AAF-cmk sensitizes tumor cells to trail-mediated apoptosis

Leukemia Research 28 (2004) 53–61
AAF-cmk sensitizes tumor cells to trail-mediated apoptosis
Izabela Młnarczuk a,b,c , Paweł Mróz a , Grażyna Hoser d , Dominika Nowis a , Łukasz P. Biały b ,
Halina Ziemba f , Tomasz Grzela b , Wojciech Feleszko e , Jacek Malejczyk b , Cezary Wójcik b ,
Marek Jakóbisiak a , Jakub Goł˛ab a,∗
a
b
Department of Immunology, Center of Biostructure, The Medical University of Warsaw, Chałubińskiego 5, 02004 Warsaw, Poland
Department of Histology and Embryology, Center of Biostructure, The Medical University of Warsaw, Chałubińskiego 5, 02004 Warsaw, Poland
c Institut fur Biochemie, Medical Faculty, Charite, Humboldt University, Monbijoustrasse 2, 10117 Berlin, Germany
d Department of Clinical Cytology, Postgraduate Center of Medical Instruction, Marymoncka 99, 01813 Warsaw, Poland
e Department of Pediatric Pneumonology, Allergic Diseases and Hematology, The Medical University Children’s Hospital,
Działubińdowska 1, 01184 Warsaw, Poland
f Department of Peridontology, The Medical University of Warsaw, Miodowa 18, 02246 Warsaw, Poland
Received 13 December 2002; accepted 30 April 2003
Abstract
TRAIL is a member of the tumor necrosis factor (TNF) superfamily. This cytokine is cytotoxic for a high proportion of tumor cells,
but could be also toxic for normal cells. There is a need to find other agents able to potentiate the antitumor effects of this cytokine. In
our study, we found that Ala-Ala-Phe-chloromethylketone (AAF-cmk) augmented cytotoxic activity of TRAIL or TNF against human
leukemic cells. Flow cytometry studies and electron microscopy revealed that apoptosis was primarily responsible for this potentiation.
Altogether, our studies indicate that AAF-cmk might effectively sensitize human leukemia cells to apoptosis induced by TRAIL and TNF.
© 2003 Elsevier Ltd. All rights reserved.
Keywords: AAF-cmk; TRAIL; TNF; Lymphoma; Apoptosis; Tripeptidylpeptidase II
1. Introduction
Regulated cytosolic proteolysis is indispensable for many
cellular functions, including activation of transcription factors, regulation of the cell cycle, processing of peptides presented by the major histocompatibility complex (MHC) class
I molecules and removal of incorrectly folded or damaged
proteins [1]. The ubiquitin- and proteasome-dependent system of protein degradation is thought to be the principal machinery responsible for the cytosolic proteolysis. The culture
of tumor cells in the presence of proteasome inhibitors (PSI)
induces apoptosis in these cells [2,3] and causes a block in
G1 phase of the cell cycle [4]. Clinical trials with at least one
class of proteasome inhibitors are already being conduced in
terminally ill cancer patients [5–7]. However, not all tumor
cell lines are equally susceptible to proteasome inhibitors
[8,9]. Even those tumor cells, which are susceptible, may
become resistant to normally lethal doses of proteasome inAbbreviations: FBS, fetal bovine serum; TPPII, tripeptidylpeptidase II
Corresponding author. Tel.: +48-22-622-63-06;
fax: +48-22-622-63-06.
E-mail address: [email protected] (J. Goł˛ab).
∗
0145-2126/$ – see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0145-2126(03)00122-X
hibitors as was shown with EL-4 cells growing in the constant presence of low doses of proteasome inhibitors [10].
It was reported that the function of proteasome could be
replaced by tripeptidylpeptidase II (TPPII) in cells adapted
to high doses of proteasome inhibitors [11,12], but some
more recent findings revealed the residual activity of proteasomes remains essential for protein degradation, antigen
presentation and survival. Therefore, the physiological role
of this protease still awaits explanation. It was hypothesized that in the cell TPPII further degrades oligopeptides
produced by the proteasome into tripeptides. The latter are
then proteolysed into free aminoacids by aminopeptidases
[13], that can be used for new protein synthesis or other
metabolic purposes.
The native form of TPPII has a remarkably high molecular
mass (>1000 kDa) and, like the proteasome, can assemble
into a higher-order structure composed of multiple 138 kDa
subunits that has an internal channel [14]. The enzyme has
been isolated from, for example human erythrocytes [14],
rat brain [15], pig [16] and Drosophila melanogaster [17].
