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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3583-3592
Liposomal ET-18-OCH3 Induces Cytochrome c-Mediated
Apoptosis Independently of CD95 (APO-1/Fas) Signaling
By
Olivier Cuvillier,
Eric Mayhew,
Andrew S. Janoff, and
Sarah Spiegel
From the Department of Biochemistry and Molecular Biology, Georgetown
University Medical Center, Washington, DC; and The Liposome Co, Inc,
Princeton, NJ.
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ABSTRACT |
ELL-12, a liposome formulation of the ether-lipid
1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine
(ET-18-OCH3), is a nonmyelosuppressive antiproliferative
agent that is more effective and less toxic than the ether lipid itself
in tumor model systems. We found that ELL-12 induced apoptosis in
Jurkat, H9, and U-937 cells that was preceded by activation of
executioner caspases. In addition, ELL-12 triggered release of
cytochrome c from mitochondria to the cytoplasm before
caspase-9 activation. Apoptosis, activation of caspases, and cytochrome
c release were blocked by Bcl-xL overexpression in
Jurkat T cells, suggesting a critical role for mitochondria in
ELL-12-triggered cell death. Furthermore, ELL-12 had no effect on
expression of CD95 ligand, and inhibition of the Fas signaling pathway
with antagonistic anti-CD95 antibody did not affect apoptosis induced
by ELL-12. Hence, ELL-12 could be a promising adjunct for the treatment
of tumors in addition to myelosuppressive chemotherapeutic drugs and/or
those that use the CD95-ligand/receptor system to trigger apoptosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
ALKYL-LYSOPHOSPHOLIPIDS, such as
the ether lipid ET-18-OCH3
(1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine), are antitumor agents that, unlike many other cancer therapeutic agents,
do not directly target cellular DNA and are not myelosuppressive in
animal models.1-3 Although ET-18-OCH3 has
antitumor activity in several animal tumor models, its clinical use has
been hampered by systemic toxic effects, including
hemolysis.4-6 Several reports have documented the
advantages of incorporating anticancer agents into liposomes to improve
therapeutic efficiency while markedly reducing nonspecific toxicity in
vivo.7-12 A stable, well-characterized liposome-based
formulation of ET-18-OCH3, known as ELL-12, has been shown
to be acutely less hemolytic both in vitro and in vivo13-15 and is currently in clinical trials.16 In mice bearing P388 leukemia, Lewis lung carcinoma, or B16/F10 melanoma, ELL-12 had a
therapeutic index at least 4-fold higher than
ET-18-OCH3.13 By preventing the serious side
effects of ET-18-OCH3, ELL-12 could potentially complement
standard anticancer chemotherapeutic agents that target DNA.
The mechanism of the growth-inhibitory effects of
ET-18-OCH3 has been extensively studied and appears to
involve modulation of signal transduction events, including protein
kinase C activity, interaction with cell membranes, inhibition of
phospholipase C, influences on calcium flux, and modification of the
cellular oxidative state.17-26 Recent work has established
that the antineoplastic effect of ET-18-OCH3 could be due
to its ability to promote apoptosis, both in human tumor cell lines and
in primary tumor cell cultures from cancer patients.27-30
Although the antiproliferative effect of ELL-12 has also been
associated with induction of apoptosis,15 little is known
of the mechanism of action of ELL-12.
Understanding of the biochemical events in apoptosis was significantly
advanced by the identification of a family of aspartate-specific cysteine proteases, named caspases, which are involved in the initiation and amplification of the cell death
machinery.31,32 Each caspase is synthesized as an inactive
zymogen (30 to 50 kD) and is converted by proteolytic cleavage to yield
an active enzyme composed of approximately 20-kD and approximately
10-kD subunits. The caspase family has been divided into initiator
caspases (eg, caspases-8 and -10) that can activate downstream
executioner caspases (eg, caspases-3, -7, and -6) responsible for the
cleavage of a limited set of proteins resulting in the disassembly of
the cell.31,32 Accumulating evidence also suggests that
mitochondria have an essential role in the apoptotic program, and
cytochrome c (cyt c) release from mitochondria is now
emerging as an important step in the apoptotic
pathway.33,34 Diverse apoptotic stimuli, including UVB,
etoposide, staurosporine, ionizing radiation, cisplatin, ara-C,
doxorubicin, betulinic acid, photodynamic therapy, and cytokines,
induce cyt c release, which can be prevented by overexpression of Bcl-2 or Bcl-xL.33,34 Released cyt
c, in turn, binds to Apaf-1, a mammalian homologue of the
Caenorhabditis elegans death-promoting protein
CED-4,35 inducing it to associate with
procaspase-9,36 thereby triggering its autoactivation into
a mature form, which in turn can directly cleave procaspases-3 and
-7.37
In the present study, we investigated the molecular mechanisms of
apoptosis triggered by ELL-12, in particular its relationship with the
caspase network and cyt c release. Our findings indicate that
ELL-12 induces caspases activation through release of cyt c in
a Bcl-xL-sensitive manner but independently of the CD95
(APO-1/Fas) ligand/receptor system.
