepared for Western blot analysis.

Cell viability was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) as previously described (Johnson et al. [13]). Briefly, after treatments where indicated, aliquots of 100 µl Jurkat T cell suspension (1 × 105 cells) were transferred to wells in a 96-well flat-bottomed tissue culture plate followed by the addition of 20 µl freshly prepared MTS/PMS solution. The plate was incubated at 37 °C in an incubator with 5% CO2 in air for 2 h before reading the absorbance at 490 nm using a Tecan 200 ELISA plate reader. All samples were assayed in triplicate.

Following treatments, Jurkat T cells (1 × 106 cells) were washed and resuspended in 100 µl of ice-cold annexin V binding buffer. The cells were incubated with 5 µl each of annexin V-FITC and PI on ice in the dark for 15 min. Following incubation, 400 µl of binding buffer was added to the cells prior to flow cytometry analysis using a FACSCalibur flow cytometer (Becton Dickinson). Excitation wavelength 488 nm (FL-1 channel) for annexin V-FITC and 595 nm (FL-2 channel) for PI were used for sample analysis.

Following treatments, Jurkat T cells were washed with ice-cold PBS and incubated with 0.1 µM TMRE at room temperature in the dark for 15 min. Cells were subsequently washed twice using ice-cold PBS and resuspended in 1 ml PBS prior to flow cytometry (FACSCalibur, Becton Dickinson) analysis using excitation wavelength 595 nm (FL-2 channel). The changes in MMP are reflected by the relative decrease in TMRE fluorescence intensity.

Nuclear morphological changes in Jurkat T cells during apoptosis were examined using UV microscopy as previously described by using Hoechst 33342 (Johnson et al. [13]). Following treatments, Jurkat T cells were washed with PBS before fixing in 50 µl paraformaldehyde (4%) for 30 min at room temperature. The fixed cells were stained with 50 µl Hoechst 33342 (2.5 µg/ml) for 30 min and centrifuged down. The cell pellets are then resuspended in glycerol/PBS (50:50 v/v) before mounting onto slides and viewed using an Olympus fluorescence microscope (Model BX51). Apoptotic cells were identified based on morphological hallmarks such as nuclei condensation and/or nuclear fragmentation into apoptotic bodies.

DNA fragmentation during apoptosis was visualized using agarose gel electrophoresis as previously described by Liow and Chow ([18]). Following treatments, 2 × 106 cells (live and dead) were harvested by centrifugation and resuspended in 500 µl Chelex®100 resin in solution (5% v/v) with proteinase K (2.5 mg/ml) and incubated at 55 °C for 45 min. After centrifugation at 10,000 rpm for 10 min, the supernatant containing the cellular DNA was collected and 2 µg of DNA was mixed with loading buffer and analyzed using a GelRed™ precast 2% agarose gel. Electrophoresis was performed at 100 V for 1 h followed by DNA visualization using the Bio-Rad gel imaging system (Gel Doc™ EZ Imager) under UV illumination.

Caspase-3 activity in Jurkat T cells was determined using the synthetic fluorescent substrate, Ac-DEVD-AFC. Following treatments, Jurkat cells were washed with PBS and resuspended in 25 µl lysis buffer (0.1 M NaCl, 1 mM Tris-HCl at pH7.6, 1 mM EDTA, 1% Triton-X-100, 1 mM PMSF). The cell suspension was taken through 3 freeze-thaw cycles using liquid nitrogen. The protein concentration of the cell lysates was measured using the Bradford Protein Assay, and 30 µg of protein was used to determine the caspase-3 activity. The assay was performed in final concentration of 100 µM substrate in 20 mM HEPES, 10% glycerol, and 10 mM DTT. The fluorescence changes were measured following incubation at 37 °C for 15 min using a Tecan 200 ELISA plate reader at 400-nm excitation wavelength and 595-nm emission wavelength.