Thus, like the proteasome, the enzyme appears to have a
ubiquitous distribution and its structure is highly conserved
[18]. TPPII and the proteasome have different substrate
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I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61
spectrum and inhibition patterns. It was suggested that the
activity of TPPII may be required for the survival of Burkitt’s
lymphoma cells, and inhibition of TPPII induced apoptosis
in these cells [8]. Also TPPII could be responsible for the
breakdown of myofibrillar proteins during muscle cachexia
[19]. Ala-Ala-Phe-chloromethylketone (AAF-cmk) has been
used in various studies as an irreversible and specific inhibitor of TPPII, however more recent data indicate that it
also blocks to some extent the chymotrypsin-like activity of
the proteasome [11,20].
Tumor necrosis factor (TNF) is one of the main inflammatory cytokines, capable of inducing apoptosis in susceptible tumor cells. However, its administration in patients
is limited by profound toxicity. More promising antitumor
agent is the recently discovered TNF-related apoptosis inducing ligand (TRAIL), which belongs to the TNF ligand
superfamily and induces apoptosis in a broad range of tumor cells with apparently no cytotoxic activity against most
non-transformed cells [21–23]. The apoptosis induced by
TRAIL and TNF can be blocked by a pan-caspase inhibitor,
benzoxycarbonyl–VAD–fluoromethylketone (zVAD-fmk)
[24,25].
Recombinant soluble TRAIL has shown promising antitumor effectiveness in numerous human and murine tumor
cell lines and in some in vivo models [21,23,26]. However,
it was reported that TRAIL could be toxic for normal human hepatocytes [27], therefore there is an urgent need for
agents, which could synergistically potentate antitumor activity of TRAIL reducing its effective doses and thus attenuating its toxicity.
Since we have recently shown that a proteasome inhibitor
(N-benzyloxycarbonyl-Ile-Glu-(o-t-butyl)-Ala-leucinal) is
capable of sensitizing tumor cells to the cytotoxic effects
of TRAIL [28] or TNF [29], we have now decided to
evaluate the antitumor effectiveness of these cytokines in
combination with an inhibitor of tripeptidylpeptidase II, a
protease that can either replace or complement the function
of proteasome.
2. Materials and methods
2.1. Reagents
Ala-Ala-Phe-chloromethylketone (Sigma, St. Louis,
MO) was dissolved in dimethylsulfoxide, DMSO and
stored as a 10 mM stock solution at −20 ◦ C. Recombinant human LZ-TRAIL, thereafter referred to as TRAIL,
was a gift from the Immunex Corporation, Seatle, WA
[30]. Stock solution was prepared according to the manufacturer instruction and kept at −70 ◦ C. Recombinant
human TNF (rhTNF produced in Escherichia coli was
kindly provided by Dr. W. Stec (Department of Bioorganic Chemistry, Center of Molecular and Macromolecular
Studies, Łódź, Poland). The specific activity of the rhTNF
was (4.3 ± 1.1) × 107 U/mg. The broad-spectrum cas-
pase inhibitor benzoyxycarbonyl-VAD-fluoromethylketone
(Sigma) was dissolved in DMSO and kept as a 20 mM stock
solution at −70 ◦ C [31].
2.2. Cell culture
U937 human promonocytic leukemia cells were routinely cultured in a humidified atmosphere of 5% CO2
at 37 ◦ C in RPMI-1640 medium supplemented with 10%
heat-inactivated fetal bovine serum (FBS), antibiotics, and
l-glutamine (2 mM) (all from Gibco BRL, Paisley, UK).
Cells were kept in 25 cm2 tissue flasks (Nunc, Roskilde,
Denmark) and passaged every 2–3 days.
2.3. Cytostatic/cytotoxic assay
The cytostatic/cytotoxic effects of TRAIL or TNF
with/without AAF-cmk on U937 cells in vitro was assayed
by oxidation of the tetrasolium salt MTT (Sigma). Leukemia
cells (5 × 103 ) in 100 ␮l of medium were dispensed into
96-well microtiter plates (Corning, Bibby Sterlin Ltd.,
Staffordshire, UK). Then, the serial dilutions of AAF-cmk
(50 ␮l; final concentration 5–20 ␮M) and TRAIL (50 ␮l;
final concentration 10–100 ng/ml) or human TNF (50 ␮l;
final concentration 125–500 ng/ml) were added in quadruplicate to a final volume of 200 ␮l. Appropriate volumes of
culture medium, supplemented with DMSO (<0.1%) were
added as controls.