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MATERIALS AND METHODS |
Cell culture.
Human T-cell leukemia Jurkat, human monocyte-like histiocytic lymphoma
U-937 (ATCC, Rockville, MD), and H9 human T-cell lymphoma (gift of Dr
Marcus Peter, German Cancer Research Center, Heidelberg, Germany) cells
were maintained in RPMI 1640 supplemented with 10% fetal bovine serum
(20% for H9 cells). Bcl-xL-transfected and neomycin
control vector (Neo)-transfected Jurkat (gift of Dr Charles Zacharchuk,
National Institutes of Health, Bethesda, MD) were maintained under the
same conditions in media supplemented with 1 mg/mL G418. For induction
of apoptosis, logarithmically growing cells were washed twice and
resuspended in serum-free RPMI 1640 medium (0.75 to 1 × 106 cells/mL), unless indicated otherwise.
Materials.
ET-18-OCH3, DOPC, and DOPE-GA were from Avanti Polar Lipids
(Alabaster, AL). Anti-Fas IgM (clone CH-11) and neutralizing anti-Fas IgG (clone ZB4) were from Upstate Biotechnology (Lake Placid, NY).
Ac-DEVD-CHO and Ac-DEVD-AMC were from Bachem (King of Prussia, PA).
Liposome preparation and characterization.
ET-18-OCH3 liposome formulation, ELL-12
[DOPC/Chol/DOPE-GA/ET-18-OCH3] (4:3:1:2, molar ratio),
was prepared using the solvent evaporation method.38
Briefly, DOPC, Chol, DOPE-GA, and ET-18-OCH3 were dissolved
in chloroform/methanol (2:1, vol/vol) and mixed to give a final molar
ratio of 4:3:1:2. After thorough mixing, the organic solvent was
vacuum-evaporated at 45°C, and the thin dried film was hydrated
with Dulbecco's phosphate-buffered saline (PBS) without
Ca2+ or Mg2+ for 1 hour at room temperature or
above the transition temperature for the lipids. The resulting
multilamellar preparations were extruded 10 times through 100-nm
double-stacked Nucleopore filters using an extruder device (Lipex
Biomembranes, Vancouver, British Columbia, Canada). The liposomes were
examined by light microscopy to check morphology, and size was
determined using a Nicomp Model 370 Submicron Particle Sizer system
(Santa Barbara, CA). Liposome size was about 100 nm.
ET-18-OCH3 and other lipid concentrations in the final
preparations were determined using reverse-phase high-performance
liquid chromatography with an evaporative light-scattering detector
with 4% water in methanol as the mobile phase.
Non-ET-18-OCH3-containing liposomes were prepared in the
same way, except that the liposome composition was DOPC:Chol:DOPE-GA in
a 6:3:1 mole ratio.
Staining of apoptotic nuclei.
Apoptosis was assessed as previously described39 by
staining cells with bisbenzimide trihydrochloride (8 µg/mL in 30%
[vol/vol] glycerol/PBS; Hoechst #33258; Calbiochem, San Diego, CA)
for 10 minutes. Cells were counted using a Zeiss Photoscope II
fluorescent microscope (Petersburg, VA). The percentage of
apoptotic cells was calculated as number of apoptotic cells per the
number of total cells counted.
Preparation of mitochondria and Western blot analysis for cyt c.
After treatment, cells were harvested by centrifugation at
1,000g for 5 minutes at 4°C. After washing twice with
ice-cold PBS, mitochondrial and cytosolic fractions were prepared by
resuspending cell pellets in 5 vol of ice-cold buffer A (20 mmol/L
HEPES-KOH [pH 7.5], 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L sodium EDTA, 1 mmol/L sodium EGTA, 1 mmol/L dithiothreitol, 0.1 mmol/L phenylmethylsulfonylfluoride, 20 µg/mL leupeptin, 10 µg/mL
aprotinin, and 10 µg/mL pepstatin) containing 250 mmol/L sucrose.
After swelling for 15 minutes on ice, the cells were homogenized by 15 to 20 passages through a 26-gauge needle, and the homogenates were
centrifuged at 1,000g for 5 minutes at 4°C. The
supernatants were centrifuged again at 12,000g for 15 minutes
at 4°C, and the resulting mitochondria pellets were resuspended in
cold buffer A and frozen in multiple aliquots at  80°C. The
12,000g supernatants were further centrifuged at
100,000g for 1 hour, and the resulting cytosolic fractions were
aliquoted and frozen at 80°C. For Western blot analysis, equivalent amounts of mitochondrial and cytosolic fractions were loaded
on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Primary antibodies were the 7H8.2C12 cyt c
monoclonal antibody (MoAb; Research Diagnostics Inc, Flanders, NJ)
and the 12C4-F12 cyt c oxidase subunit II MoAb (Molecular
Probes, Eugene, OR).