Briefly, following treatments, Jurkat cells (2 × 106) were lysed and protein concentration determined. Protein (30 μg) from whole-cell lysates was diluted in loading buffer (2% SDS, 10% glycerol, 50 mM Tris-HCl pH 6.8, 0.2% bromophenol blue, and 100 mM DTT and 4 M Urea for PARP analysis) and resolved using 13% SDS-PAGE. The separated proteins were transferred onto Hybond C membrane (Amersham, UK) and probed for caspase-2, -3, -6, -8, -9, PARP, and β-actin using antibodies. Detection was performed using appropriate secondary antibodies conjugated to horseradish peroxidase followed by chemiluminescence (Amersham, UK) and visualized using a chemiluminescence Western blot scanner (LI-COR C-DiGit).

The intracellular GSH levels in Jurkat T cells were determined as described by Kamencic et al. ([14]). Following treatments, Jurkat T cells (1 × 105) were centrifuged at 3500 rpm for 10 min and washed with 100 µl of PBS before resuspended in 100 µl of MCB (100 µM) in PBS. The unbound MCB is nonfluorescent but forms a highly blue fluorogenic adduct when bound to GSH. The fluorescence intensity was measured following incubation at 37 °C for 30 min in the dark, at excitation wavelength of 390 nm and emission wavelength of 460 nm using a Tecan 200 ELISA plate reader. Complete media with MCB was used as background fluorescence control.

The production and intracellular accumulation of ROS in Jurkat T cells were detected using DHE, a redox sensitive fluorogenic probe that reacts with superoxides to form 2-hydroxyethidium, a red fluorescent product which intercalates with nuclear DNA (Owusu-Ansah et al. [22]). Following treatments where indicated, Jurkat T cells were washed and resuspended in 500 µl serum-free RPMI medium and incubated with 10 µM DHE at 37 °C in the dark for 30 min. Cells were subsequently washed twice using ice-cold PBS and resuspended in 500 µl PBS prior to flow cytometry analysis (FACSCalibur, Becton Dickinson) using excitation wavelength 518 nm (FL-2 channel).

The release of HMGB1 protein from Jurkat T cells following treatments was determined using dot-blot analysis. Following treatments, the Jurkat T cell culture supernatants were collected and centrifuged at 1500 rpm for 15 min at 4 °C for 10 min. Aliquots of 100 µl of culture supernatants were loaded into wells of the dot blot apparatus. The culture supernatants (100 µl) were filtered onto Hybond C membrane (Amersham, UK) using vacuum suction. The membrane was subsequently probed with antibodies to HMGB1 followed by HRP-conjugated secondary antibodies before chemiluminescence (Amersham) detection and visualization using chemiluminescence Western blot scanner (LI-COR C-DiGit).

All the experimental data were analyzed using Graph-pad PRISM version 6.04 (GraphPad Software, San Diego, California). Results shown represent mean ± SEM where statistical comparison between treatment groups was performed using one-way ANOVA followed by Post-hoc Dunnett's Test. The statistical p value of <.05 is regarded as statistically significant.

We have shown previously that z-A-CMK, an aminopeptidase inhibitor, was toxic and readily induce cell death in Jurkat T cells (Liow and Chow [18]). In the present study, the toxicity of another aminopeptidase inhibitor, z-L-CMK, on Jurkat T cells was examined. As shown in Figure 1, z-L-CMK was toxic to Jurkat T cells and readily induced cell death in a dose- and time-dependent manner. Cell death mediated by z-L-CMK was apparent at ∼10 µM after 2 h and higher concentration (100 µM) induced a marked decrease in viability with only ∼13% Jurkat T cells remained viable after 2 h. At longer time points, there was a marked decreased in the IC50 as shown by the shifting of the dose-response curve to the left (Figure 1).

Graph: Figure 1. Effect of z-L-CMK on cell viability in Jurkat T cells. Jurkat T cells were treated with various concentrations of z-L-CMK and the cell viability was determined at different time points using the MTS assay. The results represent the means ± SEM from three independent experiments.