After an incubation period of 24 h with AAF-cmk and
for additional 24 h with TRAIL/TNF and/or AAF-cmk, a
standard MTT assay was performed. After solubilization of
formazan crystals in DMSO, the plates were read on an
ELISA reader (SLT-Lab Instruments Ges. M.b.H., Salzburg,
Austria) using a 550 nm filter. Cytostatic/cytotoxic effect
was expressed as relative viability of U937 cells (percentage of control U937 cells incubated with medium only) and
was calculated as follows: relative viability = (Ae − Ab ) ×
100/(Ac − Ab ), where Ab is background absorbance, Ae is
experimental absorbance and Ac is the absorbance of untreated controls.
Similar treatment regimen was applied to the experiments
with normal human monocytes isolated from peripheral
blood using a Ficoll gradient. Monocytes were plated in
triplicate at 4 × 104 cells per well. Cytostatic/cytotoxic
effect was assessed by crystal violet staining.
The tetrapeptide caspase inhibitor, zVAD-fmk was added
to the cells at a final concentration of 10 ␮M 4 h before
AAF-cmk, and then the same conditions as described above
for the cytotoxicity measurements were applied and the results were obtained using MTT assay.
2.4. Drug interaction analysis
To determine whether AAF-cmk and TRAIL exert synergistic cytostatic/cytotoxic effects against U937 cells the
isobolanalysis by Berenbaum as described elsewhere was
I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61
used [32]. Briefly, inhibition of cell proliferation was determined using MTT assay as described above. Equi-effective
concentrations (concentrations of either drug alone or in
combination, which gave equivalent inhibition of cell proliferation as compared with untreated controls at P < 0.05
Student’s t-test) were analyzed. The interaction index for
two-drug combination was computed by applying the following equation: interaction index = (TRAILc /TRAILe )
+ (AAF-cmkc /AAF-cmke ), where TRAILe and AAF-cmke
are concentrations of both drugs that produce some specified effect when used alone and TRAILc and AAF-cmkc
are concentrations of the drugs that produce the same effect when used in combination. According to this analysis,
synergy occurs when the interaction index is less than 1.0.
2.5. Apoptosis measurement using flow cytometry
Cells were treated for 24 h with AAF-cmk (20 ␮M) and
then for additional 24 h with either RPMI alone or TRAIL
(30 ng/ml) alone or with AAF-cmk.
Phosphatidylserine externalization of apoptotic cells was
visualized by staining with annexin V-FITC according to
the manufacturer’s instruction (BioSource, Camarillo, CA).
Cell death was assessed by the uptake of propidium iodide,
PI (Sigma). Briefly, the U937 cells were washed in PBS, resuspended in 300 ␮l of a calcium containing binding buffer
supplied by the manufacturer (diluted 1:10 in distilled water) with the addition of 3 ␮l annexin V-FITC (BioSource)
and 50 ␮l PI (100 ␮g/ml). After 15 min incubation at room
temperature, flow cytometry was performed. Live cells are
negative for both annexin V and PI, early apoptotic cells are
positive for annexin V only and late apoptotic and necrotic
cells are positive for both dyes [33,34]. The probes were
analyzed by FACSCalibur flow cytometer (Beckton Dickinson, San Diego, CA) and analysed by CellQuest software.
Argon laser excitation wavelength was 488 nm, while emission data were acquired at wavelength 520 nm for fluorescein and 580 nM FL-2 and 650 nM FL-3 for PI. Statistical
significance was calculated using the chi-square test.
2.6. Transmission electron microscopy
For electron microscopy after twice washing in PBS the
cells were immediately fixed in 2.5% glutaraldehyde EM
grade (Merck, Darmstadt, Germany) and 2% paraformaldehyde (Sigma) in 0.1 M cacodylate buffer (pH 7.4) for 1.5 h,
postfixed in 1% OsO4 in 0.1 M cacodylate buffer (pH
7.4) for 1 h, dehydrated in graded alcohol and embedded
in Spurr resin (Sigma). Ultrathin sections were cut with
OMU-3 ultratome (C. Reichert AG, Vienna, Austria) and
put onto copper grids and contrasted with uranyl acetate
and lead citrate (Merck). The sections were observed at
5500–6000× primary magnification with a JEOL JEM 100
S electron microscope (Jeol Ltd., Tokyo, Japan) at 80 kV.