Western blotting for caspases, Bid, PARP, Lamin B, CD95L, and
Bcl-xL.
Cell lysates preparation and Western blotting were performed as
previously described.39 Polyclonal rabbit anti-caspase-3 (gift of Dr Donald Nicholson, Merck-Frosst Centre for Therapeutic Research, Pointe Claire, Quebec, Canada), antimouse anti-caspase-8 antibody C15 (gift of Dr Marcus Peter), rabbit antisera specific for
the p20 subunit of caspase-7 (gift of Dr Edward Gelmann, Lombardi Cancer Center, Washington, DC), monoclonal rat anti-caspase-7 (gift of
Dr Junying Yuan, Harvard Medical School, Boston, MA), rabbit antisera
for PARP (Boehringer Mannheim, Indianapolis, IN), rabbit
anti-caspase-9 and rabbit anti-caspase-10 (Oncogene Research Products, Cambridge, MA), rabbit anti-caspase-2 (Santa Cruz
Biotechnology, Santa Cruz, CA), polyclonal rabbit anti-Bid
(gift of Dr Stanley Korsmeyer, Harvard Medical School), the anti-lamin
B1 (Calbiochem, San Diego, CA), and anti-Bcl-x and the anti-CD95L
(Transduction Laboratories, Lexington, KY) were used as primary
antibodies. Binding was detected with antirat (Boehringer Mannheim),
antirabbit, or antimouse (Bio-Rad, Hercules, CA) horseradish
peroxidase-conjugated IgG and detected by enhanced chemiluminescence
(SuperSignal or Ultra SuperSignal) as described by the manufacturer
(Pierce, Rockford, IL).
Fluorogenic DEVD cleavage enzyme assays.
Enzyme reactions were performed in 96-well plates with 20 µg of
cytosolic proteins and a final concentration of 20 µmol/L Ac-DEVD-AMC
substrate as previously described.39 Fluorescent aminomethyl coumarin (AMC) product formation was measured over a
30-minute period at excitation and emission wavelengths of 360 nm and
460 nm using a Cytofluor II fluorometer plate reader (PerSeptive Biosystems, Framingham, MA).
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RESULTS |
ELL-12 induces apoptosis in a time- and concentration-dependent manner
in Jurkat, H9, and U-937 cells.
Treatment of Jurkat T leukemia cells with ELL-12 for 5 hours in
serum-free medium, or for longer times in the presence of serum,
induced extensive cell death in a dose-dependent manner, as measured by
the appearance of nuclear fragmentation
(Fig 1A). In contrast, treatment of cells
with liposomes lacking ET-18-OCH3 did not induce apoptosis
(Fig 1A), whereas anti-Fas treatment induced extensive cell death (Fig
1A). Because the presence of serum did not interfere with the ability
of ELL-12 or Fas ligation to induce apoptosis (Fig 1A), but only
delayed the onset of the apoptotic process, all further experiments
were performed in serum-free media.
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| Fig 1.
Induction of apoptosis and DEVDase activation by the
liposomal ether-lipid ELL-12 in Jurkat, H9, and U-937 cells. (A) Jurkat
T cells were treated in serum-free medium for 5 hours (solid squares
and solid bars) or in the presence of serum for 24 hours (open squares
and open bars) with the indicated concentrations of ELL-12 or with
non-ET-18-OCH3 liposomes at a concentration equivalent to
the highest concentration of ELL-12 (lp) or 50 ng/mL anti-Fas antibody.
The percentage of apoptotic cells was evaluated by the DNA-specific
fluorochrome Hoechst. Nuclei were visualized by fluorescence microscopy
and a minimum of 1,000 cells were scored. Mean values ± SD from at
least 3 different experiments are shown. (B) Jurkat T cells were
treated with 10 µg/mL ELL-12 for the indicated times and the
percentage of apoptotic nuclei (solid squares) was determined as
described above. DEVDase activity (open squares) in extracts from
duplicate cultures was measured with the fluorogenic substrate
Ac-DEVD-AMC. Results are the means ± SD of at least 3 independent
experiments. (C) H9 cells (solid squares) and U-937 cells (open
squares) were treated in serum-free medium for 16 hours with the
indicated doses of ELL-12 or with non-ET-18-OCH3 liposomes
at a concentration equivalent to the highest dose of ELL-12. Cells were
then stained with the DNA-specific fluorochrome Hoechst and nuclei were
visualized by fluorescence microscopy. Apoptosis of cells incubated
with non-ET-18-OCH3 liposomes was 11.5% in H9 cells and
5.6% in U-937 cells. Data shown are representative of 3 independent
experiments. DEVDase activity in extracts from H9 (D) and U-937 cells
(E) treated for the indicated times with 10 µg/mL (open squares) or
50 µg/mL ELL-12 (solid squares) was measured with the fluorogenic
substrate Ac-DEVD-AMC. Results are the means ± SD of at least 3 independent experiments.