One of the early hallmark of apoptotic cell death involves the externalization of PS from the inner leaflet of the cell membrane, which can be detected using annexin V-FITC labeling (Zhang et al. [42]; van Engeland et al. [38]). To determine whether cell death induced by z-L-CMK involved apoptosis, Jurkat T cells were treated with various concentrations of z-L-CMK for 6 h and the binding of annexin V-FITC and uptake of PI determined using flow cytometry. As shown in Figure 2, the percentage of normal cells (annexin-FITC negative and PI negative) was found to decrease markedly with increasing concentration of z-L-CMK. Interestingly, z-L-CMK induced a transient increase in apoptotic cells (annexin V-FITC positive and PI negative) which peaks at about 12.5 µM and decreased as the concentration of z-L-CMK increases. In sharp contrast, z-L-CMK dose-dependently induced an increased in late apoptotic/necrotic cells (annexin V-FITC positive and PI positive). The results suggest that apoptosis was apparent at low concentrations of z-L-CMK (<12.5 µM), but at higher concentrations (>12.5 µM), a marked decrease in apoptotic cells was observed. This was accompanied by an increase in cells that may have undergone necrosis. Taken together, these results suggest that z-L-CMK induced apoptosis at low concentrations and necrosis at higher concentrations.

Graph: Figure 2. Effect of z-L-CMK on annexin V-FITC binding and PI uptake in Jurkat T cells. Cells were treated with various concentrations of z-L-CMK for 6 h. Following treatment, the treated cells were stained with annexin V-FITC and PI prior to flow cytometry analysis as described in Materials and methods section. The results represent the means ± SEM from three independent experiments.

It is well established that the collapse of the MMP is an early event that precede the externalization of PS during apoptosis (Ding et al. [5]). As the MMP decreases, cytochrome c from the mitochondrial inner membrane space leaks out into the cytosol. In the presence of cytosolic ATP, cytochrome c interacts with caspase-9 to form the apoptosome, which in turn will activate caspase-9 and initiate the intrinsic apoptotic pathway (Hengartner [12]; Fulda and Debatin [8]). To this end, the effect of z-L-CMK on Jurkat T cell MMP was examined. Jurkat T cells were treated with 10 and 50 μM of z-L-CMK, and at various time points the treated cells were incubated with TMRE prior to flow cytometry analysis. As illustrated in Figure 3, 10-μM z-L-CMK induced a time-dependent decrease in the MMP in Jurkat T cells. The loss of MMP was apparent after 1 h and by 6 h the cells have lost more than 50% of their MMP. In sharp contrast, the MMP in cells treated with 50 μM z-L-CMK was markedly reduced within 1 h and no further changes seen after 6 h.

Graph: Figure 3. Effect of z-L-CMK on Jurkat T cell MMP. Cells were treated with z-L-CMK (10 µM or 50 µM) for different time points where indicated. Following treatments, the treated cells were incubated with TMRE prior to flow cytometry analysis as outlined in the Materials and methods section. The results represent the mean fluorescence intensity ± SEM from three independent experiments.

Apoptotic cells display classical nuclear morphological changes such as nuclei shrinkage and chromatin condensation as well as formation of apoptotic bodies. To further determine whether z-L-CMK-induced cell death is via apoptosis, the nuclear morphology of treated Jurkat cells was examined using UV microscopy following staining with Hoechst 33342 dye. As illustrated in Figure 4(A), untreated Jurkat cells displayed normal nuclei morphology. However, Jurkat cells treated with z-L-CMK (10 μM) do not display the normal nuclear morphology as control cells (Figure 4(C)). Instead the treated cells displayed atypical nuclei morphology with numerous cavities which is neither normal nor apoptotic nuclei morphology. These nuclear morphological changes are distinct and different from the condensed nuclear chromatin characteristic of apoptosis or fragmented apoptotic nuclei as seen in staurosporine-treated Jurkat T cells which are hallmarks of apoptotic cell death (Figure 4(B)). Following treatment with 10 µM z-L-CMK, ∼90% of the treated cells displayed the distinct nuclei morphology, compared to control cells which exhibit mainly normal nuclei (Figure 4(D)). In contrast, 89% of staurosporine-treated Jurkat T cells (positive control) have condensed apoptotic nuclei morphology. Since the distinct nuclear morphological change in z-L-CMK is different from the classical apoptotic nuclei, we examined whether the chromatin would be cleaved into internucleosomal fragments, another hallmark of apoptotic cells. As illustrated in Figure 4(E), only chromatin from staurosporine-treated cells have DNA laddering, whereas both control and z-L-CMK treated cells have no DNA laddering, indicating that the nuclear chromatin in z-L-CMK-treated cells remained intact and have not been cleaved to the oligonucleosomal fragments.