The apoptotic cells were detected according to the ultrastructural criteria of alterations in the nuclei and in the cy-
55
toplasm. These alterations consisted of chromatin condensation, formation of nuclear blebs and buds with condensed
chromatin that pinched off to produce components of apoptotic bodies, leaks in nuclear envelopes, and vacuolization of
the cytoplasm [35–38]. The sequence of ultrastructural findings allowed us to divide this process into two stages: the
first—early apoptosis and the second—advanced apoptosis.
Early apoptosis was characterized by chromatin condensation in peripheral regions of the nuclei and by the presence
of numerous vacuoles in the cytoplasm, while the cytoplasm
itself was electron dense but the number of its organelles was
reduced. In advanced apoptosis the condensation of chromatin was more pronounced the nuclei were either forming
buds pinching off to form components of apoptotic bodies
or were already breaking up. In advanced apoptosis the tendency to enlargement and clustering of vacuoles could be
observed, while the cytoplasm was disorganized.
2.7. SDS-PAGE and Western blotting
Western blotting analysis was performed as described
elsewhere [28]. Briefly, leukemic cells (4 × 106 ) were lysed
in SDS-sample buffer. Equal amounts of protein were separated by 7.5% SDS-PAGE on MiniProtean II electrophoresis system (Biorad, Hercules, CA). As molecular weight
marker, kaleidoscope prestained marker set was used (Biorad). Semi-dry electrophoretic transfer was made and membranes were blocked with 5% non-fat dry milk in TBST with
0.5% fetal calf serum, overnight and probed with the rabbit
polyclonal anti poly(ADP-ribose) polymerase (PARP) antibody, (Santa Cruz, CA, USA). After washing in TBST, the
membranes were incubated with alkaline phosphatase conjugated donkey anti-rabbit polyclonal serum 1:2500 (Jackson Immunoresearch Laboratories Inc., West Grove, PN).
Alkaline phosphatase was detected by a standard method using BCIP/NBT (Sigma). After drying, the membranes were
scanned with Plustek Opticpro 9636T flatbed scanner.
2.8. Statistical analysis
Data are presented as means ± standard deviation (S.D.).
Differences between groups were analyzed for significance by the Student’s t-test. Significance was defined as a
two-sided P < 0.05. The statistical analysis for flow cytometry results was performed with use of the two-sided chisquare test. A series of independent experiments were
performed and the results presented in the paper are representative.
3. Results
3.1. Potentiated antitumor effects of AAF-cmk used in
combination with TRAIL or TNF
U937 leukemia cells were found to be susceptible to
the cytostatic/cytotoxic activity of both TRAIL (Fig. 1A)
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Fig. 2. Isobologram analysis depicting the interaction between AAF-cmk
and TRAIL in inhibiting the growth of U937 cells. Solid line concentrations of both drugs that cause a 60% inhibition of cell proliferation
(IC60 ). Hyphenated line, the hypothetical amounts of both drugs required
to cause the same decrease in cell proliferation as if the interactions were
additive. The number in the box indicate the interaction index calculated
as described in “Section 2”.
Fig. 1. U937 cells were exposed for 24 h to various concentrations of
AAF-cmk followed by 24 h incubation with AAF-cmk and/or TRAIL (A)
or with TNF (B). Cytostatic/cytotoxic effects were tested in a standard
MTT assay. Data are presented as a relative viability (percentage of
untreated controls). Each bar represents mean ± S.D. All groups marked
with (∗) are significantly different from the controls (P < 0.05).
and human TNF (Fig. 1B) as well as AAF-cmk (Fig. 1A
and B). Preincubation of U937 cells with AAF-cmk for 24 h
followed by incubation with both AAF-cmk and each of the
two cytokines for additional 24 h resulted in the potentiation
of growth inhibitory cytostatic/cytotoxic effects of TRAIL
as well as TNF (Fig. 1A and B). Isobologram analysis according to Berenbaum revealed synergistic activity of the
combination of TRAIL and AAF-cmk at the synergism index of 0.55 (Fig. 2).
Importantly, the combination treatment with AAF-cmk
and TRAIL was not toxic towards normal human monocytes
(Fig. 3).
3.2. AAF-cmk increases TNF- and TRAIL-induced
apoptosis
To investigate whether the cytotoxic/cytostatic effect of
AAF-cmk with TRAIL was due to increased apoptosis, the
U937 cells were labeled with annexin V-FITC and PI before
FACScan analysis. TRAIL in experimental concentrations
induced 19.4% apoptosis while the inhibitor of TPPII alone
induced 24.7% apoptosis in U937 cells. The combination of
AAF-cmk/TRAIL increase the percentage of apoptotic cells
to 55.8%, however there were also some necrotic cells in the
combination group. The combined treatment left 17.9% of
viable U937 cells (Table 1). Similar results were obtained
for AAF-cmk/TNF treatment (data not shown).