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At 10 µg/mL of ELL-12, nuclear fragmentation became significant after
4 to 5 hours of treatment (Fig 1B). Blebbing of cell membranes was
observed in almost all cells, which were fragmented into characteristic
condensed nuclei and apoptotic bodies, whereas untreated cells did not
exhibit any morphological changes (data not shown). Consistent with the
results in Jurkat T cells, treatment of H9 T-cell lymphoma and U-937
monoblastic leukemia cells with ELL-12 displayed a time-dependent (data
not shown) and dose-dependent nuclear apoptosis (Fig 1C). Liposomes
lacking ET-18-OCH3 did not induce cell death (data not shown).
Executioner caspases mediate ELL-12-induced apoptosis in Jurkat, H9,
and U-937 cells.
We used the fluorogenic substrate Ac-DEVD-AMC, which corresponds to the
cleavage site found in numerous executioner caspases-3 and -7 targets,
to measure the activity of these caspases in ELL-12-induced apoptosis.
ELL-12 treatment of Jurkat cells resulted in a time-dependent increase
in DEVDase proteolytic activity (Fig 1B), which correlated with the
onset of apoptosis and preceded the appearance of fragmented nuclei by
approximately 1 hour (Fig 1B). Likewise, a time-dependent DEVDase
activity increase was found in H9 (Fig 1D) and U-937 cells (Fig 1E). In
agreement with this finding, pretreatment of Jurkat, H9, and U-937
cells with the inhibitor of caspases-3 and -7 activity, Ac-DEVD-CHO,
completely reversed apoptosis induced by ELL-12 (data not shown).
Proteolytic processing of procaspases-3 and -7 induced by ELL-12
treatment was examined using specific antibodies against their active
subunits. Caspase-3 is synthesized as a 32-kD precursor that is cleaved
to generate the mature form composed of 17-kD (p17) subunits through
intermediary 20-kD (p20) and 12-kD (p12) subunits.40-42 As
shown in Fig 2A, ELL-12 activated caspase-3
in Jurkat cells, as shown by the appearance of the p17 subunit within 2 to 3 hours after the addition of ELL-12. Its level increased progressively thereafter in a time-dependent fashion similar to the
increase in DEVDase activity (Fig 1B), suggesting a correlation between
the appearance of the active form of caspase-3 and the onset of
apoptosis. Similarly, processing of caspase-3 was detected in H9 (Fig
2D) and U-937 cells (Fig 2E). These data suggest that activation of
caspase-3 plays an important role in apoptosis induced by ELL-12.
Activation of caspase-7 requires cleavage of the 35-kD precursor into
subunits of 20 and 12 kD.43,44 Similar to caspase-3, processing of caspase-7 into its active form was apparent after 2 to 3 hours of treatment with ELL-12 in Jurkat cells (Fig 2B). In H9 cells
and U-937 cells, caspase-7 was also cleaved in response to ELL-12 (data
not shown).
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| Fig 2.
Caspases activation by ELL-12 treatment. Extracts from
Jurkat T cells treated with 10 µg/mL ELL-12 for the indicated times
were resolved by SDS-PAGE and probed with anti-caspase-3 (A),
anti-caspase-7 (B), and anti-caspase-8 (C) antibodies. Cytosolic
extracts from H9 cells (D) and U-937 cells (E) treated with 10 and 50 µg/mL ELL-12 or 50 ng/mL anti-Fas antibody for 16 hours were resolved
by SDS-PAGE and probed with anti-caspase-3 antibody. The migration
position of full-length caspase-3, the cleavage intermediate p20, the
active subunit p17, the active caspase-7 subunit p20, full-length
caspase-8, the cleavage intermediates p43 and p41, and the active
subunit p18 are indicated.
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Bcl-xL completely inhibits apoptosis, DEVDase activity,
and activation of executioner caspases-3, -6, and -7, PARP, and lamins
in Jurkat cells.