Graph: Figure 4. Effect of z-L-CMK on nuclear morphological changes and DNA in Jurkat T cells. Cells were treated with 10 µM z-L-CMK or 1 µM of STS for 6 h, fixed and stained with Hoechst 33342 dye as described in Materials and methods section. The nuclear morphological changes (A-C) were examined under 400× magnification using fluorescence microscopy and 200 cells were scored for each treatments (D). The results represent the means ± SEM from three independent experiments. DNA fragmentation (E) was resolved using 2% agarose gel electrophoresis using DNA extracted from treated Jurkat T cells.

Since the activation of caspases-3 is a critical mediator for the downstream execution of apoptosis (Degterev et al. [4]), the caspase-3 activity in these cells was determined. Jurkat T cells were treated with various concentrations of z-L-CMK or 1 µM staurosporine (positive control) for 6 h and their caspase-3 activities determined using the caspase-3 substrate, Ac-DEVD-AFC. As illustrated in Figure 5(A), the cell lysates from control cells which have little or no apoptotic cells have low caspase-3 activity, whereas staurosporine-treated cells showed marked increased in caspase-3 activity. Interestingly, z-L-CMK treatment induced a transient increased in caspase-3 activity in Jurkat T cells as the concentration of z-L-CMK increases. The increased in caspase-3 activity peaked at 6.25 µM z-L-CMK, which gradually reduced to below that of the control cells at 50 and 100 µM. The caspase-3 activity induced by z-L-CMK correlates well with the annexin V-FITC results (Figure 2), suggesting that z-L-CMK induces apoptosis at low concentration whereas higher concentration causes necrosis. Because caspase-3 activity was activated in Jurkat T cells treated with 10 µM of z-L-CMK, the effect of the caspase inhibitor z-VAD-FMK was examined. To this end, Jurkat T cells were incubated with various concentrations of z-VAD-FMK for 30 min prior to treatment with 10 µM and 50 µM of z-L-CMK. As shown in Figure 5(B), z-VAD-FMK (25-100 µM) dose-dependently inhibited cell death in Jurkat T cells mediated by 10 µM z-L-CMK indicating that caspases were indeed activated in these cells, and that these cells were undergoing apoptotic cell death. In sharp contrast, z-VAD-FMK has no effect on cell death mediated by 50 µM z-L-CMK in Jurkat T cells (Figure 5(C)). These results confirmed that cells treated with high concentration of z-L-CMK were undergoing necrosis, which correlate with the annexin V-FITC binding results (Figure 2) and the lack of caspase-3 activity (Figure 5(A)).

Graph: Figure 5. Role of caspases in z-L-CMK-mediated cell death in Jurkat cells. Cells were treated with various concentrations of z-L-CMK for 6 h and cell lysates were prepared and assayed for caspase-3 activity (A). Cells treated with staurosporine (STS) serve as positive control. In (B) and (C), the cells were treated with 10 µM and 50 µM of z-L-CMK, respectively, for 6 h either alone or in the presence of various concentrations of z-VAD-FMK. The results represent the means ± SEM from three independent experiments. RFU (relative fluorescence units).

Our results suggest that caspases are activated only in Jurkat T cells treated with 10 µM and not with 50 µM of z-L-CMK. We next examined the caspases activated in Jurkat T cells exposed to 10 µM or 50 µM of z-L-CMK using Western blotting. As shown in Figure 6, all the caspases (-2, -3, -6, -8, and -9) examined remained intact in their pro-form in control untreated cells. Following treatment with 10-µM z-L-CMK, there was a marked reduction of these pro-form caspases in Jurkat T cells. The pro-form of the initiator caspases (-2, -8, and -9) was markedly reduced, where caspase-8 and caspase-9 were cleaved into their p43/41 and p37/35 subunits, respectively. On the other hand, effector caspases, such as caspase-3, were processed to the catalytically active p17 subunit while the pro-form of caspase-6 was markedly decreased. The caspase-3 substrate, PARP (116 kDa), was cleaved to the 85 kDa fragment. In the presence of 50 µM z-VAD-FMK, the processing of caspase-3, caspase-6, and caspase-8 was inhibited whereas processing of caspase-2 and caspase-9 was only partially blocked. The cleavage of PARP was also completely abrogated in the presence of z-VAD-FMK. Collectively, these results demonstrated that cell death mediated by low concentration of z-L-CMK is caspase-dependent and via apoptosis. In sharp contrast, the processing of caspases and PARP in Jurkat T cells exposed to 50 μM of z-L-CMK remained intact in the absence or presence of z-VAD-FMK.