The electron microscopy analysis of U937 cells was provided according to the ultrastructural apoptotic criteria as
described in Section 2. This analysis revealed increased
number of cells with characteristic apoptotic morphology in
groups treated with AAF-cmk and both TRAIL or TNF compared to each group treated alone. Moreover, these findings
were more pronounced and advanced in cells undergoing
the combination treatment (Fig. 4).
3.3. A pan-caspase inhibitor zVAD-fmk prevents the
cytotoxic effects of the combination of AAF-cmk with either
TRAIL or TNF
To determine whether the cytostatic/cytotoxic activity of
the combination of AAF-cmk with either TRAIL or TNF
was mediated through caspase activation, the U937 cells
were incubated with the drugs in the presence or absence
of a pan-caspase inhibitor, zVAD-fmk. Caspase inhibition
by zVAD-fmk significantly reduced the cytotoxicity of
AAF-cmk at 10 ␮M in combination with TRAIL at 30 ng/ml
against U937 cells (cell viability averaged 35 ± 13.6 and
77 ± 4.6% in the absence and presence of zVAD-fmk, respectively; n = 4) (Fig. 5A). Preincubation of the cells in
I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61
57
Fig. 3. Normal human monocytes were exposed for 24 h to various concentrations of AAF-cmk followed by 24 h incubation with AAF-cmk and/or
TRAIL. Cytostatic/cytotoxic effect was assessed by crystal violet staining. Data are presented as a relative viability (percentage of untreated controls).
Each bar represents mean ± S.D. All groups marked with (∗) are significantly different from the controls (P < 0.05).
the presence of the pan-caspase inhibitor zVAD-fmk also
diminished the cytotoxicity caused by the combination of
AAF-cmk at 10 ␮M with TNF at 500 ng/ml in U937 cells
(cell viability averaged 40 ± 7.6 and 70 ± 3.5% in the
absence and presence of zVAD-fmk, respectively; n = 4)
(Fig. 5B).
lanes 3 and 6). Cleavage of PARP from 116 to 85 kDa, was
also the strongest in the combined group. Each drug given
alone in experimental concentrations showed only weak or
no degradation of PARP (Fig. 6, lanes 2 and 5).
4. Discussion
3.4. PARP cleavage analysis
Cleavage of the nuclear repair enzyme poly(ADP-ribose)
polymerase is considered a very sensitive maker of caspase
activation and apoptosis [39]. To examine whether PARP
cleavage is enhanced following combination treatment of
AAF-cmk with TRAIL or TNF, an immunoblotting analysis
was performed. The cells constitutively expressed PARP, but
after treatment with combination of AAF-cmk with TRAIL
or TNF, proteolytic fragments of PARP were found (Fig. 6,
Table 1
Percentage of apoptotic, necrotic and living U937 cells, after 24 h pretreatment with AAF-cmk and 24 h incubation with a control solvent or
AAF-cmk and/or TRAIL
Living
Apoptotic
Necrotic
Control
AAF-cmk
(20 ␮M)
TRAIL
(30 ng/ml)
TRAIL +
AAF-cmk
95.8
2.8
1
70.1
24.7
4.1
74.2
19.4
5.52
17.9
55.8
26.3
Data was obtained in each group for 10 000 cells separated from other
events based on light scatter characteristics. Apoptotic, necrotic and living cells were discriminated as described in “Section 2”. The combinations of AAF-cmk and TRAIL were significantly different from single
agent-treated groups (chi square <0.005).
In this report, we demonstrate that AAF-cmk, an inhibitor
of TPPII, potentiates cell death induced by TNF and TRAIL
in human leukemic U937 cells. Increased cell death is primarily caused by an increased apoptosis induction as shown
by flow cytometric analysis, caspase inhibitor studies, PARP
cleavage and electron microscopy. Necrosis was also shown
in the combination group as shown in Table 1, which could
mean that after 24 h treatment the apoptoic cells deteriorated to the extent that they took up propidium iodide. The
viability of tumor cells treated with AAF-cmk is somewhat
lower in cytostatic/cytotoxic assays with MTT (Fig. 1A
and B) as compared to cytometric analyses (Table 1). This
discrepancy can be explained by the fact that MTT assay shows both antiproliferative and cytotoxic effects of
AAF-cmk, while cytometric analysis refers only to the
latter.
The physiological role of TPPII in mammalian cells is still
unresolved. It was suggested that this protease could replace
some of the functions of proteasome [11,12]. Although very
attractive, this hypothesis was recently challenged [20,40].