Cytoprotective Bcl-2 family members, Bcl-2 and Bcl-xL,
protect against diverse apoptotic stimuli.45,46 It was thus
of interest to determine whether apoptosis induced by ELL-12 was also
under the control of these proteins. Jurkat cells stably transfected with bcl-xL expressed high levels of
Bcl-xL protein (Fig 3A) and were found to be completely resistant to apoptosis induced by ELL-12
(Fig 3B). Similar results were obtained with the breast adenocarcinoma
MCF-7 cell line overexpressing Bcl-xL where ELL-12-induced apoptosis was prevented (data not shown). Bcl-xL similarly
prevented the ELL-12-induced increase in DEVDase activity in Jurkat T
cells (Fig 3C). Correspondingly, processing of the executioner
caspase-3 (Fig 4A) and caspase-7 (Fig 4B)
into their mature forms was completely prevented in
Jurkat/Bcl-xL cells. Cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), one of the known substrates for
caspases-3 and -7,41 into apoptotic 89- and 24-kD fragments was also abrogated in Jurkat/Bcl-xL cells (Fig 4C). In
vitro, caspase-3 can process caspase-6,47 which is
responsible for proteolysis of lamins, intranuclear intermediate
filament proteins forming a stratum interposed between the chromatin
and the nuclear envelope, whose cleavage is required for fragmentation
of the nucleus into multiple apoptotic bodies.48 ELL-12
induced activation of caspase-6 (Fig 4D) and subsequent degradation of
lamins (Fig 4E) that were blocked in Jurkat/Bcl-xL cells.
These results suggest that ELL-12 induces apoptosis and activation of
caspases-3, -6, and -7 in a Bcl-xL-sensitive manner.
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| Fig 3.
Bcl-xL blocks apoptosis and DEVDase activity
in Jurkat cells. (A) Expression of Bcl-xL in transfected
Jurkat cells. Cellular proteins from Jurkat stably transfected with
empty vector (neo) or Bcl-xL expression vector were
separated by SDS-PAGE and immunoblotted using anti-Bcl-x antibody. (B)
Jurkat cells transfected with vector control or Bcl-xL
expression vector were treated without or with 10 µg/mL ELL-12 for 5 hours. Apoptosis was assessed by Hoechst staining. Results are the
means ± SD of at least 3 independent experiments. (C) Activation of
DEVD-specific caspases was measured by the cleavage of the fluorogenic
substrate Ac-DEVD-AMC. Results are the means ± SD of at least 3 independent experiments.
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| Fig 4.
Bcl-xL overexpression inhibits caspases-2,
-3, -6, -7, -8, and -10 and Bid activation induced by ELL-12 in Jurkat
cells. Cytosolic extracts from Jurkat/neo or Jurkat/Bcl-xL
cells treated for 5 hours without or with 10 µg/mL ELL-12 were
subjected to SDS-PAGE and immunoblotted with anti-caspase-3 (A),
anti-caspase-7 (B), anti-PARP (C), anti-caspase-6 (D), anti-lamin B
(E), anti-caspase-2 (F), anti-caspase-8 (G), anti-caspase-10 (H), or
anti-Bid (I). The migration position of full-length caspase-3, cleavage
intermediate p20, active subunit p17, full-length caspase-7, active
subunit p20, full-length PARP, cleaved forms p89 and p24, full-length
caspase-6, full-length lamin B, cleaved form p28, full-length
caspase-2, active form p12, full-length caspase-8, cleavage
intermediates p43 and p41, active subunit p18, full-length caspase-10,
cleavage intermediate p23, active subunit p17, and full-length Bid are
indicated.
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ELL-12 induces processing of caspases-2, -8, and -10 and Bid cleavage
that are inhibited by Bcl-xL overexpression in Jurkat
cells.
Caspases with long prodomains, such as caspases-2, -8, and -10, are
commonly considered to be upstream caspases in apoptosis due to their
ability to associate with the cell surface death receptor molecules,
CD95 and TNFR1.31 Nevertheless, caspase-2 shares with
executioner caspases-3 and -7 the optimal peptide recognition motif
DExD.49 Moreover, processing of caspase-2 has been reported
to be dependent of executioner caspase-3 in diverse cell
lines,50,51 occurring later than activation of caspase-3-like proteases in Jurkat cells undergoing
apoptosis.50 Here, ELL-12 induced activation of caspase-2
into its mature form (p12 subunit), which was totally prevented by
Bcl-xL overexpression (Fig 4F).
Caspase-8 cleavage often represents the first detectable event in death
receptor-mediated apoptosis.52 However, it has been recently demonstrated that, in type II cells, such as Jurkat, caspase-8
activation can also require involvement of mitochondria, inasmuch as
Bcl-2 or Bcl-xL overexpression can inhibit its
processing.53 Caspase-8 is expressed as a 55-kD precursor
that is cleaved to generate the mature form composed of 18-kD (p18)
subunits, through 2 intermediate cleavage products of 43 and 41 kD, and
10-kD (p10) subunits.54 As shown in Fig 4G, ELL-12
activated caspase-8. The appearance of 2 cleavage intermediates p43/41
and the p18 active subunit occurred within 3 hours after addition of
ELL-12 (Fig 2C). In Jurkat cells overexpressing Bcl-xL,
processing of caspase-8 induced by ELL-12 was totally inhibited (Fig
4G). These results show that, in type II cells, caspase-8 can
effectively be activated in a Bcl-xL-sensitive fashion and
in a time-frame similar to activations of caspases-3 and -7 (Fig 2A
through C).