Graph: Figure 6. The effect of z-L-CMK on the activation of caspases and cleavage of PARP in Jurkat T cells. Cells were treated with z-L-CMK (10 μM or 50 μM) for 6 h in the presence or absence of z-VAD-FMK (50 μM). Following treatment, caspase activation and cleavage of PARP were examined using Western blotting as described in the Materials and methods section. Staurosporine-treated cells served as a positive control for apoptosis in Jurkat T cells. Results are one representative of at least three independent experiments.

Since all the evidence suggested that high concentration of z-L-CMK-induced direct necrosis rather than secondary necrosis following apoptosis in Jurkat T cells, we examined the release of the HMGB1 protein from these cells. HMGB1 is a DNA binding protein found in the nucleus and is only released during necrosis but not from cells undergoing secondary necrosis (Oppenheim and Yang [20]; Taylor et al. [37]). Following treatments with various concentrations of z-L-CMK for 6 h, the culture supernatants were collected and probed for the presence of HMGB1 using dot blot. As depicted in Figure 7(A), the supernatant from control cells have little HMGB1 protein while the supernatant from freeze-thawed (FT) cells (positive control for necrosis) has a large amount of HMGB1 protein. Following treatments with increasing concentrations of z-L-CMK, there was no increase in HMGB1 protein in the supernatants from cells exposed to low concentrations (up to 12.5 µM). However, as the concentration of z-L-CMK increases above 12.5 μM, a gradual increase in HMGB1 protein in the culture supernatants was observed (Figure 7(B)). The release of HMGB1 into the culture supernatants from cells treated with high concentration of z-L-CMK (>12.5 μM) correlates with the induction of necrosis (Figure 2). These results confirmed that cell death mediated by high concentration of z-L-CMK in Jurkat T cells is via necrosis and not via secondary necrosis.

Graph: Figure 7. Immunodetection of HMGB1 protein released from z-L-CMK-treated Jurkat T cells. Cells were treated with various concentration of z-L-CMK for 6 h and the culture supernatants were filtered onto nitrocellulose membrane in a dot blot apparatus using vacuum. Immunodetection of HMGB1 in the membrane was carried out as described in Materials and methods section. (A) Dot blot of culture supernatants derived from z-L-CMK-treated cells. Results are one representative of at least three separate experiments. (B) Dot blot membrane images were captured and the relative signal intensity for HMGB1 protein was analyzed using LI-COR C-DiGit® Blot Scanner. The results are the means ± SEM of the relative intensity of the HMGB1 protein released from z-L-CMK-treated cells from three independent experiments, where * indicates p < .05 versus control. Freeze thaw (FT) cell lysates were used as positive controls.

We have recently reported that the peptidyl methylketones such as z-VAD-FMK and z-FA-FMK were capable of inducing oxidative stress in activated T cells via the depletion of intracellular GSH and increase in ROS production (Rajah and Chow [25], [26]). We therefore examined whether z-L-CMK toxicity also involves oxidative stress. To this end, Jurkat T cells were treated with various concentrations of z-L-CMK in the presence of 1 mM NAC. As illustrated in Figure 8, cell death induced by z-L-CMK up to 100 μM was inhibited by the presence of NAC, suggesting that oxidative stress is involved. To corroborate this, we determined the intracellular GSH and ROS levels in z-L-CMK-treated cells. As shown in Figure 9(A), z-L-CMK (10 µM and 50 µM) induced a rapid depletion of intracellular GSH levels in treated Jurkat T cells. At 10 µM z-L-CMK, ∼45% intracellular GSH in the treated cells were depleted within the first hour and >90% after 6 h, whereas at 50 µM, ∼90% of the intracellular GSH in the treated cells were depleted within 1 h. This rapid depletion of intracellular GSH in z-L-CMK-treated cells was accompanied by a corresponding increase in ROS accumulation (Figure 9(B)). At 10 µM z-L-CMK, ROS accumulation in treated Jurkat T cells reaches ∼4-fold above control after 6 h. In the presence of 50 µM z-L-CMK, the increase in ROS reached ∼5-fold within 2 h. Collectively, these results suggest that z-L-CMK-induced cell death involves oxidative stress through the depletion of GSH and generation of ROS.