However, it could be suspected that the function of TPPII inhibitors could also imitate the activity of proteasome
inhibitors. Moreover, inhibition of the proteasome by PSI
potentiated the antitumor effects of both TRAIL and TNF
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Fig. 4. Ultrastucture of U937 cells treated for 24 h with AAF-cmk (20 ␮M) followed by 24 h incubation with AAF-cmk and/or TRAIL (100 ng/ml) and/or
with TNF (500 ng/ml) (magnification 5200×). Control group (A): The utlrastructure of U937 cell—the polymorphic nucleus with eu- and heterochromatin
and the cytoplasm with well preserved organelles. TRAIL group (B): The apoptotic cell with peripheral nuclear chromatin condensation and reduced
amount of organelles in cytoplasm. TNF group (C): The cell in more advanced apoptosis than in Fig. 4B. The chromatin is condensed the cytoplasm is
vacuolised with few organelles. AAF-cmk/TRAIL group (D): Two apoptotic cells, left—the early apoptotic cell with peripheral chromatin condensation
and cytoplasm vacuolization; right—the advanced apoptotic cell with condensed chromatin and disorganized cytoplasm. AAF-cmk/TNF group (E): The
advanced apoptotic cell. The nucleus is budding and breaking up to form components of apoptotic bodies. AAF-cmk group (F): The U937 cell with no
evident ultrastructural features of apoptosis.
I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61
Fig. 5. The influence of caspase inhibition with zVAD-fmk on the
AAF-cmk/TRAIL (A) and the AAF-cmk/TNF (B) mediated cytostatic/cytotoxic activity revealed by a standard MTT assay on U937 cells.
Data are presented as a relative viability (percentage of untreated controls). Each bar represents mean ± S.D. All groups marked by (∗) are
significantly different from the control (P < 0.05).
59
[28]. Despite unknown physiological role of TPPII, there is
some data indicating that it could complement or mimic the
proteolytic function of proteasome. Thus, it can be speculated that inhibition of TPPII could also induce similar
antitumor effects as proteasome inhibitors.
AAF-cmk irreversibly blocks TPPII and reversibly the
lysosomal TPPI [41]. It also blocks, to a certain degree, the
chymotrypsin-like activity of the proteasome [20]. When
TPPII is blocked and oligopeptides are not effectively degraded it could lead to accumulation of these peptides and
disturb the cell’s environment and interfere with cellular
metabolic pathways.
In our experiments, the cytostatic/cytotoxic activity of
AAF-cmk with TRAIL and AAF-cmk with TNF could
be reduced by incubation with zVAD-fmk suggesting that
activation of caspases could be responsible for the cell
death induced by this combination treatment. The results
obtained with flow cytrometric studies found also confirmation in ultrastructural studies where increased number
of apoptotic cells has been observed in the combination
groups.
Altogether, our studies indicate that AAF-cmk is capable
of sensitizing human leukemic cells to proapoptotic effects
of TRAIL and TNF. The present study provides the first evidence that, in addition to the proteasome, also TPPII might
play a role in TRAIL and TNF mediated apoptotic pathways. Whatever mechanism is responsible for this activity,
AAF-cmk seems to be a potent sensitiser for antitumor effects of TNF-family cytokines.
Fig. 6. SDS-PAGE (7.5%) of the caspase-specific substrate PARP in either control U937 whole lysates (lane 1), or the same cells treated with: 100 ng/ml
TRAIL, lane 2; AAF-cmk (20 ␮M), lane 4; TNF 500 ng/ml), lane 5 and combination of TRAIL/AAF-cmk, lane 3; as well as combination of TNF
with AAF-cmk, lane 6. Treatment of U937 cells by combination of AAF-cmk and TRAIL/TNF correlated with PARP cleavage. Filled arrows indicate
uncleaved forms of PARP and opened arrows indicate the cleaved forms of these protein. Actin was used as the loading control.
60
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Acknowledgements
This work has been supported by KBN Grant 4 P05A
084 16 to CW, by the Medical University Grant 01-1M15NS2-2002 to IM and by the CMKP grant 501-2-1-03-48/02
to GH.
References
[1] Hershko A, Ciechanover A. The ubiquitin system. Annu Rev
Biochem 1998;67:425.
[2] Wójcik C, Stokłosa T, Giermasz A, Goł˛ab J, Zagożdżon R, Kawiak J,
et al. Apoptosis induced in L1210 leukemia cells by an inhibitor of the
chymotrypsin-like activity of the proteasome. Apoptosis 1997;2:455.