Caspase-10 has been proposed to bind to Fas receptor-like
caspase-8.55,56 Using an antibody specific for the p23/p17
processed forms, we demonstrated that the 55-kD precursor caspase-10
was activated in response to ELL-12 (Fig 4H). In agreement with
previous data,54 caspase-10 activation occurs in a
Bcl-xL-sensitive manner in Jurkat T cells (Fig 4H).
We next asked whether ELL-12 treatment could lead to cleavage of Bid, a
pro-apoptotic Bcl-2 family member, which is cleaved by caspase-8 into a
C-terminal fragment that can directly act on mitochondria to trigger
cyt c release.57-60 In agreement with the
Bcl-xL effect on ELL-12-induced activation of caspase-8,
Bid activation by ELL-12 was impeded by Bcl-xL (Fig 4I).
These results suggest that Bid activation may also be under the control
of Bcl-xL.
ELL-12 induces translocation of mitochondrial cyt c into the cytosol
and activation of caspase-9, effects that are blocked by
Bcl-xL overexpression in Jurkat cells.
Because ELL-12-triggered apoptosis was tightly controlled by
Bcl-xL, we therefore asked whether ELL-12 could also
trigger mitochondrial cyt c release, because Bcl-2 and
Bcl-xL interfere with cyt c release.61
As shown in Fig 5A, cytosolic cyt c
levels were markedly increased as early as 1 hour after treatment of Jurkat cells with ELL-12. These results demonstrate that cyt c release is an early event in ELL-12-treated Jurkat cells, occurring before activation of caspase-3, -7, and -8 (Fig 2A through C). Cyt
c release induced by ELL-12 was inhibited in mitochondria from
Bcl-xL-overexpressing cells (Fig 5C). Similarly, ELL-12
treatment triggered cyt c release in H9 cells (Fig 5E) and
U-937 cells (data not shown).
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| Fig 5.
ELL-12 triggers cytosolic accumulation of cyt c and
caspase-9 activation that are inhibited by Bcl-xL
overexpression. Jurkat T cells treated with 10 µg/mL ELL-12 were
harvested at the indicated times, and cytosolic and mitochondrial
proteins were separated by 15% SDS-PAGE and analyzed by immunoblotting
with anti-cyt c (A) or anti-cyt c oxidase (subunit II;
B). Cyt c oxidase serves as a marker for mitochondrial
contamination of cytosolic extracts. A mitochondrial extract from
nontreated cells (mit. fr. Con) was used as a positive control for cyt
c and cyt c-oxidase (subunit II). Jurkat/neo and
Jurkat/Bcl-xL cells, treated without or with 10 µg/mL
ELL-12 for 2 hours, were harvested and cytosolic proteins were
separated by 15% SDS-PAGE and analyzed by immunoblotting with anti-cyt
c (C). Cytosolic extracts from Jurkat/neo or
Jurkat/Bcl-xL cells treated for 5 hours without or with 10 µg/mL of ELL-12 were subjected to SDS-PAGE and immunoblotted with
anti-caspase-9 (D). Cytosolic and mitochondrial proteins from H9 cells
treated with 10 and 50 µg/mL ELL-12 for 16 hours were separated by
15% SDS-PAGE and analyzed by immunoblotting with anti-cyt c
(E) or anti-cyt c oxidase (subunit II; F). Cytosolic extracts
from H9 cells treated for 16 hours without or with 10 and 50 µg/mL of
ELL-12 were subjected to SDS-PAGE and immunoblotted with
anti-caspase-9 (G). The migration positions of full-length caspase-9,
cleavage intermediate p35, and active subunit p10 are indicated.
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Recent studies have shown that caspase-9 is activated by cyt c
due to clustering of caspase-9 by Apaf-1, leading to activation of
caspases-3 and -7.35-37 Because ELL-12 induced cyt
c release and caspases-3 and -7 activation in a
Bcl-xL-dependent pathway, we then examined whether
caspase-9 was also activated by ELL-12. Caspase-9 exists as a pro-form
of 46 kD that is processed into 35- and 10-kD forms. Caspase-9 was
processed into its active forms in response to ELL-12 treatment in
Jurkat (Fig 5D) and H9 cells (Fig 5G), and Bcl-xL
overexpression totally blocked this activation in Jurkat cells (Fig
5D).
Induction of apoptosis by ELL-12 is not mediated through the CD95
receptor/ligand system in Jurkat and H9 cells.