Graph: Figure 8. The effect of NAC on z-L-CMK-induced cell death in Jurkat T cells. Cells were treated with various concentrations of z-L-CMK in the presence and absence of NAC (1 mM) for 6 h and the cell viability determined using the MTS assay. The results represent the means ± SEM from three independent experiments.

Graph: Figure 9. The effect of z-L-CMK on intracellular GSH and ROS levels in Jurkat T cells. Cells were incubated with z-L-CMK (10 µM or 50 µM) for various time points where indicated and the intracellular GSH levels (A) and ROS accumulation (B) were determined using the fluorescent dye MCB and DHE probes, respectively as outlined in the Materials and methods section. The results represent the means ± SEM from three independent experiments.

We have previously shown that the cathepsin B inhibitor, z-FA-CMK, was toxic and induced cell death in Jurkat T cells (Liow and Chow, [18]). Structure activity studies revealed that z-A-CMK, an analog of z-FA-CMK having the phenylalanine in the peptide sequence moiety removed, was equally toxic. N-protected A-CMK was developed as an inhibitor to block the activity of leucine aminopeptidase, one of many aminopeptidases that play important roles in the regulation of protein maturation, degradation, and regulation of hormonal and nonhormonal peptides (Birch et al. [2]). In the present study, we found that z-L-CMK, another leucine aminopeptidase inhibitor, was also toxic and induce cell death in Jurkat T cells in a dose- and time-dependent manner.

Using several apoptosis hallmarks to distinguish apoptotic cell death from necrosis, our results showed that z-L-CMK at low concentrations readily induced apoptosis in Jurkat T cells. However, when exposed to higher concentrations of z-L-CMK, the cells die of classical necrosis. Although apoptotic cell death in Jurkat T cells mediated by z-L-CMK displayed some distinctive apoptotic features, such as the externalization of PS, collapse of MMP, and activation of caspases, there was no nuclear condensation or nuclear fragmentation into apoptotic bodies in the treated cells. Instead the nuclei of the treated cells appeared convoluted and creased which are similar to our previous reports showing apoptotic cell death in the absence of nuclear condensation or DNA fragmentation (Johnson et al. [13]; Liow and Chow [18]). It is well established that during apoptosis the activation of caspase-3 is associated with downstream activation of the DNA fragmentation factor (DFF), which exist as a complex consisting of a 40 kDa (DFF40) and a 45 kDa (DFF45) subunit (Liu et al. [19]; Enari et al. [6]). Upon activation by caspase-3, DFF45 is cleaved and release DFF40, which translocate into the nucleus (Sakahira et al. [30]; Wolf et al. [40]). DFF40 contains intrinsic nuclease activity and cleaves nuclear chromatin into the hallmark apoptotic feature of oligonucleosomal-length DNA fragmentation (Liu et al. [19]). The marked increase in caspase-3 activity and cleavage of PARP in cells treated with low concentration of z-L-CMK suggests that the activity of processed caspase-3 in these cells were not blocked. Although z-L-CMK-treated cells have no DNA fragmentation or apoptotic bodies, cell death and other apoptotic hallmarks were completely abrogated by the broad spectrum caspase inhibitor z-VAD-FMK. Collectively, these results suggest that z-L-CMK may have also inhibited downstream events of apoptosis beside activating the caspase-dependent apoptotic cell death pathway in Jurkat T cells.