[3] Wójcik C. Proteasomes in apoptosis: villains or guardians? Cell Mol
Life Sci 1999;56:908.
[4] Wójcik C, Schroeter D, Stoehr M, Wilk S, Paweletz N. An inhibitor
of the chymotrypsin-like activity of the multicatalytic proteinase
complex (20S proteasome) induces arrest in G2-phase and metaphase
in HeLa cells. Eur J Cell Biol 1996;70:172.
[5] Adams J. Proteasome inhibition in cancer: development of PS-341.
Semin Oncol 2001;28:613.
[6] Adams J. Development of the proteasome inhibitor PS-341.
Oncologist 2002;7:9.
[7] Bonn D. Targeting protein breakdown to treat cancer. Mol Med
Today 1999;5:48.
[8] Gavioli R, Frisan T, Vertuani S, Bornkamm GW, Masucci MG.
c-myc overexpression activates alternative pathways for intracellular
proteolysis in lymphoma cells. Nat Cell Biol 2001;3:283.
[9] Pleban E, Bury M, Młynarczuk I, Wójcik C. Effects of proteasome
inhibitor PSI on neoplastic and non-transformed cell lines. Folia
Histochem Cyto 2001;39:133.
[10] Wang EW, Kessler BM, Borodovsky A, Cravatt BF, Bogyo M,
Ploegh HL, Glas R. Integration of the ubiquitin–proteasome pathway
with a cytosolic oligopeptidase activity. Proc Natl Acad Sci USA
2000;97:9990.
[11] Geier E, Pfeifer G, Wilm M, Lucchiari-Hartz M, Baumeister W,
Eichmann K, Niedermann G. A giant protease with potential to
substitute for some functions of the proteasome. Science 1999;
283:978.
[12] Glas R, Bogyo M, McMaster JS, Gaczynska M, Ploegh HL. A
proteolytic system that compensates for loss of proteasome function.
Nature 1998;392:618.
[13] Yao T, Cohen RE. Giant proteases: beyond the proteasome. Curr
Biol 1999;9:551.
[14] Balow RM, Tomkinson B, Ragnarsson U, Zetterqvist O. Purification,
substrate specificity, and classification of tripeptidyl peptidase II. J
Biol Chem 1986;261:2409.
[15] Rose C, Vargas F, Facchinetti P, Bourgeat P, Bambal RB, Bishop PB,
et al. Characterization and inhibition of a cholecystokinin-inactivating
serine peptidase. Nature 1996;380:403.
[16] Chowdhary BP, Johansson M, Gu F, Brauner-Nielsen P, Tomkinson B,
Andersson L, et al. Assignment of the linkage group EAM-TYRP2TPP2 to chromosome 11 in pigs by in situ hybridization mapping
of the TPP2 gene. Chromosome Res 1993;1:175.
[17] Renn SC, Tomkinson B, Taghert PH. Characterization and cloning of
tripeptidyl peptidase II from the fruit fly, Drosophila melanogaster.
J Biol Chem 1998;273:19173.
[18] Tomkinson B, Jonsson AK. Characterization of cDNA for human
tripeptidyl peptidase II: the N-terminal part of the enzyme is similar
to subtilisin. Biochemistry 1991;30:168.
[19] Hasselgren PO, Wray C, Mammen J. Molecular regulation of muscle
cachexia: it may be more than the proteasome. Biochem Biophys
Res Commun 2002;290:1.
[20] Bury M, Młnarczuk I, Pleban E, Hoser G, Kawiak J, Wójcik
C. Effects of an inhibitor of tripeptidyl peptidase II (Ala–Ala–
Phe–chloromethylketone) and its combination with an inhibitor of
the chymotrypsin-like activity of the proteasome (PSI) on apoptosis,
cell cycle and proteasome activity in U937 cells. Folia Histochem
Cyto 2001;39:131.
[21] Treon S, Mitsiades C, Poulaki V, Anderson K, Mitsiades N. Concepts
in the use of TRAIL/Apo2L: an emerging biotherapy for myeloma
and other neoplasias. Expert Opin Investig Drugs 2001;10:1521.
[22] Kayagaki N, Yamaguchi N, Nakayama M, Takeda K, Akiba
H, Tsutsui H, et al. Expression and function of TNF-related
apoptosis-inducing ligand on murine activated NK cells. J Immunol
1999;163:1906.
[23] Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin
M, et al. Tumoricidal activity of tumor necrosis factor-related
apoptosis-inducing ligand in vivo. Nature Med 1999;5:157.