Involvement of the CD95 receptor/ligand system has been proposed to
mediate apoptosis induced by several anticancer drugs in a variety of
tumors cells.62 Because caspase-8 is activated in response
to CD95 triggering through its recruitment to the CD95
receptor52 and because ELL-12 induced caspase-8 activation in Jurkat (Figs 2C and 4G) and also in H9 cells (see Fig 7A), we
determined whether stimulation of the CD95 system could account for
caspase-8 activation and apoptosis induced by ELL-12. ELL-12 did not
induce upregulation of CD95 ligand protein (CD95L) in Jurkat
(Fig 6A) or in H9 cells
(Fig 7B). Moreover, blocking the CD95
receptor/ligand interaction by antagonist anti-Fas antibody clone ZB4
did not inhibit ELL-12-induced cell death, whereas apoptosis triggered
by agonist anti-Fas antibody CH-11 was markedly reduced in Jurkat (Fig
6B) and H9 cells (Fig 7C). Consistent with this result, we found that
ZB4 antibody completely abrogated DEVDase activity induced by anti-Fas
CH-11, but not that induced by ELL-12 in Jurkat cells (Fig 6C).
Finally, Fas receptor blockade by ZB4 completely impeded caspases-8 and
-3 processing induced by anti-Fas CH-11 but not that induced by ELL-12
(Fig 6D and E). Together, these findings negate a role for the CD95
ligand/receptor signaling in ELL-12-induced apoptosis in Jurkat and H9
cells.
View larger version (35K):
[in this window]
[in a new window]
| Fig 6.
ZB4 anti-Fas antibody antagonizes Fas-induced but not
ELL-12-induced cell death, DEVDase activity, and caspases-8 and -3 activation in Jurkat cells. (A) Extracts from Jurkat T cells treated
with 10 µg/mL ELL-12 for the indicated times were resolved by
SDS-PAGE and probed with anti-CD95L antibody. (B) Jurkat T cells were
pretreated with 300 ng/mL antagonist anti-Fas MoAb (clone ZB4) for 1 hour and then treated with 50 ng/mL anti-Fas MoAb or 10 µg/mL ELL-12
for an additional 5 hours. Apoptosis was assessed by Hoechst staining.
Results are the means ± SD of 3 independent experiments. (C)
Activation of DEVD-specific caspases was measured by the cleavage of
the fluorogenic substrate Ac-DEVD-AMC. Results are the means ± SD of
3 independent experiments. Cytosolic extracts were subjected to 15%
SDS-PAGE and immunoblotted with anti-caspase-8 (D) or anti-caspase-3
(E). () and (+) indicate cells treated without or with 300 ng/mL
antagonist anti-Fas MoAb (clone ZB4). The migrations indicated are
full-length caspase-8, the cleavage intermediates p43 and p41, the
active subunit p18, full-length caspase-3, the cleavage intermediate
p20, and the active subunit p17.
|
|
View larger version (26K):
[in this window]
[in a new window]
| Fig 7.
ELL-12 induces caspase-8 activation and apoptosis
independently of the CD95 receptor/ligand system in H9 lymphoma cells.
Extracts from H9 cells treated with 10 and 50 µg/mL ELL-12 or 50 ng/mL anti-Fas antibody for 16 hours were resolved by SDS-PAGE and
probed with anti-caspase-8 antibody (A) or anti-CD95L antibody (B).
Migrations indicated are full-length caspase-8, the cleavage
intermediates p43 and p41, the active subunits p18 and p16, and
full-length CD95L. H9 cells were pretreated with 300 ng/mL antagonist
anti-Fas MoAb (clone ZB4) for 1 hour and then treated with 50 ng/mL
anti-Fas MoAb or 10 and 50 µg/mL ELL-12 for an additional 16 hours
(C). Apoptosis was assessed by Hoechst staining. Results are the means ± SD of 3 independent experiments.
|
|
| |
CONCLUDING REMARKS |
ET-18-OCH3 and related lipids have been administered
intravenously and orally for treatment of leukemia, lymphomas, and
solid human cancers and topically for treatment of skin
metastases.6 Although beneficial responses were observed,
especially by the topical route, intravenous and oral dosing were
limited by serious gastrointestinal, hematological, and pulmonary side
effects. Liposome-associated ET-18-OCH3 (ELL-12) was
developed to reduce systemic side effects and therefore offers the
potential of enhanced activity against various tumors concomitant with
greatly reduced side effects.13-15
In this study, we report that ELL-12 triggers apoptosis via proteolytic
processing of caspases that was blocked by caspase inhibitors.