In contrast, Jurkat T cells treated with high concentration of z-L-CMK have no apparent apoptotic hallmarks indicating that the cells die of necrosis. Interestingly, some caspase-3 and caspase-9 were cleaved to their subunits but there was no evidence of caspase-3 activity detected and PARP was not cleaved. The presence of z-VAD-FMK has virtually no effect on cell death mediated by high concentration of z-L-CMK in these cells, although both caspase-3 and caspase-9 cleavage were attenuated. This suggests that some caspase-9 may have been activated in Jurkat T cells when treated with high concentration of z-L-CMK, and the activated caspase-9 managed to cleave some of the caspase-3. The lack of any caspase-3 activity could be due to the incomplete processing of caspase-3 to the p17 and p20 fragments. Necrotic cell death in these cells was further corroborated as seen from the extracellular release of HMGB1, a protein marker for necrosis. The release of HMGB1 specifically during necrotic cell death following loss of membrane integrity have been well documented by numerous studies (Scafidi et al. [31]; Raucci et al. [28]; Yang et al. [41]; Evankovich et al. [7]). In apoptotic cells, HMGB1 is deacetylated and retained within the chromatin in the nuclei even upon compromised membrane integrity such as during late apoptosis or secondary necrosis (Scafidi et al. [31]; Evankovich et al. [7]). In contrast, during necrosis, HMGB1 is hyperacetylated and translocate from the nucleus to the cytosol, which then passively leaks into the extracellular space upon loss of membrane integrity, contributing to inflammatory responses (Yang et al. [41]; Evankovich et al. [7]). Cellular contents such as HMGB1, ATP, DNA fragments, and mitochondrial contents belong to a family of intracellular molecules known as Damage-associated molecular patterns (DAMPs) which are immunogenic in extracellular environment or may function as alarmins or cytokines which contributes to inflammatory response (Oppenheim and Yang [20]; Rock and Kono [29]).

We have previously reported that peptidyl-methylketones such as z-VAD-FMK and z-FA-FMK are able to deplete intracellular GSH in activated T cells, causing oxidative stress which was inhibited by low molecular weight thiols such GSH, NAC, and L-cysteine (Rajah and Chow [25], [26]). Interestingly, z-L-CMK toxicity was also completely blocked by NAC suggesting that this peptidyl-CMK was also able to induce oxidative stress in Jurkat cells. Indeed, z-L-CMK was able to deplete the intracellular GSH level in Jurkat T cells with a concomitant increase in ROS. Oxidative stress have been shown to play important role in the induction of apoptosis or necrosis frequently, depending on the severity of the insult, which often mediated through the depletion of GSH pools coupled by an increase in ROS (Tan et al. [36]; Chandra et al. [3]; Orrenius [21]). The mitochondrion is the main site of cellular respiration as well as the production of ROS such as superoxide anions, hydroxyl radical, and hydrogen peroxides. The production of oxygen radicals is attributed to the respiratory chain, exogenous ROS, and cytotoxic insults (Kannan and Jain [15]). Excessive ROS causes damage to vital cellular components and macromolecules, such as the mitochondria which leads to the collapse of MMP and the release of cytochrome c (Stridh et al. [35]; Kannan and Jain [15]). The released cytochrome c may then take part in the induction of apoptosis by subsequently triggering the caspase activation cascade of the apoptotic intrinsic pathway (Hengartner [12]; Fulda and Debatin [8]; Siegel [34]). However, it remains unclear whether z-L-CMK is able to react with intracellular GSH directly causing its depletion which further leads to the accumulation of ROS.

In summary, our results have shown that the aminopeptidase inhibitor, z-L-CMK, is toxic and readily induces cell death in human Jurkat T cells. Dose-response studies revealed that lower concentration of z-L-CMK readily induces apoptosis while higher concentration of z-L-CMK causes necrosis. Despite having many of the hallmarks of apoptosis, the nuclear morphological changes in z-L-CMK-induced apoptosis were atypical, and no DNA fragmentation was observed. This suggests that z-L-CMK may also inhibit downstream processes involved in DNA fragmentation and condensation. We have also demonstrated that z-L-CMK-induced cell death is mediated via oxidative stress through the depletion of intracellular GSH and accumulation of ROS. Collectively, these results suggest that z-L-CMK possesses a potent toxic side effect and careful consideration must be exercised when using it as a specific enzyme inhibitor in cells.

No potential conflict of interest was reported by the authors.