[24] Park JW, Choi YJ, Jang MA, Baek SH, Lim JH, Passaniti T, et
al. Arsenic trioxide induces G2/M growth arrest and apoptosis after
caspase-3 activation and bcl-2 phosphorylation in promonocytic U937
cells. Biochem Biophys Res Commun 2001;286:726.
[25] Medema JP, Scaffidi C, Kischkel FC, Shevchenko A, Mann M,
Krammer PH, et al. FLICE is activated by association with the
CD95 death-inducing signaling complex (DISC). EMBO J 1997;16:
2794.
[26] Griffith TS, Wiley SR, Kubin MZ, Sedger LM, Maliszewski CR,
Fanger NA. Monocyte-mediated tumoricidal activity via the tumor
necrosis factor-related cytokine, TRAIL. J Exp Med 1999;189:
1343.
[27] Jo M, Kim T, Seol D, Esplen JE, Dorko K, Billar TR, et al.
Apoptosis induced in normal human hepatocytes by tumor necrosis
factor-related apoptosis inducing ligand. Nature Med 2000;6:564.
[28] Młynarczuk I, Hoser G, Grzela T, Stokłosa T, Wójcik C, Malejczyk
J, et al. Augmented pro-apoptotic effects of TRAIL and proteasome
inhibitor in human promonocytic leukemic U937 cells. Anticancer
Res 2001;21:1237.
[29] Gołccb J, Stokłosa T, Czajka A, D˛abrowska A, Jakóbisiak M,
Zagozdzon R, et al. Synergistic antitumor effects of a selective
proteasome inhibitor and TNF in mice. Anticancer Res 2000;20:
1717.
[30] Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY,
Boiani N, et al. TRAIL-R2: a novel apoptosis-mediating receptor for
TRAIL. EMBO J 1997;16:5386.
[31] Garcia-Calvo M, Peterson E, Leiting B, Ruel R, Nicholson D,
Thornberry N. Inhibition of human caspases by peptide-based and
macromolecular inhibitors. J Biol Chem 1998;273:32608.
[32] Goł˛ab J, Nowis D, Skrzycki M, Czeczot H, Anna Baranczyk-Kuzma
A. Antitumor effects of photodynamic therapy are potentiated by
2-methoxyestradiol: a superoxide dismutase inhibitor. J Biol Chem
2003;278:407.
[33] Homburg C, Haas M, Borne A, Verhoeven A, Reutelingsperger C,
Roos D. Human neutrophils lose their surface Fc gamma RIII and
acquire Annexin V binding sites during apoptosis in vitro. Blood
1995;85:532.
[34] Boersma A, Nooter K, Burger H, Kortland C, Stoter G. Expression of
the apoptosis-associated proteins Bcl-2 and Bax was quantitated by
flow cytometry (FCM) in chemosensitive testicular germ-cell tumor
NT2 cells, and the results were compared with those obtained by
Western blotting. NT2 cells were incubated with cisplatin. Cytometry
1997;27:275.
[35] Wyllie A, Kerr J, Currie A. Cell death: the significance of apoptosis.
Int Rev Cytol 1980;68:251.
[36] Ihara T, Yamamoto T, Sugamata M, Okumura H, Ueno Y. The process
of ultrastructural changes from nuclei to apoptotic body. Virchows
Arch 1998;433:443.
[37] Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. Normotonic
cell shrinkage because of disordered volume regulation is an
early prerequisite to apoptosis. Proc Natl Acad Sci USA 2000;97:
9487.
I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61
[38] Soligo D, Servida F, Delia D, Fontanella E, Lamorte G, Caneva L,
et al. The apoptogenic response of human myeloid leukaemia cell
lines and of normal and maxlignant haematopoietic progenitor cells
to the proteasome inhibitor PSI. Br J Haematol 2001;113:126.
[39] Fulda S, Friesen C, Debatin KM. Molecular determinants of apoptosis induced by cytotoxic drugs. Klinische Padiatrie 1998;210:148.
[40] Princiotta MF, Schubert U, Chen W, Bennink JR, Myung J, Crews
CM, et al. Cells adapted to the proteasome inhibitor 4-hydroxy-5-
61
iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomes for continued survival. Proc Natl
Acad Sci USA 2001;98:513.
[41] Page AE, Fuller K, Chambers TJ, Warburton MJ. Purification
and characterization of a tripeptidyl peptidase I from human
osteoclastomas: evidence for its role in bone resorption. Arch
Biochem Biophys 1993;306:354.