Treatment of Jurkat, H9, or U-937 cells with ELL-12 results in
activation of executioner caspases, leading to cleavage of PARP or
lamin B. Activation of the CD95 ligand/receptor system has been
implicated in apoptosis triggered by anticancer drugs such as
doxorubicin,63-66 bleomycin,67,68
cisplatin,68 cytarabine,65 etoposide,69 or methotrexate65,68 in a variety
of tumor cells. In contrast, stimulation of the CD95 ligand/receptor
system did not appear to account for activation of caspases induced by
ELL-12 inasmuch as CD95L expression was unchanged and obstruction of CD95 ligand/receptor interaction had no effect on ELL-12-induced cell
death in Jurkat and H9 cells.
A wealth of reports support the view that release of mitochondrial cyt
c may play an important role in the activation of the apoptotic
machinery.33,34 Cyt c release was detected in all cell lines treated with ELL-12, and cyt c release clearly
preceded activation of caspases and nuclear fragmentation. In addition, overexpression of Bcl-xL conferred protection against
ELL-12-induced apoptosis by blocking cyt c release, caspase-9
activation, and subsequent cleavage of caspases. Although the molecular
mechanism by which ELL-12 induces cyt c release is still
unknown, the results presented here suggest that release of cyt
c is probably not mediated by Bid. This pro-apoptotic member of
the Bcl-2 family has been shown to be activated by caspase-8 and then
translocated from the cytosol to the mitochondria, where its truncated
form (tBid) mediates the release of cyt c.57-59,70
However, we found that processing of caspase-8 and subsequent Bid
cleavage occurred downstream of mitochondrial events because
Bcl-xL totally inhibited their processing. Moreover, cyt
c release distinctly preceded caspase-8 processing in Jurkat
cells, negating a primary role for the caspase-8/Bid pathway in
inducing cyt c release. It is noteworthy that several anticancer drugs, including hydroxamic acid, vinblastine, and dexamethasone, have been shown to induce apoptosis and caspase-3 activation without affecting cyt c levels,71,72
indicating that triggering of apoptosis by chemotherapeutic agents can
occur at multiple sites in the cell death pathway.
In summary, our findings suggest that ELL-12 induces caspases
activation through release of cyt c in a
Bcl-xL-sensitive manner, but independently of the CD95
(APO-1/Fas) ligand/receptor system. Therefore, ELL-12 could be useful
as an adjunct for chemotherapy in cancer cells that have a defect in
upstream apoptosis pathways acting by bypassing the requirement of the
CD95 ligand/receptor system. Moreover, ELL-12 could also enhance the
action of anticancer drugs that act by a cyt c-independent
pathway by stimulation of cyt c-dependent apoptosis.
| |
ACKNOWLEDGMENT |
The authors thank Dr Donald Nicholson for caspase-3 antibody, Dr Edward
Gelmann for caspase-7 antibody, Dr Marcus Peter for caspase-8 antibody
and H9 cells, Drs Atan Gross and Stanley Korsmeyer for anti-Bid
antibody, Dr Junying Yuan for caspase-7 antibody, and Dr Charles
Zacharchuk for Jurkat/Bcl-xL cells.
| |
FOOTNOTES |
Submitted May 10, 1999; accepted July 16, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Sarah Spiegel, PhD, Department of
Biochemistry and Molecular Biology, Georgetown University Medical
Center, 353 Basic Science Bldg, 3900 Reservoir Rd, NW, Washington, DC
20007; e-mail: spiegel{at}bc.georgetown.edu.
| |
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C. Henderson, M. Mizzau, G. Paroni, R. Maestro, C. Schneider, and C. Brancolini
Role of Caspases, Bid, and p53 in the Apoptotic Response Triggered by Histone Deacetylase Inhibitors Trichostatin-A (TSA) and Suberoylanilide Hydroxamic Acid (SAHA)
J. Biol. Chem.,
March 28, 2003;
278(14):
12579 - 12589.
[Abstract]
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O. Cuvillier and T. Levade
Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria
Blood,
November 1, 2001;
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[Abstract]
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T. Kurita-Ochiai, K. Ochiai, and K. Fukushima
Butyric Acid-Induced T-Cell Apoptosis Is Mediated by Caspase-8 and -9 Activation in a Fas-Independent Manner
Clin. Vaccine Immunol.,
March 1, 2001;
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[Abstract]
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T. Wieder, F. Essmann, A. Prokop, K. Schmelz, K. Schulze-Osthoff, R. Beyaert, B. Dorken, and P. T. Daniel
Activation of caspase-8 in drug-induced apoptosis of B-lymphoid cells is independent of CD95/Fas receptor-ligand interaction and occurs downstream of caspase-3
Blood,
March 1, 2001;
97(5):
1378 - 1387.
[Abstract]
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O. Cuvillier, L. Edsall, and S. Spiegel
Involvement of Sphingosine in Mitochondria-dependent Fas-induced Apoptosis of Type II Jurkat T Cells
J. Biol. Chem.,
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[Abstract]
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ABSTRACT
INTRODUCTION